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Primary sjogren syndrome: Clinical and immunopathologic features
Seminars in Arthritis and Rheumatism NOVEMBER
VOL XIV, NO 2
Primary
Sjogren ByRobert
Syndrome: Clinical Features I. Fox, Francis V. Howell,
1984
and Immunopathologic
Robert C. Bone, and Paul Michelson
Primary Sjogren syndrome is an autoimmune condition in which dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia) result from lymphocytic infiltration of lacrimal and salivary glands. Clinical and laboratory features of 60 primary Sjogren syndrome patients seen at our clinic during the past three years are presented. These patients illustrate the wide spectrum of extraglandular features that may occur as a result of lymphoid infiltration of lung, kidney, skin, stomach, liver, and muscle. They further emphasize the difficulty in classifying a patient as primary or secondary Sjogren syndrome (ie, sicca symptoms associated with systemic lupus erythematosus, rheumatoid arthritis, or scleroderma), particularly early in the disease course. As an initial step in understanding the pathogenesis, the lymphocytes that infiltrate the salivary glands and lymph nodes
I
N 1888 Johann von Mikulicz Radecki described a 42-year-old Prussian farmer with enlargement of lacrimal and salivary glands “consisting of small round cells.“’ Subsequently, the name “Mikulicz Syndrome” was appended to any swelling of salivary glands due to lymphocyte infiltration, including a collection of pathologies that we now realize included tuberculosis, lymphoma, sarcoid, and salivary-duct inflammation associated with bacterial infections. After these etiologies had been eliminated, there remained a group of patients whose salivary and lacrimal gland lymphoid infiltrates were associated with autoimmune features such as hyperglobulinemia and autoantibodies. In 1933, Henrik Sjogren described the association of keratoconjunctivitis sicca (KCS), dry mouth (xerostomia), and rheumatoid arthritis.* Although this association of symptoms is generally known as Sjogren syndrome (SS), it is occasionally referred to as “Gougerout-Sjogren” syndrome to acknowledge the earlier report of such symptoms by Gougerout in 1925.3 In 1953, Seminars in Arfhritis and Rheumatism, Vol 14, No 2 (November), 1984
were characterized by using monoclonal antibodies that recognize distinct lymphocyte subsets and by using in vitro functional assays. These studies have demonstrated that affected tissues have infiltrates of T cells with helper/inducer activity and with a high frequency of “activation antigens.” The immunohistologic techniques are useful in differentiating “benign” and “pseudolymphoma” lesions (both due predominantly to T cells) from non-Hodgkin lymphoma (usually due to B-cell infiltrates). Although there is no “cure” for primary Sjogren syndrome patient’s symptoms may be significantly improved by measures aimed at prevention of ocular and dental complications and by the recognition of extraglandular features that may be amenable to specific treatment.
Morgan and Castleman demonstrated that a similar histologic type of infiltrate was present in both SS and Mickulicz disease.4 Bloch et al’ suggested that SS be subdivided into primary SS and secondary SS. Secondary SS was defined as patients with sicca symptoms in association with particular diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), From the Departments of Basic and Clinical Research, Oral Pathology, Head and Neck Surgery, and Ophthalmology, Scripps Clinic and Research Foundation, La Jolla, Calif. Robert I. Fox, MD, PhD: Department of Basic and Clinical Research; Francis V. Howell, DDS: Department of Oral Pathology; Robert C. Bone, MD: Department of Head and Neck Surgery; and Paul E. Michelson, MD: Department of Ophthalmology, Scripps Clinic and Research Foundation, La Jolla, Calif Supported in part by research grant No. AM/CA 33983 from the National Institutes of Health. Address reprint requests to Robert I. Fox, MD, PhD. Department of Basic and Clinical Research, Scripps Clinic, IO666 N Torrey Rd. La Jolla, CA 92036. 0 I984 by Grune and Stratton, Inc. 0049-0172/84/1402-0001%5.00/0 77
78
FOX
scleroderma (PSS), or dermatomyositis, while primary SS patients lacked these particular diseases6 Primary SS patients may frequently have extraglandular manifestations such as thyroiditis, pneumonitis, or interstitial nephritis.s ~9 In this review, we will describe the spectrum of clinical features, possible mechanisms of pathogenesis, and approaches to treatment of patients with SS. We will emphasize findings in primary SS since these patients are usually not receiving corticosteroids, immunosuppressive drugs, or remittive agents (ie, gold, penicillamine) that may alter the underlying pathologic mechanisms. These patients provide an opportunity to safely perform biopsy procedures on the actual site of tissue damage (ie, the salivary gland) to characterize the infiltrating lymphocytes. Since primary SS patients share a similar histocompatibility leukocyte antigen, HLA-DR3, and immunopathologic features with diseases such as juvenile onset diabetes mellitus, celiac sprue, thyroiditis, and Addison disease,’ the study of primary SS may provide insight into these other diseases where the target organ is not readily accessible to biopsy. Finally, patients with SS have a significantly increased prevalence of lymphoma,‘“,” so that study of primary SS may give clues to factors which predispose to lymphoid malignancy. Clinically, increased interest has been focused on SS by (1) the recognition that xerostomia and KCS are more frequent problems than previously recognized”,” and (2) that relatively little information is available on the treatment of these patients. Therefore, the patient is often left with the discouraging feeling that “nothing can be done.” Although there is no “cure,” symptomatic relief may be achieved by early recognition of the extraglandular manifestations and by careful attention to the prevention of ocular and dental complications. We will discuss the multidisciplinary approach to evaluation and treatment used by the divisions of rheumatology, oral medicine, ophthalmology and otolaryngology at the Scripps Clinic.
using rose bengal and/or fluorescein dye,’ ” and a dry mouth. We will limit our discussion to those subjects with a minor salivary-gland biopsy with grade IV infiltrate (classified on a scale from 0 to IV as described below) (Table I). However, the patients who fulfill these criteria remain heterogeneous in their clinical and laboratory features. Several examples of the “types” of patients who might be referred for further evaluation because of a grade IV lip biopsy include the following examples. I. The “traditional” primary SS patient who is a woman (age, 30 to 65 years) with hypergammaglobulinemia, autoantibodies (usually rheumatoid factor [RF] and/or antinuclear antibody [ANA]), anemia, and elevated sedimentation rate.’ They share an increased frequency of histocompatibility antigen HLA-DR3,’ suggesting a common genetic feature of pathogenesis. They frequently have extraglandular manifestations of nonthrombocytopenic purpura, thyroiditis, interstitial nephritis, or pneumonitis. Based on the operational definition of primary SS. these patients do not have sufficient clinical features to be classified as SLE, RA, polymyositis, or PSS. 2. Elderly patients (~80 years old) with absent tears, dry mouth, and a lip biopsy with acinar atrophy and with sufficient lymphocytic infiltrate to be class IV. They usually lack autoantibodies, hyperglobulinemia, and extraglandular features. Although these patients satisfy the criteria for primary SS, their relationship to the previous group of primary SS patients
Table 1. Features of Primary and Secondary Primary
SS
SS
KCS Schwmer
test with
Positive
rose bengal
< 10 mm
wetting/5
or fluorescein
min
staining
of cornea
and conjunctiva Xerostomia Decreased ~0.5 Grade
flow
I-IV)
extraglandular
biopsy
features
pneumonttis,
associated
rate using Lashley
cups wth
min
IV (on scale
anemia.
Secondary
parotid
mLl5
Although
of minor
salwary
(thyroidltis,
neuropathies,
gland
nephntls.
etc) may occur.
no
RA, SLE, or PSS
SS
KCS and xerostomia
PROBLEMS IN THE DEFINITION OF SS
Exclusions:
To be classified as SS, patients must have dry eyes with decreased tearing (Schirmer test t10 mm wetting/5 min), KCS on ophthalmic exam
ET AL
plus RA, SLE. polymyosms,
sarcoidosls.
lymphoma,
pemphigoid.
or PSS infec-
t1ons* *Additional other
than
causes
of keratitis
SS are presented
and salivary
in Table
6.
gland
enlargement
79
SJOGREN SYNDROME
remains unclear as they usually lack other features suggesting an autoimmune process. 3. Patients with secondary SS who have sicca complaints associated with characteristic features found in RA, SLE, polymyositis, or PSS.556 Such patients originally may have been thought to have primary SS, but evaluation of their clinical course over subsequent years shows the development of characteristic features of the other diseases. The distinction between primary and secondary SS associated with RA or SLE will be discussed. 4. Patients with leukemia or lymphoma may first complain of dry eyes and dry mouth due to lymphocytic infiltration. The presence of such diseases serves to exclude the diagnosis of SS.’ However, the relationship of these lymphomas to SS is intriguing since approximately 10% of salivary-gland lymphomas occur in the setting of preexisting SS14 and patients with SS have a markedly elevated incidence of extraglandular lymphoma.9~‘0 5. Patients with prior radiation to the head and neck, frequently for Hodgkin disease, may have dry eyes and xerostoma. In several cases, we and othersI have noted a grade IV lymphocytic infiltrate in their salivary gland biopsies. The radiation often antedated these symptoms by several years, and there was no evidence for recurrence of tumor. Although the history of prior lymphoma would technically exclude the diagnosis of SS, such patients may provide insight into pathogenetic mechanisms and may benefit from a therapeutic approach similar to that in primary SS. 6. Some patients have a grade IV minor salivary gland biopsy and autoantibodies (such as ANA, Sjogren syndrome A (SS-A), and/or Sjogren syndrome B (SS-B) but do not have complaints of dry eyes. The normal Schirmer test and ophthalmologic examination do not allow them to be classified as SS. Lack of ocular complaints suggests that their lacrimal glands are not as severely injured as their salivary glands. Conversely, some patients develop severe KCS but have normal salivary-gland biopsies and parotid flow rates. Further studies are necessary to determine if these patients are forme fruste of SS or represent a distinct entity. The distinction between primary and secondary SS associated with RA is usually not diffi-
cult. RA patients have characteristic joint symptoms and radiographic features.16 However, a possible source of confusion is the patient with sicca complaints, arthralgias, and positive RF that has not developed objective synovitis on examination or erosive changes on roentgenograms during many years of clinical symptoms. Thus, it is important to recognize the diagnosis of primary SS because (1) the patient can be reassured that a positive RF occurs in conditions other than RA, (2) potentially hazardous drug therapies (ie, remittive agent used in advanced RA) may be avoided, and (3) attention is directed to conservative measures to prevent ocular and dental complications of primary SS. Several features suggest that the pathogenesis of primary SS may differ from that of secondary SS plus RA (Fig 1). Patients with primary SS have a high frequency of histocompatibility antigen HLA-DR3 and lack antibodies to salivary gland ducts; in contrast, RA patients with secondary SS have increased frequency of HLA-DR4 and possess antibodies to salivary ducts.‘,” On the other hand, the distinction between primary and secondary SS associated with SLE is often more difficult. The recently revised crite-
Overlap of Clinical Syndromes Rheumatoid
Siouren
Systemic
Arthritis
Syndrome
Erylhemalosus
Lupus
Shared Features +
I. autoantibodies 2. Msis;;opatibilily
rheumatoid HLA-OR4
laclor
I ANA, SSA, SSB HLA-OR3
Fig 1. Patients with sicca complaints may present with characteristic extraglandular features. These patients may be grouped into particular subsets that differ in genetic predisposition end mechanisms of pethogenesis. Patients with secondary SS and SLE have increased frequency of histocompatibility antigen HLA-D3. This marker has also been found in increased prevalence in patients with primary biliary cirrhosis and the CREST variant of scleroderma: these populations have a high incidence of sicca symptoms. In contrast secondary SS plus RA patients have increased HLADR4 and antibodies to salivary duct antigens, features not found in most primary SS patients.
80
FOX
ria for diagnosis of SLE are listed in Table 2. Patients with SLE and primary SS have an increased prevalence of ANAs (including SS-A and SS-B), leukopenia, anemia, and elevated sedimentation rate.s.‘8.‘y Arthralgia, myalgias, pneumonitis, and nephritis also may be present in both groups. The overlap of laboratory and clinical features in primary SS and SLE patients suggests that similar pathogenetic mechanisms may be operating, and this is supported by the Table
2.
The
1982
Revised
Criteria
of SLE 1.
Bufferfly
2.
DIscold
for
the
Classification
1982)
rash lupus
3.
Photosensitivity
4.
Oral ulcers
5.
Arthritis’
6.
Serositls a.
(ARA
rub heard
by a physician
or pleural
effusion,
or b.
Perlcardms:
documented
of pericardial
7.
8.
Renal
disorder
Persistent
b.
Cellular
casts:
tubular.
or mlxed
Neurologlc
proteinurla
Sewres:
b.
20.5
g/d
may be red cell, hemoglobin,
granular,
disorder
known
9.
by ECG or rub or ewdence
effusion
a.
a.
In the absence metabolic
Psychosis
of offending
drugs
or
derangements
in the absence
Hematologlc
of offendlng
drugs
disorder
a.
Hemolytic
b.
Leukopenla:
anemia:
with
i4.000
retlculocytosis
cells/fiL
or
on two
or more
occa-
SlO”S or c.
Lymphopenla: caslons
d.
- 1,500
Thrombocytopenta:
on two
or more
oc-
i
lOO.OOO/~L
in the absence
of
drugs
lmmunologlc
disorder
a.
Positive
b.
Anti-DNA:
LE cell preparation presence
in abnormal c.
cells/KL
or
offending 10.
Anti-Sm:
or
of antlbody
to untreated
DNA
to Sm nuclear
antl-
titer or presence
of antibody
gen or d.
False-positwe six months
11.
Armnuclear antlbody
antibody.
in ttme
to be associated NOTE.
STS
inhibttion, *The purpose
FTA
STS known
to be positwe
and confwmed
An abnormal
“drug-induced
= Serologic
test
= fluorescent
proposed
pattents
lupus”
is based in clinical
on
of the any
Reprinted
with
permtssion.‘6
during
syndrome*
TPI
= treponemal
1 1 criteria.
studies,
serially
simultaneously,
assay known
antibody.
said to have SLE if any four or more or
of drugs
for syphilis,
treponemal
classification
of identifying
ttter of antmuclear or an equwalent
and in the absence
with
for at least
by TPI or FTA tests
by immunofluorescence
at any point
finding of a high frequency of histocompatibility antigen HLA-DR3 in both primary and secondary SS associated with SLE.‘“,” However, primary SS patients lack sufficient features of SLE to meet the criteria for this diagnosis (Table 2), although it is possible that some primary SS patients represent a subset of SLE with a limited-disease expression. In summary, SS is not a single disease entity. Among the primary SS patients, only some will have particular extraglandular features or autoantibodies. It is important to recognize this heterogeneity of patients when attempting to predict prognosis or discuss mechanisms of pathogenesis. CLINICAL
Pleuntis:
a person
1 1 criteria
interval
of
For the shall be
are present observation.
ET AL
FEATURES
OF PRIMARY
SS
A brief review of the clinical features of primary SS is presented to point out the wide spectrum of extraglandular features that may occur in patients who may initially present with oral or ocular symptoms. Patient Populatioms The clinical and laboratory characteristics of 55 patients with primary SS seen in the past three years in our clinic are shown in Table 3. The median age of our patients was 56 years (range, 17 to 88 years). Their histories suggested a mean disease duration of seven years. Approximately 25% of these patients were first seen in the rheumatology clinic with complaints of arthralgia, myalgia, and fatigue, while 25% were seen in ophthalmology for ocular discomfort, and 50% in oral medicine because of xerostomia and increasing dental decay. There was a predominance of women (only one man). The age of onset and duration of symptoms for our patients was similar to those at National Institutes of Health (N IH) studied by Bloch et al5 and in Glasgow by Whaley et al.” In virtually all of these primary SS patients, medications at the time of biopsy were limited to salicylates or other nonsteroidal drugs. None had a history of prior cytotoxic drugs or irradiation. In several patients, treatment with low-dose corticosteroids (_=IO mg/d prednisone) had been used but had been discontinued at least three months prior to biopsy. The clinical characteristics of several patients with secondary SS in whom a grade IV lip biopsy
81
SJOGREN SYNDROME
Table 3. Clinical and Laboratory
Features
of Patients With Primary SS
Patient LetteE
RF
Aoe ivrl
ANA
SS-A
ss-6
+ +
+ + _
Other Features
Is%
Predominantly salivary/lacrimal symptoms GO
21
La
24
Br
68
10,246 ...
256 256
VU
71
320 ...
CP Ve
60
...
. . .
_ _
68
...
SC
70
...
... ...
_
Sa
66
...
160
160 320
2,000
Pseudolymphoma
...
4,400 1,550
Parotid
...
1,150
_
1,460
Parotid
... 615
_
Pseudolymphoma parotid
...
1,400
Myalgias/arthralgias symptoms
ct
56
Dr
53
Be
39
Ar
56
640
256
co We
77 ...
640
. . .
Me
61
MO
75
We Ch
52 ...
Pa MO
72 ...
Gr
79
... ... ...
... ... ... ...
... ...
256 256 . . .
+ _ _ _
1,250
Myalgia
1,550
Myalgia
1,052
Myalgia
2,200
Myalgia
1,250
Myalgia
1,400
Arthralgia
1,400
Arthralgia
1,500
Arthralgia
. . . . . .
256
_ _
256
+
. . .
_ _ _
...
...
CO
58
640
256
Th
60
640
256
Dermal
+ _ _ +
+ +
1,200
Arthralgia
+
1,350
Arthralgia
+ + + f +
+ + +
2,600
Purpura
3,450
Purpura
2,750
Purpura
5,220
Purpura
2,100
Purpura
+ + + + _ _ + _
+ + + + _ _ _ _
2,200
Thyroid
1,960
Thyroid
2,527
Thyroid
+ _
+ _
4.050
manifestations
BI
17
2,560
256
Cl
67
1,280
256
Pe
60
5,120
256
Si
17
2,560
256
Tr
57
...
256
Endocrme
manifestations 640
256
Sh
45
SU
49
. . .
256
Ph
48
1,280
256
Me
. . .
. . .
64
Be
39
...
Bu
70
... ...
64
Wa
72
640
64
On
52
320
. . .
Pulmonary
...
Thyroid
1,400
Thyroid
1,300
Pernicious
1,400
...
Pernicious
anemia
anemia
Diabetes
mellitus
Pseudolymphoma
thyroid
manifestations
Cr
53
1,280
256
RY
72
640
64
820
Pulmonary Nodular
fibrosis infiltrates,
pleural
pseudolymphoma Ar
68
. . .
320
St
74
2,560
256
co
49
No
60
Renal,
neurologic
640
256
2,560
256
_ + + +
+ _ +
+ _ +
_ _ + +
_ + _
1,200
Pulmonary
hypertension
1,450
Pulmonary
fibrosis
2,400
Pleural
1,400
Pulmonary fibrosis
4,050
RTA
1,500
Peripheral neuropathy
1,600
Headaches
effusions
manifestations
Fo
79
Wa
60
160
Wi
53
160
Gastrointestinal
256
5,120
+ + + +
... 320
manifestations
Th
52
. . .
160
Sa
62
. . .
160
Ha
61
Wi
61
640
...
256 256
2,200
BC
1,800
Sclerosing cholangitis
1,400
Sprue
2,000
Hepatitis
effusions,
82
FOX
Table 3.
ET AL
I Continued)
PatM?tlt LetteE Patients
RF
Age (~1 >80
yr with
primary
ANA
SS-A
SS-B
Other Features
bG
SS
Al
88
...
...
1.200
Arthralgla
MO
92
. . .
. . .
1,550
Myalgia
Wa
a2
. . .
.
1,400
8a
85
..
De
81
. . .
NOTE.
Parotid
refers
to tender,
swollen
.
. . . . parotid
gland;
thyroid
...
1,400
Thyroid
1,200
Arthralgla
to hypothyroidism;
RTA
to renal
tubular
acidosis:
and
8C
to biliary
cirrhosis.
was found are presented in Table 4. The relatively small number of secondary SS patients is related to the fact that we do not do lip biopsies in most secondary SS patients as a routine part of our research study. Many of the secondary SS patients listed in Table 5 have received corticosteroids and/or remittive drugs that may alter the underlying disease process. Autoantibodies
in SS
ANA are found in the majority (70%) of our primary SS patients. The pattern of immunofluorescence is usually fine speckled, suggesting the presence of antibody to nonhistone nuclear proteins.22 The pattern of immunofluorescence can vary with the tissue substrate (ie, mouse kidney, KB cell, or HeLa cell). Also the results may be influenced by the high solubility of some nonhistone antigens that allows them to be leached from the nuclei during washing procedures.‘X Therefore sera containing antibodies to SS-B or nRNP are sometimes reported to be negative for ANA. Further, certain nuclear antigens may be destroyed by some fixatives while other antigens maintain reactivity. This may prove important in using commercially available ANA kits where fixatives have been used as preservatives.” ANA may be directed against DNA (single or double stranded), histones, or nonhistone proteins. Antibodies to DNA or histones are relatively rare in primary SS patients and their presence ought to suggest the presence of SLE. Exciting progress has been made in recent years in characterizing autoantibodies directed against nonhistone proteins.‘* The majority of primary SS patients possess antibody to SS-A antigen and about 50% of these patients have antibody to SS-B. These antibodies may be of the IgG, IgA, or IgM isotype in serum and saliva.23,24 The
molecular weight of the SS-A antigen is not known but the SS-B antigen is approximately 50,000 daltons24m2B” and can be isolated from human thymus, spleen, liver, and lymphoblastoid cell lines. It is also found in certain tissues from dog, calf, and rat. In uninfected cells, the SS-B antigen associates with a mixture of small RNA molecules. On cellular infection with adenovirus or Epstein-Barr virus, the SS-B antigen specifically complexes with viral associated RNA.“.19
Table
Clinical and Laboratory
4.
Secondary
Features
SS or Malignancy
Pi3tlWlt Letters
of Patients With
Plus Sicca Assocmted
Am
k~l
RF
ANA
SS-A SS-B
t
IqG
DlSeaSe
1,500
RA RA
Pa
32
320
Go
42
640
80
1,400
PC
64
5,120
250
1,400
GI
43
640
160
Sh
52
5,120
64
Hal
51
2,560
16
Hag
65
5,120
.
Ka
60
1,280
~~
RA RA
+
1,100 929
Cop
77
640
.. ..
8ac
66
5.120
256
+
RA RA
1,400
RA
1,600
RA
~
1,800
RA
t
1.530
RA
Is
30
640
256
t
t
2,200
SLE
St
40
640
320
+
+
1,400
SLE
HU
29
160
160
t
Ba
39
640
256
*
La
42
320
256
+
Ja
72
1,280
Do
60
...
so
50
640
Gr
48
640
co
53
.
.. . . ...
ZI
80
De
60
... ...
... ...
MO
65
Ma
60
640 ...
De
22
Gr
22
3,100
SLE
-
1.900
SLE
+
1,800
SLE
256
1,400
PSS
256
1,200
PSS
~
~~
1,000
Dermatomyosltls
1,400
Dermatomyosttls
1,230
Dermatomyosms
1,200
T-CLL
-
1,000
T-CLL
256 ...
+
450
Lymphoma
800
Lymphoma
...
...
~
900
Lymphoma
...
...
~
800
C-GVHD
83
SJDGREN SYNDROME
The common element among these disparate RNA species is their synthesis by RNA polymerase III.3o Another interesting feature of the SS-B antigen is that its location within the cell changes during different phases of the cell cycle.3’ During Go phase, the antigen is located in the nucleoplasm, but during S phase the localization becomes nucleolar. The significance of autoantibodies to SS-B or SS-A antigens in pathogenesis is unclear. Their detection may prove helpful in suggesting the diagnosis of SS, especially in patients where multisystem involvement may overshadow the sicca complaints.25 An association between the presence of anti-SS-A and vasculitis in primary SS patients has been reported by Alexander et aL3* The presence of extraglandular features also has been correlated with antibody to SS-B.24 Detection of other nonhistone proteins such as Sm and Scl-70” is also important, since it should suggest the presence of associated SLE or PSS, respectively. Organ-specific autoantibodies, such as antisalivary gland, are infrequent in primary SS patients.33 They have been reported in the majority of RA patients with secondary SS, suggesting that they may be an epiphenomena rather than playing a role in pathogenesis. In this regard, primary SS differs from organ specific autoimmune diseases such as thyroiditis, myasthenia gravis, and diabetes mellitus where antibodies to target organs can be found early in the disease process. Finally, RFs (antibodies to IgG) are consistently found in primary SS patients’ saliva, tears, and serum.34%3sThey may be of IgM, IgG, or IgA isotypes. Their role in pathogenesis is unclear. It has been suggested that their synthesis is normally induced to amplify other weak antibodyof antigen reactions36 or to aid in clearance immune complexes.37 The persistence of high titers of RF in primary SS patients may reflect continued antigenic stimulation, elevated immune complex level, or failure of the normal suppressive mechanisms to inhibit further RF synthesis. Ocular Features Patients generally complain of a burning and/ or foreign body sensation in their eyes, although a variety of our ocular complaints and signsI can be encountered (Table 5). Fewer than 50% of the
Table 5. Clinical Features
Clinml
Features
of KCS
SS Patients
Control Patients
(N = 64)
IN = 541
Symptoms Foreign-body sensation
81
4
Burning
80
4
Tiredness with or without diffi-
67
9
67
2
culty in opening the eyes Dry feeling with or without a poor response to physical or chemical irritants and emotions Redness
48
7
Difficulty in seeing
31
11 19
Itchiness
37
Aches
36
7
Soreness or pain
23
4
Photosensitivity and excess of
31
4
31
7
secretion which may appear to be watery, ropy, or as a film over the eye Signs Dilatation of the bulbar conjunctival vessels (usually interpalpebral) Mild pericorneal injection
36
0
Photophobia
47
13
Irregularity of the cornea1 image
63
5
White and frothy or Yellow and
22
0
27
0
11
0
tenacious discharge Dullness of the conjunctiva and/ or cornea Ptosis
NOTE. Data are percentage of patients with that clinical feature. Data taken from Whaley et al.”
patients report eye pain or photosensitivity. We ask the patients to grade the symptoms on a scale from 0 to 4: 0 for no symptoms, 1-t for mild irritation and burning, 2+ for redness and itching, 3+ for pain and photophobia that mildly limit function, and 4-t for severe pain and photophobia that dramatically limit functional activity. We encourage our patients to keep a diary of their symptoms on a weekly basis to identify the general tempo of their disease and recognize factors that may lead to exacerbation. The symptoms of sicca are nonspecific and are influenced by environmental irritants, medications (especially those containing anticholinergic or antihistamine activity), and emotional upset. However, it is important to identify these factors so that they may be altered to improve the patient’s functional status3’ Signs of KCS include the dilation of bulbar conjunctival vessels (especially interpalpebral),
a4
pericorneal injection, ptosis, and tenacious discharge that may form filamentary strands. Biomicroscopic examination often shows attached cornea1 filaments, abnormal amounts of precorneal debris, and superficial punctate keratitis5,‘2 as well as larger epithelial erosions. The diagnosis of KCS should be confirmed by instilling a 1% aqueous solution of rose bengal or fluorescein dye into each eye. The eyes are then examined and with the slit lamp to determine areas of punctate keratitis (Fig 2). Staining localized to areas previously in contact with the filter paper used in Schirmer test should be discounted. Keratitis, including punctate or filamentary changes, is not specific for SS; it can occur in patients with pemphigoid, sarcoidosis, trauma (such as contact lenses), or infections of bacterial, chlamydial (ie, trachoma), and viral etiology (Table 6). Neuropathy on a congenital basis (Reily-Day syndrome) or acquired basis (ie, damage to 5th cranial nerve) can lead to keratitis. In patients with known SS, a sudden flare of ocular symptoms should always raise suspicion of cornea1 erosion or infection. Oral Features
Virtually all patients describe a dry mouth necessitating increased fluid intake, particularly when eating dry foods such as crackers. In our series, 30% gave a history of parotid gland swelling which was unilateral and intermittent. Twenty percent had an enlarged parotid and/or submandibular glands on examination on at least one occasion. Bilateral parotid glands that
Fig 2. Schematic representation of KCS in a primary SS patient as visualized by rose-bengal staining. The left panel shows increased interpalpebral staining with fine punctate lesions. In the right panel, more severe imflammation is associated with coalescent erosions and filamentary strands on the cornea. These strands should be distinguished from mucoid strands that are often present in the bulbar conjunctiva but that may occasionally migrate to the cornea region during examination. These patients may also suffer cornea1 abrasions.
FOX
ET AL
Table 6. Causes of Keratitis and Salivary Gland Enlargement
Other Than SS
Keratitis
1. Mucus 2. 3.
membrane
Infections:
virus (adenovirus,
ria, or chlymdia 4.
Trauma tant
herpes,
vaccinia),
bacte-
tie, trachoma)
(ie. after
including
lights 5.
pemphrgord
Sarcoidosis
contact
chemrcal
lens) and envrronmental burns,
exposure
in-
to ultraviolet
or roentgenograms
Neuropathy
includmg
to 5th cranial
nerve)
neurotropic and familial
keratitis
he, damage
dysantonomia
(Reily-
Day syndrome) 6.
Hypovitaminosis
7.
Erythema
Salivary
gland
1.
Sarcoidosis. Bactenal
gonococci
tie, infectious
Tuberculosis,
and syphilis)
mononucleosis,
actinomycosis.
and vwal
mumps)
histoplasmosis,
tracho-
leprosy
4.
Iodide,
5.
Hyperlrpemrc
6.
Tumors mor).
syndrome)
amylordosrs
(including
infecttons
ma,
(Stevens-Johnson
enlargement
2.
3.
A
multiforma
lead,
or copper states,
(usually
epithelial
ma, and mrxed
hypersensitwrty especially
unilateral) (adenoma. salivary
types
including
IV and V cysts
adenocarcinoma),
gland
(Warthrn
tu-
lympho-
tumors
remained enlarged for at least 12 months were seen in only seven patients: four with pseudolymphoma, two with recurrent bacterial parotitis superimposed on primary SS and only one with uncomplicated primary SS. These findings are comparable to those reported by Bloch et al and Whaley et al,‘,” where approximately 20% had a history of unilateral parotid enlargement, 10%’ had bilateral parotid enlargement, and 5% had submandibular gland swelling. Thus, the majority of primary SS patients do not have significant parotid or submandibular enlargement. Lymphocytic infiltration is present in biopsy specimens of the major salivary glands in SS patients even when swelling is not clinically apparent.j9 4’ Salivary gland enlargement can occur in diseases other than SS (Table 6). Diseases such as sarcoidosis, amyloidosis, infection (including bacterial, fungal, viral), and tumors must be considered. In previous years, reports of an unusual form of sarcoidosis called uveoparotid fever (Heerfordt disease) were common.4’ These patients had parotid enlargement and ocular symptoms but often lacked other features of sarcoidosis (hilar lymphadenopathy, diffuse lung infiltrates, skin sarcoid or hepatosplenomegaly). It is likely that some of the cases published as examples of Heerfordt disease were instances of
85
SJOGREN SYNDROME
SS4’ An SS-like syndrome with salivary gland involvement may occur after bone marrow transplantation.42 It has been proposed that this may be due to chronic stimulation of the immune system by allogeneic cells or to opportunistic viral infections.” A sudden increase in salivary gland pain or size should suggest the possibility of infection, which is more frequent in these patients due to inadequate salivary secretion and ductal lavage. The most frequent offending organisms are Staphylococcus, Streptococcus viridans, Streptococcus hemolyticus, and Pneumococcus which are normal flora of the oral cavity.4’ Cultures must be taken of the pus escaping from Stensen or Wharton duct. Salivary gland infections must be treated promptly to prevent formation of abscesses. A high frequency of stomatitis due to Candida albicans is also present in SS patients, particularly after receiving antibiotics for other illnesses.
Table 7. Extraglandular
Manifestations
Respiratory Chronic bronchitis secondary to dryness of upper and lower airway with mucus plugging Lymphocytic interstitial pneumonitis Pseudolymphoma with nodular infiltrates Pleural effusions Pulmonary hypertension Lymphoma Gastrointestinal Dysphagia associated with xerostomia Atrophic gastritis Liver disease including biliary cirrhosis and sclerosing cholangitis Skin Vaginal dryness Hyperglobulinemic purpura-nonthrombocytopenic Raynaud phenomena Vasculitis Renal Interstitial nephritis and renal tubular acidosis Glomerulonephritis-in
absence of antibodies to DNA
Endocrine, neurologic. and muscular ThYroiditis Peripheral neuropathy-symmetric
involvement of Extraglandular
Sites
Pulmonary. Involvement of exocrine glands in the upper respiratory tract leads to dryness of the nasal passages in approximately 50% of the patients (Table 7).13 In a comprehensive review, Hunninghake and Fauci43 emphasized the high incidence of pulmonary abnormalities in patients with SS, including pleurisy with and without effusion, interstitial fibrosis, dessication of tracheobroncheal mucous membrane, and lymphoid interstitial disease. Clinical studies of pulmonary function in primary SS patients have yielded conflicting results. Newball et a144 and Siegal et a14’ found evidence of mild to moderate obstructive airway disease in almost 50 of their primary SS patients. In another study, Oxholm et a146did not find obstructive changes in any of 43 patients with primary SS, but did note decreased diffusing capacity in patients with prior pneumonia or pleurisy. Pathologic changes in the lung of primary SS patients include atrophy of mucous glands, tenacious secretions, focal atelectases, recurrent infections, and increased frequency of bronchiectasis.43-45 These complications appear to result from infiltration of the bronchial glands by lymphocytes in a nodular or diffuse pattern.46A8 Although this article deals primarily with primary SS patients, it should be noted that non-
in Patients
With Primary SS
involvement of hands
and/or feet Mononeuritis multiplex Myalgias Hematologic Pseudolymphoma Angioblastic lymphadenopathy Lymphoma and myeloma Lymphopenia and neutropenia Aplastic anemia
smoking RA patients with secondary SS have a higher incidence of airway disease than nonsmoking RA patients lacking sicca sympon chest toms.49150 The presence of infiltrates roentgenogram in primary SS patients may indicate the emergence of lymphoma or infection in these patients. We have noted one primary SS patient with primary pulmonary hypertension, a condition that has not previously been reported with primary SS, but has been associated with PSS ” SLE 52and RA.53 Gktroiniestinal. Difficulty in swallowing is frequent in primary SS patients, due primarily to severe dryness of the esophagus as a result of lymphocytic infiltration and destruction of the submucosal glands.54 Abnormal esophageal motility (particularly in the upper one third of the esophagus) may also contribute to dysphagia in some primary SS patients.55 Gastric biopsy specimens show chronic atrophic gastritis and lym-
86
phocytic infiltrates.56 Patients with marked gastric involvement and hyposecretory states can be recognized by their elevated serum pepsinogen leveIs.57 Investigation of pancreatic function has shown impaired response to secretin and pancreozymin, suggesting subclinical pancreatic disease.5R,59 Clinical and/or biochemical evidence of liver disease is found in 5% to 10% of primary SS patients.60 Patients with positive antibody to smooth muscle or antibody to mitochondrial were found to have a higher prevalence of hepatomegaly, splenomegaly, and abnormal liver function tests.@‘* Liver biopsy specimens in these patients show nodular lymphoid infiltrates and cholangitis. These findings suggest that the pathogenetic process responsible for salivary gland destruction and damage to the exocrine hepatic apparatus may be similar. The high incidence of SS in patients with primary biliary cirrhosish”63 lends support to this concept. Drugs such as methyldopa, isoniazid, nitrofurantoin, oxyphenasatin, aspirin, and other nonsteroidal antiinflammatory drugs may contribute to liver dysfunction. Cutaneous, renal, and endocrine. Dryness of the vagina and consequent vulvar pruritis are common problems and often contribute to disturbing symptoms of dyspareunia. This has been attributed to chronic inflammation of the exocrine sweat glands.6,h4 Dependent nonthrombocytopenic purpura was present in about 10% of our patients and was associated with hyperglobulinemia.h5 Raynaud phenomena occurred in approximately 20% of our primary SS patients; these patients also have an increased frequency of telangiectasia although nail-fold changes, calcinosis or digital ulceration were rare. Renal tubular acidosis (especially type II) has been associated with SS; histologic examination of kidney shows interstitial infiltration of lymphocytes.66.h7 The lymphocytes appear to invade the renal parenchyma and immunologically damage the tubules. Similar infiltrates have been noted in patients receiving renal allografts.6x Another renal manifestation in some SS patients is immune complex, hypocomplementemic glomerulonephritis.69 These patients, in contrast to SLE, do not have antibodies to DNA in their serum.‘7,h9 Clinically apparent hypothyroidism has been
FOX ET AL
reported in 10% to 15% of SS patients6.“.” and occurred in a similar proportion of our patients. Antibody to thyroglobulin, antibody to thyroid microsomal, and thyroid-stimulating hormone levels may be elevated in 40% to 50% of patients”.” suggesting that subclinical thyroid damage may be relatively common. Thus endocrine as well as exocrine glandular cells may be targets for immune attack in primary SS. The known similarity in genetic background, HLADR3, and histologic similarities between Hashimoto thyroiditis and primary SS supports the hypothesis that a similar mechanisms may be operating in each disease. Insulin dependent diabetes mellitus’ and pernicious anemia” occur with similar frequency in primary SS to that noted in the general population. Hematologic abnormalities. Many primary SS patients have a normocytic. normochronic anemia,‘,’ with low serum iron and transferrin levels as in other chronic inflammatory diseases. Occasionally bone marrow aspiration shows decreased iron stores that probably result from chronic gastorintestinal blood loss associated with use of salicylates and nonsteroidal antiinflammatory drugs. Leukopenia (total WBC count ~~4,000 cells/ pL) was present in 30% of our patients; previous reports have noted leukopenia in 6% to 33%:. of their patients.‘.” The mechanism of this leukopenia remains unclear but may involve antibodies to leukocytes, splenic sequestration, or abnormal bone marrow maturation in certain patients.” Certain patients have marked lymphadenopathy and/or salivary gland swelling 5.6.‘o,7’~74 (Fig 3). Since their biopsies do not fulfill criteria for malignancy, these patients’ syndrome has been termed “pseudolymphoma.” Other forms of nonmalignant lymphoid proliferation in primary SS patients include thymomah and angioimmunoblastic lymphadenopathy.” In both pseudolymphoma and angioblastic lymphadenopathy, there appears to be a high frequency of progression to frank lymphoma.‘0~7h The relative risk of developing lymphoma for primary SS patients has been estimated to be approximately 40-fold higher than age-, sexmatched control subjects by investigators at the NIH.” However, Whaley et al” in Glasgow did not find such an increased prevalence. These discrepancies may be attributed to several fac-
SJOGREN SYNDROME
87
mucosa are more likely to form scar tissue and to encounter anatomic structures such as blood vessels and nerves. As shown in Fig 4, the lower lip is reflected and local anesthetic (0.5% marcaine with epinephrine as a vasoconstrictor), administered at least 1.5 cm posterior to the site where the biopsy will be taken (frame 1). With a scalpel, a l-cm incision was made through the mucosa (but not
Fig 3. Massive bilateral parotid gland enlargement in patient IA. with pseudolymphoma and SS. Clinical and histologic features of this patient and three additional pseudolymphoma are presented in reference 74.
tors including the relatively small numbers of patients reported with lymphoma and ascertainment bias in patient referral patterns. However, these differences also may reflect the difficulty in distinguishing lymphoma from extensive infiltrates due to “benign” SS or pseudolymphoma.74 We are currently using specific monoclonal antibodies (described below) to help make this distinction. Finally, the differences may be related to treatment patterns since some patients may have been treated with cytotoxic agents or irradiation that may increase the frequency of lymphoma.“,” THE SALIVARY GLAND BIOPSY
Procedure for Obtaining Minor Salivary Gland Biopsies We obtain minor salivary biopsies from the intraoral mucosal surface of the lower lip just below the corner of the mouth (ie, the commissure). Biopsies from the midline of the oral
Fig 4. Procedure for obtaining minor salivary gland biopsy. Frame 1 shows the site where the biopsy will be taken after local anesthesia. In frame 2, the glands are everted through the skin by gentle pressure and separated from underlying muscle by careful disection. Frame 3 shows black silk sutures to close the incision.
88
to enter the submucosa), and the glands were everted by gentle pressure on the outer lip (frame 2). The scalpel undermines the mucosa and the protruding glands are removed by separation from the muscle with care to avoid the sensory nerves. Black silk sutures are used because absorbable sutures may loosen or be absorbed too rapidly. The sutures are removed in approximately one week. No significant morbidity has been noted (eg, sagging of lip); however, 10% to 20% may note hypoesthesia in the region of the biopsy. Because of potential hazard, biopsy of the major salivary gland is recommended only when there is a suggestion of intraparotid malignancy or serious diagnostic doubt. Changes in the minor glands usually mirror changes in the major glands” so that biopsy of the minor gland is the procedure of choice. To help determine which patients should have a minor salivary gland biopsy and to assess the major salivary gland’s function, a Lashley cup (Fig 5) is placed over the opening of Stensen duct by gentle pressure on the outer concentric ring. Salivary flow is stimulated by a sugarless lemon drop, and the rate of flow is determined. A flow rate of >3 mL/5 min is normal; most primary SS patients have a rate ~0.5 mL/5 min. Histologic Structure Histologically salivary glands appear as a number of lobules separated from each other by
FOX ET AL
connective tissue septa. The entire structure is surrounded by a connective tissue capsule. The ducts are arranged in a tree-like pattern (shown schematically in Fig 6). A acinus consists of a number of pyramidal cells grouped around a small central lumen. The light- and electronmicroscopic structure of these cells varies with their product. Production of saliva begins in the terminally located acini. Two major types of acinar cells can be distinguished: serous and mucous. On routine hematoxylin-eosin stained slides, serous cells have round nuclei and basophilic cytoplasm. Mucous cells have pale cytoplasm with nuclei compressed against the cell bases. Each acini is surrounded by a basement membrane. Between the acinar cell and the basement membrane is a myoepithelial cell that consists of a cell body and several long cytoplasmic processes that contain myofilaments similar to those found in smooth muscle ceils. The contraction of myoepithelial cells causes a sudden release of secretory granules from the acinar cells and propels acinar contents into the ducts. The most distal ducts are termed “striated” while the larger, more proximal ducts are termed “intercalated.” The human salivary gland system consists of paired major glands (ie, parotid, submandibular, and sublingual) as well as numerous minor salivary glands located in the buccal submucosa. These glands differ in their composition of mucous, serous, and seromucous acini (Table 8). The parotid glands contain predominantly serous glands and the minor salivary glands (obtained by routine labial biopsy) contain predominantly
(a) Fig 5. Lashley cup for measurement of parotid flow rate. The plastic cap consists of concentric rings. The outermost ring is attached to a suction cup and is used to anchor the cup over the opening of the parotid gland. After stimulation with a lemon drop, the flow enters through the inner concentric ring into the collecting tube.
8eroum flw
(b) mucous Wnd
(c) hWrcalmted ducts
(d) rtrlatod ducts
excretory act
Microanatomy of the salivary gland. The excreFig 6. tory ducts are located in the septal connective tissue linterlobular). All other components (a through d) are located within the parenchymatous lobules fintralobular).
89
SJOGREN SYNDROME
Table 8.
Characterization
of Major and Minor
Salivary Glands Characterwation Histologic
Submandibular
Minor Salivan,
Mucous
Mucous
Sections
Predominant of acinar Presence Flow
Parotid
rate
SWOUS
type cell
and
serous
of ducts
Prominent
(mL/5
3.5
and
SerOm”cO”S
Rare
Rare
3.0
(1.5)
min) Proteins
present
in saliva
from
each type
of gland
Amylase
High
Low
. . .
Lysozyme
Low
High
...
Glycoprotein
Cationic
Anionic’
. . .
Lactoferrin
High
High
. . .
Kallikrein
High
High
...
Low
High
. . .
IgG
Low
Low
IsA
High
High
Epidermal
growth
factor
Complement *Due
C3
to high content
Low of sialidated
Low
... ... ...
glycoproteins.
mucous glands. The submandibular and sublingual glands contain roughly equal proportions of mucous and serous glands. Most of the saliva is produced by the parotid and submandibular glands, approximately 15% to 20% is produced by the labial submucosal (ie, minor) salivary glands. The protein composition and flow rates of the major glands can be accurately determined since it is possible to collect fluid directly from their opening (ie, Stensen or Wharten duct). In contrast, the minor glands discharge their contents through multiple small ducts directly into the oral cavity and precise quantitation and characterization of these fluids is difficult. Significant differences in the protein content of the saliva from major glands have been noted (Table 8) including amylase, lysozyme, and cationic proteins. Kallikrein, lactoferrin, lactoperoxidase, and lipoproteins are found in the secretions of both major glands. Thus, in analyzing possible targets for the immune response in SS, it is important to remember the heterogeneity of different salivary (or lacrimal) glands. Many biologically active polypeptides are located in the submandibular gland, including epidermal growth factor, endodermal growth factor, neural growth factor, erythropoietin, renin, and somatostatin.56’78 There are relatively low levels of these peptides in the parotid or minor salivary glands. Also, lower levels of these
factors are present in the submandibular gland of women than men.79 It is possible that this difference in distribution of biologically active peptides may contribute to the female predominance of patients with SS. Immunoglobulin in saliva is predominantly due to secretion by plasma cells that surround the intralobular ducts. Many of these plasma cells originate in Peyer patches in the intestine and subsequently migrate to the salivary gland.*&** For example, intragastric feeding of particular antigens leads initially to appearance of “blast” cells in Peyer patches that then migrate through the thoracic duct and localize in salivary or mammary glands.83 This pattern of “homing” is strongly influenced by female sex hormones.84 Thus, the salivary gland is part of a common mucosal immune system that includes at least the salivary gland, bronchial glands, intestine, and cervix.83-s5 This localization pattern of lymphocytes may help explain the distribution of organ involvement in patients with primary SS. IgA in salivary secretions may also result from immunoglobulin synthesized at nonsalivary gland sites and secreted into the saliva.58 The secretion of serum IgA into saliva is facilitated by “secretory component,” a low molecularweight protein synthesized by serous acinar and intercalated ductal cells.*’ This molecule serves as a specific receptor that binds polymeric IgA in the serum and participates in its endocytosis and vesicular transport to the ductal lumen. The mechanisms for transport of IgA into saliva are thought to be similar to those described for the mammary gland and biliary system.*’
THE SALIVARY GLAND IN SS
Light and Electron Microscopy In SS, focal lymphocytic infiltrations are initially observed in a periductal distribution of the minor salivary gland. The majority of lobules are involved and infiltrates are more prominent in the central portion of the lobule than the periphery. Well-defined germinal centers are infrequent in minor salivary gland biopsy specimens but occasionally present in parotid and submandibular infiltrates. Normal parenchymal structures such as acini and ducts may be extensively replaced by these infiltrates. “Activated” lymphocytes with large, vesicular nuclei and promi-
90
nent nucleoli are frequently present and may be difficult to distinguish from the lymphocytes in lymphoma.9~“~72-74 In the early stages of the SS lesion, the acini and ductal cells undergo degenerative changes, and there is hyperplasia of the myoepithelial cells. The ducts often become filled with inspissated material and become dilated. Since the stroma of the salivary gland remains, the lobular architecture is preserved, which differentiates SS from lymphoma of the salivary gland. In Fig 7, frame A shows a low-power photomicrograph of a minor salivary-gland biopsy specimen with acini surrounded by lymphocytes. At higher power, some acini are relatively intact (frame B), but occasional lymphocytes are present within the acinar structure (arrow). Frames C and D show other acinar and ductal structure with increasing numbers of infiltrating lymphocytes. These photographs suggest an active role of
FOX
ET AL
lymphocytes in the destruction of salivary gland acini and ducts. The histopathologic changes that occur in the minor salivary glands are similar to those in the major glands. However “myoepithelial islands” are rarely present in minor salivary gland biopsies, although are found in 40% of the major salivary gland biopsies in SS.Xh These islands are thought to arise from proliferation of ductal myoepithelial cells that obliterate the lumen, forming a mass of cells that becomes surrounded by lymphocytes. Our results using monoclonal antibodies support this hypothesis since myoepithelial islands do not react with antibodies to lymphocytes*’ but do react with tubular and myoepithelial cells antibodies.” Morgan and Castleman found that myoepithelial islands helped distinguish SS from malignant lymphoma.x’ At the electron microscopic level, most acinar
Photomicrograph of a minor salivary gland biopsy. Frame A shows e low-power view while frames B through D Fig 7. show different acinar and ductal structures with infiltrating lymphocytes (shown by arrows).
SJOGREN
91
SYNDROME
cells are collapsed and the amount of secretory granules are markedly decreased in glands from patients with primary SS.9&92 Mitochondria are decreased in number and the remaining ones were swollen. Nuclei of the acinar cells appeared relatively unremarkable except for prominent nucleoli. Tubuloreticular structures may be present and may represent a nonspecific response to epithelial cell injury.93,94 No electron dense deposits at the basement membrane of the ducts or blood vessels were noted in minor salivary gland, major salivary gland, or lung biopsy specimens.9’m93 These results suggest that immune complexes may not play a direct role in the destruction of salivary gland, and the presence of lymphocytic infiltrates (as shown in Fig 7) suggests that cellular mechanisms are more important. Grading Systems for Labial Salivary Gland Biopsies In 1968, Chrisholm and Mason” reported a grading scale to indicate the relative number of mononuclear cells infiltrating minor salivary gland tissue (Table 9). A “focus” was described as an aggregate of at least 50 mononuclear cells (lymphocytes, plasma cells, or histocytes). For the diagnosis of primary SS, a grade IV biopsy Table 9. Grading Systems for Minor Salivary Gland Biopsies Chisholm
and Masong
Grade
Lymphocytes/4
0
mm2
(ie, greater than or equal to two foci/4 mm2) was required. However, Chisholm and Mason required only a grade III biopsy for the diagnosis of secondary SS plus RA. However, since a grade III biopsy occurs in up to 20% of “normal” elderly individuals,95v96 we require a grade IV biopsy before classifying our patients as secondary SS. In 1974, Greenspan et a196 revised the Chisholm and Mason scale by presenting the actual number of foci/4 mm* (Table 10). The highest possible score was 12, where confluent lymphocytic infiltrates were noted. Although other parameters (number of pyrinophilic cells, number of mast cells, degree of acinar depletion, fatty infiltration and fibrosis) were described, they were not incorporated into the grading system. A third classification system was reported by Tarpley et a198who used five classes to assess the chronic inflammatory cell infiltration and acinar destruction. Ductal and acinar destruction were graded on a 1 to 4 scale independent of the lymphocytic infiltration scale but were judged not to be of additional value. The grading system used by Chisholm and Mason is sensitive at the low end of the scale; four of the five grades (grades 0 through 3) denote infiltrates of less than two foci and thus are sensitive in evaluating biopsies that are negative for SS. However, this scale is a poor discriminator at the high end of the scale where anywhere from 2 to 12 foci/4 mm* are lumped into a single category. The addition of the focus score
None Slight
Moderate
3
1 Focus
4
r2
Greenspan Focus
infiltrate
score
2
Cell-Surface Antigens
1 focus)
Leu Antibody
Foci Mature
Lymphocytes/4
T cells
mm2
No foci
T cell subset
1 Focus
T helper
2 Foci
including
lymphocytic
infiltrate
aI’*
including
T suppressor B cells
Normal
2
More
Plasma
than
Diffuse partial Diffuse 4
0KT3
Leu 3
0KT4
Leu 2
OKT8
IgD
0KB2
Leu cells
1 or 2 foci
3
OKTl
Leu 4
cells
Criteria
0
acinar
12
round
HLA-DR
cell infiltration
acinar
destruction
infiltration
with
destruction
with
(la-antigen)
0KB7
Cytoplasmic kappa,
2 foci
OKT Antibody
Leu 1
cells
T cell subset
Confluent
Class
(but
et a?’
0
Tarple:Tkt
Table 10. Monoclonal Antibodies to Lymphocyte
infiltrate
2
lambda
L243
Okla- 1
Monocytes
Leu M3
OKM-
Natural
Leu 7
killer cells
complete
Leu Immature
T cells
11
Leu 6
0KT6
1
FOX ET AL
92
by Greenspan et a19’ increases the sensitivity of grade 4 by creating 11 subgrades (ie, focus scores 2 through 12). The Tarpley scale is intermediate between the other two systems since it provides three categories for biopsy specimens with two or more foci. IMMUNOLOGIC
EVOLUTION OF PERIPHERAL
BLOOD AND SALIVARY GLAND LYMPHOCYTES
Cell-Surface Antibodies
Antigens Dejined by Monoclonal
Based on studies in animal systems, it was recognized that immune responses depend on the interaction of different subsets of mononuclear cells: T cells, B cells, and monocytes.94 T ceils represent a class of cells that originate in bone marrow but migrate to the thymus to differentiate into functionally mature cells. B cells represent a different population that arises in the bone marrow that differentiate to become antibodyforming cells. Since this differentiation step occurred in the “bursa of Fabricius” in birds, such cells were termed B cells. In general, T cells provide regulatory function such as T helper cells that instruct B cells to make antibody or T suppressor cells that inhibit further antibody production. Some T cells regulate other T cells; T inducer cells are necessary for the production of additional T helper or T suppressor cells. Another subpopulation of T cells, termed cytotoxic lymphocytes, can lyse certain virally infected or neoplastic target cells; the differentiation of these cells is also controlled by T inducer cells. Additional subsets of mononuclear cells play important roles in fighting bacterial and viral infection: natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) cells each provide a first line of defense against potential pathogens. Finally, monocytes and macrophages are important in processing foreign antigens and presenting them in an immunogenic form to T cells or B cells. The interaction between these cell types depends on direct cellcell interaction but also involves the release of soluble factors.” Each of these interactions (as sites of factor production) represents a potential site where abnormality can lead to either autoimmunity or immunodeficiency. To further understand these regulatory systems, attempts have been made to identify characteristic cell-surface markers that would allow
recognition and separation of distinct mononuclear subsets. About ten years ago, initial studies showed that T cells could be identified by their ability to interact with sheep erythrocytes (ie, to form rosettes), B cells by their cell-surface immunoglobulin, and monocytes by their phagocytic-adherence properties. Histochemical stains for a-napthyl acid esterase (for most T cells) and nonspecific esterase (for monocytes) also were helpful. Recent attention has been directed to the use of monoclonal antibodies to detect particular subsets. Monoclonal antibodies are produced by immunizing mice with human lymphocytes; this leads to production of mouse B cells making antibodies to human lymphocytes antigens. These murine B cells are “immortalized” by fusion with a murine myeloma; the resulting “hybridoma” cell line secretes a single antibody (ie, a myeloma protein) that reacts with a single human cell surface antigen and thus can provide an unlimited amount of standardized reference reagent. Examples of monoclonal antibodies to human lymphocytes are shown schematically in Table 10 and in Fig 8. We list the commercially available preparations that are most widely used: the “Leu” series’“” from Becton-Dickinson (Mt. View, Calif) and the “OKT” series’“’ from Ortho Diagnostics (Raritan. NJ). The entire population of T cells can be enumerated with Leu 5; this consists of mature T cells (reactive with Leu I or Leu 4) or immature (T cells, Leu 6). Mature T
RECOGNIZED
SUBSETS OF PBL BY MONOCLONAL ANTIBODIES T-cells
. .. -0KT4
Non T-cell
SClp4 OKT3+O,KM’+lg-1 --+-OKT8+1 (Leu 3) bu
2)
-v
I ; l-la, I
Fig 8. Schematic representation of lymphocyte subsets. Total T cells are defined by the presence of receptor for sheep erythrocytes (detected by Leo 51. Mature T cells are defined by antibody SC1 (similar to Leu 1) or 0KT3. Reciprocal T cell subsets react with 0KT4 (same as Leo 3) and OKTS (same as Leu 2).
SJOGREN SYNDROME
cells express either Leu 3 (T helper/inducer cells) or Leu 2 (T suppressor/cytotoxic cells) (Fig 8). Although there are significant excep~~ons,102~104 there is generally good correlation between Leu 3 or Leu 2 phenotype and functional properties. Additional monoclonal antibodies identify NK cells (Leu 7, Leu 1 l)‘05-‘07 monocytes (Leu M3) and B cells. As more is learned about lymphocyte function, additional monoclonal antibodies will be developed to better define lymphocyte subsets. However, at the present time, the available markers permit an adequate assessment of whether lymphocytes in blood or tissue infiltrates are polyclonal or monoclonal. Characterization of Blood Lymphocytes in Primary SS Welo and others23 found that the number of Leu 3 T cells/pL (the helper/inducer subset) was relatively normal. While the number of Leu 2’ T cells (suppressor/cytotoxic subset) was significantly decreased in many primary SS patient’s blood, the mechanisms responsible for this decreased number of Leu 2 cells remain unclear. We did not find autoantibodies in primary SS patients directed against “normal” T cells, ‘Oxin contrast to some SLE patients where autoantibodies to suppressor cells have been reported.“’ Immunohistologic Characterization of Salivary Gland Lymphocytes To characterize salivary gland biopsy specimens, frozen tissue sections are fixed in acetone and specific monoclonal antibodies are applied. After rinsing, horseradish peroxidase conjugated goat antibody to mouse IgG is added, followed by the enzyme substrate 3’3’diaminobenzidine. This series of reactions leads to the deposition of an insoluble brown product (3’3’diaminobenzidine) at the site where the original antibody was bound. In some instances, additional amplification of the original antibody-binding signal is obtained by using biotin conjugated-goat antibody to mouse Ig and avidin-conjugated horseradish peroxidase.‘08,“0 An example is shown in Fig 9 (frame A) where the antibody to T cells, Leu 4, was used. When a control myeloma protein with no known antihuman activity (MOPC 21) was used, no staining
93
was evident. The majority of T cells belong to the Leu 3a subset (that includes T helper/inducer cells; frame C). Some Leu 2a+ T cells (T cytotoxic/suppressor cells) were also noted (frame D). B cells (detected by antibody to IgD) were also present in the gland (frame B). Of particular interest, a B cell subset defined by antibody B532 was present in salivary gland and lymph nodes A high degree of staining but not in blood.““” with antibody to HLA DR (anti-Ia) was present, including an interstitial pattern as well as in an intracytoplasmic distribution in some cells. When T cells were prepared from suspensions of salivary gland lymphocytes, approximately 50% of the T cells were Ia + . In contrast, < 10% of T cells are Ia+ in normal peripheral blood lymphocytes (PBL) or primary SS PBL. Together, these results emphasize that both T cells and B cells at the site of inflammation differ significantly from those in the same patient’s blood. In addition to minor salivary gland biopsies in primary SS, parotid gland (in six patients) and submandibular gland (in three patients) biopsy specimens were characterized. The majority of lymphocytes were again T cells of the Leu 3a subset although some Leu 2a cells were present. 74~‘08 Small clusters of germinal center like B cells (detected by antibody B532) were present but large lymphoid follicles were infrequent. Thus the lymphoid infiltrates in minor salivary gland biopsies appear representative of the process affecting the major salivary glands. To confirm these results and to obtain quantitation of lymphocyte subsets, suspensions of salivary gland were stained with monoclonal antibodies and analyzed cytofluorimetrically to demonstrate that >75% were Leu 3a+ and ~25% were Leu 2a+~llo.lll In several primary SS patients, we were able to study muscle biopsy specimens and transbronchial lung biopsy specimens. We found that infiltrates were generally present in the perivascular region and contained predominantly T cells similar to those in the salivary gland biopsy specimens described above. Monoclonal antibodies have proved most useful in distinguishing pseudolymphoma from lymphoma in biopsies of salivary glands and lymph node. In four patients with pseudolymphoma,74 we found a diffuse infiltration of T cells that were predominantly Leu 3a’. In contrast, most non-
94
FOX ET AL
Fig 9. Microanatomy of the salivary gland. The excretory ducts are located in the septal connective tissue (interlobular). All other components (A through D) are located within the parenchymatous lobules (intralobular).
Hodgkin lymphomas are characterized by B cell infiltrates.” Although T cell lymphomas rarely occur in adults, these patients experience rapidly enlarging lymph nodes and tissue lesions accompanied by a poor prognosis. In contrast, the pseudolymphomatous lesions may remain stable for years,74 and the karyotype of lymphocytes from blood and salivary glands of pseudolymphoma was normal, in contrast to patients with non-Hodgkin lymphoma.74 These results suggest that the cellular content of the pseudolymphoma lesions is similar to that found in the “benign” lesions. The factors stimulating this extensive proliferation of lymphocytes remain to be determined. The progression from pseudolymphoma (similar to benign lesions with polyclonal T cells and B cells) to lymphoma (monoclonal B cells with either kappa or lambda) has been reported.“* Also, it might be expected that some biopsies will show an intermediate stage during which a predominant B cell emerges.“2m”5
Our finding of predominantly T cell infiltrates in the minor salivary gland biopsy specimens of SS patients are in contrast to several previous studies. Greenspan et a19’ reported a preponderance of plasma cells based on pyrinophilic staining. Although this histochemical stain was thought specific for plasma cells, recent studies demonstrated pyrinophilic staining of metabolically active T cells due to their increased RNA and production ‘I6 in cases of T cell lymphoma’” immunoblastic lymphadenopathy.“’ Konttinen et ,]“9,‘20 have used staining for acid alphanapthyl acetate esterase since peripheral blood T cells give a “dot-like” pattern of staining while B cells are negative. They found that the majority of salivary gland lymphocytes lacked the dot-like staining pattern and concluded that these were B cells. Data now show that mitogen activated T cells lose their alpha-napthyl acetate esterase activity.‘2’m’23 Thus, presence of pyrinophilic cells and alpha-napthyl acetate esterase-negative cells
95
SJOGREN SYNDROME
in salivary gland may result from in vivo activation of T cells, a supposition that is consistent with our finding of activated T cells reactive with antibodies OKTlO and antibody to Ia. Other methods to enumerate T cells and B cells in the salivary gland have included specific antiserum to immunoglobulin and rosetting assays with complement-coated erythrocytes.‘24*‘25 These studies have yielded conflicting results. Using heteroserum to thymocytes Talal et a1’24found a predominance of T cells only in very large foci while we found a majority of T cells regardless of focus size. This difference from our study may result from different antibodies used as detecting reagents. Also our patient selection was totally primary SS while the other studies were predominantly secondary SS plus RA. In our experience, the enumeration of B cells in tissue sections using monoclonal antibodies is difficult. Antibodies to IgG, IgA, IgM, kappa, or lambda are suitable for staining plasma cells but the large amount of interstitial immunoglobulin prevent the enumeration of surface Ig+ B cells. Relatively few B cells were noted with antibody to IgD, a marker present on the majority of peripheral blood B cells. However, IgD cannot be used as a measure of total B cells since it is known that germinal center B cells in lymph node lack IgD.99 However, germinal center B cells can be detected by monoclonal antibody B532llO.lli; therefore, we have used the combination of IgD and B532 antibodies to enumerate the B cell subpopulations. It remains possible that additional B cell subpopulations lacking these markers are present in primary SS biopsy specimens and studies are currently in progress using newly developed monoclonal antibodies to nonimmunoglobulin antigens on B cells including the complement C3 receptors. Using these methods 10% to 25% of lymphoid cells in primary SS salivary gland biopsies have B cell markers.74 This includes 5% to 10% of cells bearing cytoplasmic IgG or IgA and having a plasmacytoid appearance.“‘*‘26 Also, we have noted a higher proportion of B cells in secondary SS biopsies than in primary SS biopsies and that these secondary SS B cells are more likely to be located in germinal center-like structures (authors’ unpublished observations). These differences in the immunohistologic architecture between primary and secondary SS may
represent different mechanisms of pathogenesis or may reflect prior treatment in the secondary SS patients.
In Vitro Functional Studies of Lymphocyte Function PBL from primary SS patients can be stimulated by mitogens to secrete immunoglobulin in vitro (Table 11). Production of IgG and IgM was comparable to that of PBL from normal subof these T cells (E’ = sheep jects.‘08 Treatment erythrocyte receptor-positive cells) with monoclonal antibody 0KT4 plus complement abolished IgM and IgG synthesis whereas antibody 0KT4 alone or complement alone was without with antibody OKT8 plus effect.74”08 Treatment complement did not alter the amount of Ig synthesized or lead to the production of RF. Thus, T helper activity in the blood of primary SS patients is included in the OKT4 subset; this is similar to results obtained with normal blood lymphocytes. Similar to PBL, the salivary gland lymphocyte (SGL) could be induced to secrete immunoglobulin in vitro (Table 11). However, the SGL produced RF while the PBL did not. Lysis of the T cells with antibody 0KT4 plus complement blocked this production. This suggests that tissue lymphocytes are the major source of RF production and that this synthesis remains under normal immunoregulation by T cells. Also the OKT4’ cells were noted to possess T inducer activity necessary for the generation of T cytotoxic cells.74 T suppressor and T cytotoxic activ-
Table 11. Production of Antibody In Vitro by SGL and PBL of Primary SS Patients IgM*
IgG’
BFt
SGL
310 ? 80 25 + 5 60 + 4
Total SGL
E+ EE+ (OKT4
+ C’) + E-
E+ IOKT8
+ C’) + E-
69 + 7
670
2 110
27+
10
110 + 15 cl0
85 k 25
60 + 25
76 2 21
295
k SO
416
? 104
371
+ 161
700
+ 186
PBL Total PBL
19 k 7
342
13
E-
27 + 5
39k
10
cl0
38 + 7
46 + 7
E+ (OKT4
+ C’) + E-
E+ (OKTE + C’) + E‘ng antibody 743
E+
t
315
* 152
698
+ SO for six normal PBL, IgM = 297
188.
tRF = IgM RF (ng k SO).
+ 179
+ 122 and IgG =
96
ity generated from primary SS PBL and SGL were included in the OKT8’ subset. Since 0KT4’ T-cells from normal PBL have recently been shown to possess NK and ADCC activities under certain circumstances,‘02~‘04 we examined SGL for these activities. Little NK activity (ie, lysis of “Cr-K562 cells) or ADCC function (ie, lysis of 5’Cr Chang cells coated with antiserum to Chang cells) was noted.74 The NK and ADCC levels in these patients blood was at the lower limit of normal range for this age group; the SGL’s activity was even lower than their respective PBL. In summary, our results suggest that the OKT4 and OKT8 T-cell subsets of PBL and SGL of primary SS patients possess functions similar to those in normal blood or tonsillar lymph node. However, the presence of “activation” antigens on the SGL (defined by monoclonal antibodies as described above) suggested that the initial stages of lymphocyte activation have already occurred in vivo for SGL. Further studies are in progress to determine the response of these PBL and SGL to soluble factors derived from lymphocytes. The ability to augment or inhibit these T cell responses may provide additional avenues for future therapy. Most previous studies on the functional properties of PBL from primary SS patients have indicated similar immunoregulatory properties as found in normal subjects. No increase in spontaneous IgG production or anti-TNP plaque forming cells was present;‘,‘*’ these findings contrasted with SLE patients where a marked increased in such activated B cells were presNormal conconavalin A suppressor cell ent. ‘27m’29 function was noted* and ADCC levels were within normal limits in one study13’ although low in a subsequent report.13’ Miyasaka et al’32.‘33 recently reported that some primary SS patients have a deficient response in autologous and allogeneic mixed lymphocyte reaction; this might reflect abnormality in T cell-macrophage interactions. In assessing functional studies of primary SS patients, it may be important to compare these patients to disease-free control subjects that bear histocompatibility antigens HLA-DR3.‘34 Ambinder et al’35 showed that such normal control subjects have increased IgG secretion in vitro as compared to HLA-DR3-negative normal sub-
FOX ET AL
jects. Defects in clearance of immune complexes or IgG-sensitized erythrocytes have been noted in HLA-DR3 positive individuals with SS’36 but also in HLA-DR3 normal control subjects.‘37.‘38 SPECULATIONS
ON PATHOGENESIS
In a general sense, the presence of autoimmune disease implies the presence of T-B cell and T-T cell interactions that allow responses against host tissues. This is shown schematically in Fig 10, where an extrinsic injury probably leads to release of tissue antigens. T helper and B cells are activated to produce autoantibodies; such T cells and B cells may be induced directly within the salivary gland or may migrate (ie, “home”) to this site after their encountering antigen in some of other lymphoid regions. In addition to antigenspecific responses, many antigen nonspecific responses may also result as a consequence of the release of soluble factors that enhance immunologic responses. Under normal circumstances, this “autoreaction” would be limited by suppressor cells. Therefore, the presence of autoantibodies implies either insufficient T-suppressor responses or failure of other cells to respond to these signals. The crucial questions to be answered are whether this represents an exaggerated form of normal immune response or whether there exists intrinsic defects in the T or B cells responsible for the disordered immunoregulation. The initial inciting lesion in SS remains unknown. Leading candidates include Epstein-
Extrinsic Injury
I
I
---Lhost cell disruption with release of autoantigens
1
7 generate T-suppressor
I generate l-helper B-cells
I
I
I
I
I
I
oolvclonal B-cell ktivation (RF, ANA)
I
s:A,
I
T-cells infiltrate salivary gland
I I
-
I
-- failure of T-suppressor cells tb'modulate
I
--__
I Tissue injury
I
I 1_
Fig tion
I
__
Release of autoantigens
10.
Schematic
in SS.
representation
for
immunoregula-
97
SJOGREN SYNDROME
Barr virus (EBV) and cytomegalovirus. These viruses are thought to reside in the salivary EBV can be glands normally.‘39*‘40 Infectious cultured from the opening of Stensen duct in 20% of normal subjects and in >50% of patients receiving corticosteroids or immunosuppresand sives.‘4’ Thus, these viruses are ubiquitous already present at the relevant target organ.‘4o9’42 Further, EBV can stimulate polyclonal antibody production and autoantibody synthesis such as RF.‘43 Recently, Yang et a1299’44reported that antibody SS-B found in many SS patients was directed against a ribonuclear protein that selectively associate with EBV-encoded RNA. In nasopharyngeal carcinoma, a disease that is known to be associated with EBV transformation of epithelial cells,‘45-‘47 an intense lymphocytic response occurs that has been termed a “lymand these lesions resemble phoepithelioma”,‘48 the changes found in primary SS. Taken together, these findings suggest a role for EBV initiating SS, but direct evidence for the presence of viral genomes and gene products still must be established. Since HLA-DR 3 is more prevalent in primary SS, genetic factors may be important. Evidence for three possible mechanisms for the role of histocompatibility antigens and disease have been presented. These genes may predispose patients to an exaggerated or deficient response to certain infections such as EBV.‘49-‘5’ The gene products of the HLA-DR gene (termed Iaantigens for “immune associated” proteins with molecular weight 34,000 and 28,000) may serve as a “target” for autoimmune lymphocytes. This is presumed to be the mechanism in the salivary gland involvement in mice with chronic graftversus-host disease.ls2 Finally, HLA-DR3 has been associated with defective clearance of immune complexes by the reticuloendothelial system in liver and spleen.‘37m’38 Possibly allowing immune complexes to localize at other target sites such as salivary gland where they could stimulate immune responses. After the “initial lesion” in a genetically susceptible individual, lymphocytic infiltration of T cells and B cells occurs. These cells are predominantly T-helper cells, and they induce local active immunoglobulin synthesis.‘53*‘54 These immunoglobulins are broken down to form Fc-
fragments that further stimulate antibody production’5s*‘56 or form immune complexes that activate complement, which in turn can further activate lymphocyte function.‘57*‘58 It is therefore possible that chronic stimulation of B cells within lymph nodes or other tissues may result from exposure to antigen(s), T cell derived soluble factors, and fragments derived from immune complexes and complement. The original initiating antigen need no longer be present by the time that disease is clinically apparent. Since the MRL/l mouse shows tissue lesions similar to human SS (ie, salivary glands infiltrated by Lyt l+, 2- T cells, hyperglobulinemia, and RF),‘59*‘62 it is important to note that these animals have marked abnormalities in production of their T cell derived lymphokines; they have diminished levels of interleukin 2 production but high levels of factors that drive B cell growth and differentiation.‘60.‘6’ The actual mechanism of destruction of the salivary and lacrimal gland also remains unknown. The cells infiltrating the acini and tubules are probably responsible for cellmediated destruction. Although ADCC mechanisms remain possible, antibodies to salivary duct are infrequent in primary SS patients.60 The actual antigens being detected may be a viral antigen or perhaps a host antigen crossreactive with a viral gene product.‘63 TREATMENT OF PATIENTS WITH SS
The treatment of SS is aimed at symptomatic relief and limiting the damaging local effects of chronic xerophthalmia and xerostomia. They may have specific problems such as thyroiditis, nephritis, gastritis, or pneumonitis. In addition many have symptoms suggestive of a chronic pain syndrome that includes myalgias, arthralgias, and depression. The goals of therapy must be to develop programs to address organ specific as well as organ nonspecific manifestations of this disease. The Dry Mouth Clinical management of the dry mouth is a difficult problem. In addition to rampant dental caries and chronic pain, patients have troublesome intraoral soft tissue problems that include infections due to Candida and difficulty with dentures due to dryness. Before any treatment
98
FOX
program is started, it is important to identify contributing factors such as mouth breathing, heavy smoking, stress, depression, and drugs that have anticholinergic side effects. The most frequently implicated drugs are the phenothiazines, tricyclic antidepressants, antispasmodics, antiparkinsonian, and decongestant medications. Home remedies and nonprescription medications may possess anticholinergic side effects even though the patients may not recognize these agents as “drugs.” Many of the treatments (or approaches to treatment) suggested below have initially been used by radiotherapists for patients with prior radiation of the head and neck, and we have found similar treatments helpful in our patients. Most patients discover that it is helpful to carry water with them, and to avoid dry foods, alcohol, tobacco, and mouth breathing. Dental prophylaxis by their dentists is supplemented by frequent use of a toothpick or Water Pik device. Sugarless chewing gum and sugarless lemon drops are helpful in some cases. A variety of saliva substitutes are available (Table 12). These differ in their flavoring agents and preservatives. Salivart spray has the theoretical advantage that it contains no preservatives since these agents may be responsible for topical irritation in some patients. After administration of these sprays, parotid flow rates are increased for seven to eight minutes in both normal subjects and SS patients.‘64m’66 However, the patient’s sense of Table
12.
Commercial Tears
Preparation Artificial
Preparations
of Artificial
and Saliva
Manufacturer
PR?SerVstlVe
saliva
Saliment
Ferring
Parahydroxybenzoate
Xerolube
Scherer
Paraben
Saliva
Roxane
Paraben
Westoort
None
Substitute
Salivart Artificial
tears
Liquifilm Liquifllm Tears
Forte
Plus
Allergan
Chlorbutanol
Allergan
Thimerosol
Allergan
Chlorbutanol
Hypotears
cooper
Benzalkonium
chloride
Tears
Alcon
Benzalkonium
chloride
Adsorbotears
Alcon
Thimerosol
Mum
Muro
Benzalkonium
Duolube
Muro
None
Duratears
Alcon
Methylparaben
Lacrilube
Allergan
Chlorbutanol
Lacrlserts
Merke
None
Ocular
Naturale
Tears ointments
chloride
ET AL
xerostomia may be decreased for up to several hours. Treatment with a 0.4% stannous fluoride has been suggested to enhance dental remineralization of damaged tooth surfaces. Previous studies have reported increased salivary flow rates after administration of parasympathomimetic agents such as piocarpine or neostigmine as either a mouthwash or as a systemic medication. Others have used potassium iodide solutions (SSKI) for this purpose. Varying degrees of success have been noted. Our experience and that reported at the NIH” indicates that these medications may help some patients with relatively early or mild xerostomia but not patients with severe xerostomia. Treatment
of Dry Eves
The treatment of dry eyes is not only symptomatic but also designed to prevent scarring secondary to KCS or infection. The administration of artificial tears containing methylcellulose (Table 12) gives considerable relief to most patients, but disabling symptoms remain present in some cases. Failure of artificial tears has been attributed in some cases to the preservatives in the artificial tears (such as benzalkonium chloride, chlorbutanol, or thimerosal) that provoke further topical irritation.“’ We routinely ask patients to try several different preparations sequentially to identify those that seem most tolerable. Lacrilube or Duolube ointment is prescribed for use at bedtime. In patients who have mild dry eyes, Lacriserts have been helpful, but our patients with marked xerophthalmia have frequently noted increased ocular irritation on inserting the pellets or visual blurring leading them to discontinue this medication. In all patients, symptoms tend to wax and wane, particularly in association with the seasons when dry winds are prevalent. When patients can identify exacerbating problems increased frequency of artificial tear application should be started before symptoms develop in the hope of preventing objective eye findings. The use of humidifiers at night, wrap around glasses, and even goggles during flares often is helpful. Sudden worsening of ocular symptoms should always suggest possible ocular infection. In patients with associated diseases such as RA, nodular corneai lesions and vasculitic lesions also must be considered.
99
SJOGREN SYNDROME
European studies have suggested that bromhexine, a synthetic alkaloid derivative originally used as a mucolytic agent in cough remedies, may have beneficial effects in increasing lacrimal and salivary gland function.‘68*‘69 Doses of at least 48 mg/d were required since no benefits were reported at lower doses. This medication is not yet available in the United States. In patients at our clinic who have been refractory to commercially available artificial tears and ointments, we have recently begun a study of using artificial tears prepared from the patient’s own serum. The serum is diluted with two volumes of sterile normal saline and aliquoted into 1 mL vials (in the absence of added preservative). These vials are then stored at -20 “C, and the patient removes one vial per day to use as a source of artificial tears. In 15 consecutive KCS patients we observed improvement in symptoms and in rose bengal and/or fluorescein staining. None of the patients had complete resolution of symptoms but each described a generally soothing effect of the autologous serum tears.“’ Patients could easily distinguish between these eye drops and normal saline or their commercially available tears. However, double-blind prospective studies must be conducted before this approach can be advocated for general use. Finally, in patients in whom response to commercially available (or serum) tears remains too transient, consideration can be given to closure of the puncta and canaliculi by surgery or cautery. This opening is surrounded by dense, relatively avascular connective tissue and can be occluded by electrocautery as an outpatient. Theoretically, soft contact lenses might help prevent the tear film by preventing evaporation. However, great care must be taken to maintain frequent lubrication with a low-viscosity artificial tear preparation or severe abrasions may result. Rarely, a partial tarsorrhaphy (sewing the lateral portion of the eyelids together) may be required. Dryness of the Nasal Passage and Sinusitis Many patients complain of nasal dryness and symptoms of sinusitis with postnasal drip. Our initial approach is to provide increased moisture to this region by use of normal saline sprays (Ocean Spray) and humidifiers at night. In some cases, nasal irrigation with normal saline proves helpful. This can be easily performed by the
patient using an irrigation syringe or a Water Pik device (set for the lowest volume-pressure delivery level). The diagnosis of sinusitis is confirmed by sinus roentgenogram with air-fluid levels and CBC with leukocytosis. In these cases, it may be necessary to establish drainage, obtain definitive cultures, and treat with antibiotics. Role of Diet Patients frequently ask about the role of diet either in causing their disease or in their treatment. No definite answers are known but environmental agents (perhaps food antigens) may play a role. One of the best examples of diet-related autoimmune disease is celiac sprue, where autoimmune reaction against gliadin (a wheatderived product) plays an important role.“’ These patients have a high frequency of histocompatibility antigen HLA-DR3, similar to primary SS patients. Another example is dermatitis herpetaformis patients who possess iodide sensitivity and HLA-DR3.‘72 Therefore, some undetected antigen in food may play an analogous role in primary SS. If this analogy is correct, then a high titer of antibodies to the particular food antigens should be detectable. However, caution must be exercised in looking for “anti-food” antibodies since antibodies to milk protein have been found in normal individuals’73 as well as those with apparent food allergies. Recent interest has centered on the possible role of linoleic acids since these fatty acids are precursors of prostaglandins and/or leukotrienes that are important in the inflammatory response. One preliminary report suggests a deficiency of prostaglandin E, in SS that was treated with dietary supplement of fatty acids.‘74*‘75 Vitamin A deficiency has also been suggested since patients with this deficiency may have xerophthalmia.“‘j However, these reports must be considered preliminary. Many of our patients have tried these treatments and various elimination diets without benefit. In our clinic, we have suggested that patients take a supplemental multivitamin of their choice. We suggested yogurt or buttermilk added to diet since some patients have noted beneficial responses and had an apparently lower incidence of oral candida. Based on reports that zinc was helpful in reducing stomatitis in patients after
FOX ET AL
head and neck irradiation’76 we tried zinc sulfate (220 mg/d) without significant improvement in most cases. In summary, double-blind studies on large numbers of primary SS patients will be required before the role of dietary factors can be assessed. Approach to Treatment of the Myalgias. Arthralgias. and Fatigue in SS Patients Many patients complain of profound myalgias. In most cases, normal (or only slight elevation) of muscle enzymes such as aldolase are present. Several patients had electromyographic studies with mild myopathic changes and their muscle biopsy specimens showed perivascular lymphocyte accumulations (mostly T cells) without frank changes of muscle necrosis. Similar perivascular changes have been noted in muscle biopsy specimens in RA or SLE that were performed because of myalgias. However, the significance of these biopsies remains unclear until results are available on biopsies from primary SS patients who lack myalgias. Frequently, the patient has already been told that their symptoms are due to depression. Although this may in part be true, it often leads to anger and loss of the therapeutic physician-patient relationship. When antidepressants are used, medications with minimal anticholinergic side effects (Amoxapine, Norpramin, Trazadone) are preferable since amitrypthine (Elavil) may cause significant ocular and mucous membrane drying that exacerbates underlying sicca symptoms. The Role of Corticosteroids and Immunosuppressive Agents In general, we try to avoid these medications except in cases where visceral disease (pneumonitis, nephritis, CNS symptoms) requires their use. Since our patients do not frequently have clinically significant nephritis or CNS disease, the major use has been for pneumonitis or polyserositis refractory to nonsteroidal agents. The role of immunosuppressive and/or cytotoxic drugs in the development of lymphoma has been extensively discussed.‘0,“,‘7.74.‘77 Some groups believe that these treatments predispose to development of lymphoma.” Other groups assume that only the most severe patients are treated with cytotoxics and these patients were more likely to develop lymphoma because of
severe disease.” Because of the increased incidence of lymphoma in animals treated with cyclophosphamide and in other patients receiving this drug, we try to avoid its use. In many cases, patients have been inactive for many months and have marked “deconditioning” of their cardiovascular and musculoskeletal system. After reassurance that muscle necrosis has not occurred, we emphasize physical therapy and gradually increasing exercise programs. Lowdose salicylates or nonsteroidal drugs are frequently helpful. Surgical Approach to the Salivary Gland In certain patients, biopsy of the major salivary glands may be required to rule out lymphoma. The basic approach is a superficial parotid lobectomy or removal of the entire submandibular gland. The frequent occurrence of scarring and cyst formation due to the disease process may make identification of the facial nerve difficult. In other patients, parotid infections or abscess may require surgical drainage. In this type of case, a facial flap may be raised and then a tunnel created parallel to the branches of the facial nerve to break the septate connections that exist within the gland and to allow adequate drainage. Ultrasound may aid in identifying abscesses within the gland preoperatively. SUMMARY
In this review, we have concentrated on the immunohistologic features of primary SS. The precise relationship between primary and secondary SS and SLE or RA is unclear in many patients, particularly early in the disease. Nevertheless, recognition of sicca components is important in patient management. The extraglandular features found in primary SS were reviewed to emphasize the reported histologic changes. Routine studies on tissue biopsy specimens can be extended by using monoclonal antibodies that detect particular subsets of mononuclear cells. Studies of lymphocyte function show in primary SS patients that salivary glands contain predominantly T-helper/inducer cells. Based on these results, speculations in the pathogenesis of primary SS are presented that incorporate current concepts of soluble factor (ie, lymphokine) function. Finally, we outline the mul-
101
SJOGREN SYNDROME
tidisciplinary approach to therapy followed by our divisions of rheumatology, opthalmology, otolaryngology, and oral medicine. ACKNOWLEDGMENT We thank the members of the division of rheumatology (Drs K. Hench, J. Vaughan, E. Tan, C. Robinson, J. Curd, and F. Kozin), otolaryngology (Drs D. Bell and R. Fantozzi), and ophthalmology (Drs J. Michelson and R. Chan) for allowing us to study their patients. We appreciate the technical assistance of D. Harlow, R. Burhrow, S. Brewster, and F.
Kral in performing these experiments and preparing the manuscript. Special thanks to Carol Peebles, who evaluated autoantibody profiles on some of our patients, and to Drs Tom Adamson, Scott Carsten, and Carol Young who helped perform immunohistochemical studies during their rheumatology fellowships. Generous gifts of monoclonal antibodies were made available by Drs N. Warner (Becton-Dickinson, Mt View, Calif) and G. Goldstein (Ortho Diagnostic, Raritan, NJ). We appreciate the helpful discussion with Drs S. Fong, C. Tsoukas, D. Carson, E. Morgan, and A. Theofilopoulos on the mechanisms and immunoregulation. Finally, we thank our primary SS patients for participating in our studies.
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158. Morgan E, Weigle W, Hugh T: Anaphlatoxinmediated regulation of the immune response. I. C3a mediated suppression of human and murine humoral immune responses. J Exp Med 155:1412-1426, 1982 159. Hang L, Theofilopoulos A, Dixon F: A spontaneous rheumatoid arthritis-like disease in MRL/ 1 mice. J Exp Med 155:1690-1701,1982 160. Altman A, Theophilopoulos A, Weiner R, et al: Analysis of T-cell function in autoimmune murine stains. Defects in production of and responsiveness to interleuken 2. J Exp Med 154:791-808, 1981 161. Lu C, Unanue E: Spontaneous T-ceil lymphokine production and enhanced macrophage expression and tumoricidal activity in MRL-lpr mice. Clin Immunol Immunopathol 25:213-222, 1982 162. Prud’homme G, Park C, Fieser T: Identification of a B-cell differentiation factor(s) spontaneously produced by proliferating T-cells in murine lupus stains of the Ipr/lpr genotypes. J Exp Med 157:730-742, 1983 163. Haspel M, Onodera T, Prabhakar B, et al: Virusinduced autoimmunity: Monoclonal antibodies that react with endocrine tissues. Science 220:304-306, 1983 164. Matzker J: Synthetic saliva: A new therapeutic agent. Deutches Anzteblatt 7 1:704-706, 1974 165. Weisz A: The use of a saliva substitute as treatment for xerostomia in Sjogren’s syndrome. Oral Surg 52:38&396, 1981 166. Shannon I, McCrary R, Starcke E: A saliva substitute (or use by xerostomic patients undergoing radiotherapy to the head and neck. Oral Surg 44:656-66 1, 1977 167. Wilson F: Adverse external ocular effects of topical ophthalmic medications. Surv Ophthalmol 24:57-86, 1979 168. Avisar R, Savin H, Machtey I, et al: Clinical trial of bromhexine in Sjogren’s syndrome. Ann Ophthalmol 13:97 l973, 1981 169. Manthorpe R, Frost-Larsen K, Hoj L, et al: Bromhexine treatment of Sjogren’s syndrome. Effect on lacrimal and salivary secretion and on proteins in tear fluid and saliva. Stand J Rheumatol 10: 177-I 80, 198 1 170. Fox R, Michelson J, Chan R: Beneficial effect of artificial tears made from autologous serum. Arthritis Rheum 27~459-462, 1984 171. Weiss J, Austin R, Schanfield M, et al: Gluten sensitive enteropathy. Immunoglobulin G heavy chain (Gm) allotypes and the immune response to wheat gliaden. J Clin Invest 72:96-101, 1983 172. Alexander HO (ed): Dermatitis Herpetiformis. Philadelphia, WB Saunders, 1975 173. Ziff M: Diet in the treatment of rheumatoid arthritis. Arthritis Rheum 26:457-461, 1983 174. Yu J, Burns S, Schneyer C: Prostaglandin E induced salivary secretion. Experientia 38:1077-1078, 1982 175. Horrabin D, Campbell A, McEwen C: Treatment of the sicca syndrome with EFA, pyridoxine and vitamin C, essential fatty acids and prostoglandins. Holman RT, ed. Prog Lipid Res 20:253-254, 1981 176. Silverman J, Weber C, Silverman S: Zinc supplementation and taste in head and neck cancer patients undergoing radiation therapy. J Oral Med 38:1&16, 1983 177. Klein G, Purtillo D: Summary: Symposium on Epstein-Barr virus induced lymphoproliferative diseases in immunodeficient patients. Cancer Res 41:4302-4308, 198 1
VOL XIV, NO 2
Primary
Sjogren ByRobert
Syndrome: Clinical Features I. Fox, Francis V. Howell,
1984
and Immunopathologic
Robert C. Bone, and Paul Michelson
Primary Sjogren syndrome is an autoimmune condition in which dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia) result from lymphocytic infiltration of lacrimal and salivary glands. Clinical and laboratory features of 60 primary Sjogren syndrome patients seen at our clinic during the past three years are presented. These patients illustrate the wide spectrum of extraglandular features that may occur as a result of lymphoid infiltration of lung, kidney, skin, stomach, liver, and muscle. They further emphasize the difficulty in classifying a patient as primary or secondary Sjogren syndrome (ie, sicca symptoms associated with systemic lupus erythematosus, rheumatoid arthritis, or scleroderma), particularly early in the disease course. As an initial step in understanding the pathogenesis, the lymphocytes that infiltrate the salivary glands and lymph nodes
I
N 1888 Johann von Mikulicz Radecki described a 42-year-old Prussian farmer with enlargement of lacrimal and salivary glands “consisting of small round cells.“’ Subsequently, the name “Mikulicz Syndrome” was appended to any swelling of salivary glands due to lymphocyte infiltration, including a collection of pathologies that we now realize included tuberculosis, lymphoma, sarcoid, and salivary-duct inflammation associated with bacterial infections. After these etiologies had been eliminated, there remained a group of patients whose salivary and lacrimal gland lymphoid infiltrates were associated with autoimmune features such as hyperglobulinemia and autoantibodies. In 1933, Henrik Sjogren described the association of keratoconjunctivitis sicca (KCS), dry mouth (xerostomia), and rheumatoid arthritis.* Although this association of symptoms is generally known as Sjogren syndrome (SS), it is occasionally referred to as “Gougerout-Sjogren” syndrome to acknowledge the earlier report of such symptoms by Gougerout in 1925.3 In 1953, Seminars in Arfhritis and Rheumatism, Vol 14, No 2 (November), 1984
were characterized by using monoclonal antibodies that recognize distinct lymphocyte subsets and by using in vitro functional assays. These studies have demonstrated that affected tissues have infiltrates of T cells with helper/inducer activity and with a high frequency of “activation antigens.” The immunohistologic techniques are useful in differentiating “benign” and “pseudolymphoma” lesions (both due predominantly to T cells) from non-Hodgkin lymphoma (usually due to B-cell infiltrates). Although there is no “cure” for primary Sjogren syndrome patient’s symptoms may be significantly improved by measures aimed at prevention of ocular and dental complications and by the recognition of extraglandular features that may be amenable to specific treatment.
Morgan and Castleman demonstrated that a similar histologic type of infiltrate was present in both SS and Mickulicz disease.4 Bloch et al’ suggested that SS be subdivided into primary SS and secondary SS. Secondary SS was defined as patients with sicca symptoms in association with particular diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), From the Departments of Basic and Clinical Research, Oral Pathology, Head and Neck Surgery, and Ophthalmology, Scripps Clinic and Research Foundation, La Jolla, Calif. Robert I. Fox, MD, PhD: Department of Basic and Clinical Research; Francis V. Howell, DDS: Department of Oral Pathology; Robert C. Bone, MD: Department of Head and Neck Surgery; and Paul E. Michelson, MD: Department of Ophthalmology, Scripps Clinic and Research Foundation, La Jolla, Calif Supported in part by research grant No. AM/CA 33983 from the National Institutes of Health. Address reprint requests to Robert I. Fox, MD, PhD. Department of Basic and Clinical Research, Scripps Clinic, IO666 N Torrey Rd. La Jolla, CA 92036. 0 I984 by Grune and Stratton, Inc. 0049-0172/84/1402-0001%5.00/0 77
78
FOX
scleroderma (PSS), or dermatomyositis, while primary SS patients lacked these particular diseases6 Primary SS patients may frequently have extraglandular manifestations such as thyroiditis, pneumonitis, or interstitial nephritis.s ~9 In this review, we will describe the spectrum of clinical features, possible mechanisms of pathogenesis, and approaches to treatment of patients with SS. We will emphasize findings in primary SS since these patients are usually not receiving corticosteroids, immunosuppressive drugs, or remittive agents (ie, gold, penicillamine) that may alter the underlying pathologic mechanisms. These patients provide an opportunity to safely perform biopsy procedures on the actual site of tissue damage (ie, the salivary gland) to characterize the infiltrating lymphocytes. Since primary SS patients share a similar histocompatibility leukocyte antigen, HLA-DR3, and immunopathologic features with diseases such as juvenile onset diabetes mellitus, celiac sprue, thyroiditis, and Addison disease,’ the study of primary SS may provide insight into these other diseases where the target organ is not readily accessible to biopsy. Finally, patients with SS have a significantly increased prevalence of lymphoma,‘“,” so that study of primary SS may give clues to factors which predispose to lymphoid malignancy. Clinically, increased interest has been focused on SS by (1) the recognition that xerostomia and KCS are more frequent problems than previously recognized”,” and (2) that relatively little information is available on the treatment of these patients. Therefore, the patient is often left with the discouraging feeling that “nothing can be done.” Although there is no “cure,” symptomatic relief may be achieved by early recognition of the extraglandular manifestations and by careful attention to the prevention of ocular and dental complications. We will discuss the multidisciplinary approach to evaluation and treatment used by the divisions of rheumatology, oral medicine, ophthalmology and otolaryngology at the Scripps Clinic.
using rose bengal and/or fluorescein dye,’ ” and a dry mouth. We will limit our discussion to those subjects with a minor salivary-gland biopsy with grade IV infiltrate (classified on a scale from 0 to IV as described below) (Table I). However, the patients who fulfill these criteria remain heterogeneous in their clinical and laboratory features. Several examples of the “types” of patients who might be referred for further evaluation because of a grade IV lip biopsy include the following examples. I. The “traditional” primary SS patient who is a woman (age, 30 to 65 years) with hypergammaglobulinemia, autoantibodies (usually rheumatoid factor [RF] and/or antinuclear antibody [ANA]), anemia, and elevated sedimentation rate.’ They share an increased frequency of histocompatibility antigen HLA-DR3,’ suggesting a common genetic feature of pathogenesis. They frequently have extraglandular manifestations of nonthrombocytopenic purpura, thyroiditis, interstitial nephritis, or pneumonitis. Based on the operational definition of primary SS. these patients do not have sufficient clinical features to be classified as SLE, RA, polymyositis, or PSS. 2. Elderly patients (~80 years old) with absent tears, dry mouth, and a lip biopsy with acinar atrophy and with sufficient lymphocytic infiltrate to be class IV. They usually lack autoantibodies, hyperglobulinemia, and extraglandular features. Although these patients satisfy the criteria for primary SS, their relationship to the previous group of primary SS patients
Table 1. Features of Primary and Secondary Primary
SS
SS
KCS Schwmer
test with
Positive
rose bengal
< 10 mm
wetting/5
or fluorescein
min
staining
of cornea
and conjunctiva Xerostomia Decreased ~0.5 Grade
flow
I-IV)
extraglandular
biopsy
features
pneumonttis,
associated
rate using Lashley
cups wth
min
IV (on scale
anemia.
Secondary
parotid
mLl5
Although
of minor
salwary
(thyroidltis,
neuropathies,
gland
nephntls.
etc) may occur.
no
RA, SLE, or PSS
SS
KCS and xerostomia
PROBLEMS IN THE DEFINITION OF SS
Exclusions:
To be classified as SS, patients must have dry eyes with decreased tearing (Schirmer test t10 mm wetting/5 min), KCS on ophthalmic exam
ET AL
plus RA, SLE. polymyosms,
sarcoidosls.
lymphoma,
pemphigoid.
or PSS infec-
t1ons* *Additional other
than
causes
of keratitis
SS are presented
and salivary
in Table
6.
gland
enlargement
79
SJOGREN SYNDROME
remains unclear as they usually lack other features suggesting an autoimmune process. 3. Patients with secondary SS who have sicca complaints associated with characteristic features found in RA, SLE, polymyositis, or PSS.556 Such patients originally may have been thought to have primary SS, but evaluation of their clinical course over subsequent years shows the development of characteristic features of the other diseases. The distinction between primary and secondary SS associated with RA or SLE will be discussed. 4. Patients with leukemia or lymphoma may first complain of dry eyes and dry mouth due to lymphocytic infiltration. The presence of such diseases serves to exclude the diagnosis of SS.’ However, the relationship of these lymphomas to SS is intriguing since approximately 10% of salivary-gland lymphomas occur in the setting of preexisting SS14 and patients with SS have a markedly elevated incidence of extraglandular lymphoma.9~‘0 5. Patients with prior radiation to the head and neck, frequently for Hodgkin disease, may have dry eyes and xerostoma. In several cases, we and othersI have noted a grade IV lymphocytic infiltrate in their salivary gland biopsies. The radiation often antedated these symptoms by several years, and there was no evidence for recurrence of tumor. Although the history of prior lymphoma would technically exclude the diagnosis of SS, such patients may provide insight into pathogenetic mechanisms and may benefit from a therapeutic approach similar to that in primary SS. 6. Some patients have a grade IV minor salivary gland biopsy and autoantibodies (such as ANA, Sjogren syndrome A (SS-A), and/or Sjogren syndrome B (SS-B) but do not have complaints of dry eyes. The normal Schirmer test and ophthalmologic examination do not allow them to be classified as SS. Lack of ocular complaints suggests that their lacrimal glands are not as severely injured as their salivary glands. Conversely, some patients develop severe KCS but have normal salivary-gland biopsies and parotid flow rates. Further studies are necessary to determine if these patients are forme fruste of SS or represent a distinct entity. The distinction between primary and secondary SS associated with RA is usually not diffi-
cult. RA patients have characteristic joint symptoms and radiographic features.16 However, a possible source of confusion is the patient with sicca complaints, arthralgias, and positive RF that has not developed objective synovitis on examination or erosive changes on roentgenograms during many years of clinical symptoms. Thus, it is important to recognize the diagnosis of primary SS because (1) the patient can be reassured that a positive RF occurs in conditions other than RA, (2) potentially hazardous drug therapies (ie, remittive agent used in advanced RA) may be avoided, and (3) attention is directed to conservative measures to prevent ocular and dental complications of primary SS. Several features suggest that the pathogenesis of primary SS may differ from that of secondary SS plus RA (Fig 1). Patients with primary SS have a high frequency of histocompatibility antigen HLA-DR3 and lack antibodies to salivary gland ducts; in contrast, RA patients with secondary SS have increased frequency of HLA-DR4 and possess antibodies to salivary ducts.‘,” On the other hand, the distinction between primary and secondary SS associated with SLE is often more difficult. The recently revised crite-
Overlap of Clinical Syndromes Rheumatoid
Siouren
Systemic
Arthritis
Syndrome
Erylhemalosus
Lupus
Shared Features +
I. autoantibodies 2. Msis;;opatibilily
rheumatoid HLA-OR4
laclor
I ANA, SSA, SSB HLA-OR3
Fig 1. Patients with sicca complaints may present with characteristic extraglandular features. These patients may be grouped into particular subsets that differ in genetic predisposition end mechanisms of pethogenesis. Patients with secondary SS and SLE have increased frequency of histocompatibility antigen HLA-D3. This marker has also been found in increased prevalence in patients with primary biliary cirrhosis and the CREST variant of scleroderma: these populations have a high incidence of sicca symptoms. In contrast secondary SS plus RA patients have increased HLADR4 and antibodies to salivary duct antigens, features not found in most primary SS patients.
80
FOX
ria for diagnosis of SLE are listed in Table 2. Patients with SLE and primary SS have an increased prevalence of ANAs (including SS-A and SS-B), leukopenia, anemia, and elevated sedimentation rate.s.‘8.‘y Arthralgia, myalgias, pneumonitis, and nephritis also may be present in both groups. The overlap of laboratory and clinical features in primary SS and SLE patients suggests that similar pathogenetic mechanisms may be operating, and this is supported by the Table
2.
The
1982
Revised
Criteria
of SLE 1.
Bufferfly
2.
DIscold
for
the
Classification
1982)
rash lupus
3.
Photosensitivity
4.
Oral ulcers
5.
Arthritis’
6.
Serositls a.
(ARA
rub heard
by a physician
or pleural
effusion,
or b.
Perlcardms:
documented
of pericardial
7.
8.
Renal
disorder
Persistent
b.
Cellular
casts:
tubular.
or mlxed
Neurologlc
proteinurla
Sewres:
b.
20.5
g/d
may be red cell, hemoglobin,
granular,
disorder
known
9.
by ECG or rub or ewdence
effusion
a.
a.
In the absence metabolic
Psychosis
of offending
drugs
or
derangements
in the absence
Hematologlc
of offendlng
drugs
disorder
a.
Hemolytic
b.
Leukopenla:
anemia:
with
i4.000
retlculocytosis
cells/fiL
or
on two
or more
occa-
SlO”S or c.
Lymphopenla: caslons
d.
- 1,500
Thrombocytopenta:
on two
or more
oc-
i
lOO.OOO/~L
in the absence
of
drugs
lmmunologlc
disorder
a.
Positive
b.
Anti-DNA:
LE cell preparation presence
in abnormal c.
cells/KL
or
offending 10.
Anti-Sm:
or
of antlbody
to untreated
DNA
to Sm nuclear
antl-
titer or presence
of antibody
gen or d.
False-positwe six months
11.
Armnuclear antlbody
antibody.
in ttme
to be associated NOTE.
STS
inhibttion, *The purpose
FTA
STS known
to be positwe
and confwmed
An abnormal
“drug-induced
= Serologic
test
= fluorescent
proposed
pattents
lupus”
is based in clinical
on
of the any
Reprinted
with
permtssion.‘6
during
syndrome*
TPI
= treponemal
1 1 criteria.
studies,
serially
simultaneously,
assay known
antibody.
said to have SLE if any four or more or
of drugs
for syphilis,
treponemal
classification
of identifying
ttter of antmuclear or an equwalent
and in the absence
with
for at least
by TPI or FTA tests
by immunofluorescence
at any point
finding of a high frequency of histocompatibility antigen HLA-DR3 in both primary and secondary SS associated with SLE.‘“,” However, primary SS patients lack sufficient features of SLE to meet the criteria for this diagnosis (Table 2), although it is possible that some primary SS patients represent a subset of SLE with a limited-disease expression. In summary, SS is not a single disease entity. Among the primary SS patients, only some will have particular extraglandular features or autoantibodies. It is important to recognize this heterogeneity of patients when attempting to predict prognosis or discuss mechanisms of pathogenesis. CLINICAL
Pleuntis:
a person
1 1 criteria
interval
of
For the shall be
are present observation.
ET AL
FEATURES
OF PRIMARY
SS
A brief review of the clinical features of primary SS is presented to point out the wide spectrum of extraglandular features that may occur in patients who may initially present with oral or ocular symptoms. Patient Populatioms The clinical and laboratory characteristics of 55 patients with primary SS seen in the past three years in our clinic are shown in Table 3. The median age of our patients was 56 years (range, 17 to 88 years). Their histories suggested a mean disease duration of seven years. Approximately 25% of these patients were first seen in the rheumatology clinic with complaints of arthralgia, myalgia, and fatigue, while 25% were seen in ophthalmology for ocular discomfort, and 50% in oral medicine because of xerostomia and increasing dental decay. There was a predominance of women (only one man). The age of onset and duration of symptoms for our patients was similar to those at National Institutes of Health (N IH) studied by Bloch et al5 and in Glasgow by Whaley et al.” In virtually all of these primary SS patients, medications at the time of biopsy were limited to salicylates or other nonsteroidal drugs. None had a history of prior cytotoxic drugs or irradiation. In several patients, treatment with low-dose corticosteroids (_=IO mg/d prednisone) had been used but had been discontinued at least three months prior to biopsy. The clinical characteristics of several patients with secondary SS in whom a grade IV lip biopsy
81
SJOGREN SYNDROME
Table 3. Clinical and Laboratory
Features
of Patients With Primary SS
Patient LetteE
RF
Aoe ivrl
ANA
SS-A
ss-6
+ +
+ + _
Other Features
Is%
Predominantly salivary/lacrimal symptoms GO
21
La
24
Br
68
10,246 ...
256 256
VU
71
320 ...
CP Ve
60
...
. . .
_ _
68
...
SC
70
...
... ...
_
Sa
66
...
160
160 320
2,000
Pseudolymphoma
...
4,400 1,550
Parotid
...
1,150
_
1,460
Parotid
... 615
_
Pseudolymphoma parotid
...
1,400
Myalgias/arthralgias symptoms
ct
56
Dr
53
Be
39
Ar
56
640
256
co We
77 ...
640
. . .
Me
61
MO
75
We Ch
52 ...
Pa MO
72 ...
Gr
79
... ... ...
... ... ... ...
... ...
256 256 . . .
+ _ _ _
1,250
Myalgia
1,550
Myalgia
1,052
Myalgia
2,200
Myalgia
1,250
Myalgia
1,400
Arthralgia
1,400
Arthralgia
1,500
Arthralgia
. . . . . .
256
_ _
256
+
. . .
_ _ _
...
...
CO
58
640
256
Th
60
640
256
Dermal
+ _ _ +
+ +
1,200
Arthralgia
+
1,350
Arthralgia
+ + + f +
+ + +
2,600
Purpura
3,450
Purpura
2,750
Purpura
5,220
Purpura
2,100
Purpura
+ + + + _ _ + _
+ + + + _ _ _ _
2,200
Thyroid
1,960
Thyroid
2,527
Thyroid
+ _
+ _
4.050
manifestations
BI
17
2,560
256
Cl
67
1,280
256
Pe
60
5,120
256
Si
17
2,560
256
Tr
57
...
256
Endocrme
manifestations 640
256
Sh
45
SU
49
. . .
256
Ph
48
1,280
256
Me
. . .
. . .
64
Be
39
...
Bu
70
... ...
64
Wa
72
640
64
On
52
320
. . .
Pulmonary
...
Thyroid
1,400
Thyroid
1,300
Pernicious
1,400
...
Pernicious
anemia
anemia
Diabetes
mellitus
Pseudolymphoma
thyroid
manifestations
Cr
53
1,280
256
RY
72
640
64
820
Pulmonary Nodular
fibrosis infiltrates,
pleural
pseudolymphoma Ar
68
. . .
320
St
74
2,560
256
co
49
No
60
Renal,
neurologic
640
256
2,560
256
_ + + +
+ _ +
+ _ +
_ _ + +
_ + _
1,200
Pulmonary
hypertension
1,450
Pulmonary
fibrosis
2,400
Pleural
1,400
Pulmonary fibrosis
4,050
RTA
1,500
Peripheral neuropathy
1,600
Headaches
effusions
manifestations
Fo
79
Wa
60
160
Wi
53
160
Gastrointestinal
256
5,120
+ + + +
... 320
manifestations
Th
52
. . .
160
Sa
62
. . .
160
Ha
61
Wi
61
640
...
256 256
2,200
BC
1,800
Sclerosing cholangitis
1,400
Sprue
2,000
Hepatitis
effusions,
82
FOX
Table 3.
ET AL
I Continued)
PatM?tlt LetteE Patients
RF
Age (~1 >80
yr with
primary
ANA
SS-A
SS-B
Other Features
bG
SS
Al
88
...
...
1.200
Arthralgla
MO
92
. . .
. . .
1,550
Myalgia
Wa
a2
. . .
.
1,400
8a
85
..
De
81
. . .
NOTE.
Parotid
refers
to tender,
swollen
.
. . . . parotid
gland;
thyroid
...
1,400
Thyroid
1,200
Arthralgla
to hypothyroidism;
RTA
to renal
tubular
acidosis:
and
8C
to biliary
cirrhosis.
was found are presented in Table 4. The relatively small number of secondary SS patients is related to the fact that we do not do lip biopsies in most secondary SS patients as a routine part of our research study. Many of the secondary SS patients listed in Table 5 have received corticosteroids and/or remittive drugs that may alter the underlying disease process. Autoantibodies
in SS
ANA are found in the majority (70%) of our primary SS patients. The pattern of immunofluorescence is usually fine speckled, suggesting the presence of antibody to nonhistone nuclear proteins.22 The pattern of immunofluorescence can vary with the tissue substrate (ie, mouse kidney, KB cell, or HeLa cell). Also the results may be influenced by the high solubility of some nonhistone antigens that allows them to be leached from the nuclei during washing procedures.‘X Therefore sera containing antibodies to SS-B or nRNP are sometimes reported to be negative for ANA. Further, certain nuclear antigens may be destroyed by some fixatives while other antigens maintain reactivity. This may prove important in using commercially available ANA kits where fixatives have been used as preservatives.” ANA may be directed against DNA (single or double stranded), histones, or nonhistone proteins. Antibodies to DNA or histones are relatively rare in primary SS patients and their presence ought to suggest the presence of SLE. Exciting progress has been made in recent years in characterizing autoantibodies directed against nonhistone proteins.‘* The majority of primary SS patients possess antibody to SS-A antigen and about 50% of these patients have antibody to SS-B. These antibodies may be of the IgG, IgA, or IgM isotype in serum and saliva.23,24 The
molecular weight of the SS-A antigen is not known but the SS-B antigen is approximately 50,000 daltons24m2B” and can be isolated from human thymus, spleen, liver, and lymphoblastoid cell lines. It is also found in certain tissues from dog, calf, and rat. In uninfected cells, the SS-B antigen associates with a mixture of small RNA molecules. On cellular infection with adenovirus or Epstein-Barr virus, the SS-B antigen specifically complexes with viral associated RNA.“.19
Table
Clinical and Laboratory
4.
Secondary
Features
SS or Malignancy
Pi3tlWlt Letters
of Patients With
Plus Sicca Assocmted
Am
k~l
RF
ANA
SS-A SS-B
t
IqG
DlSeaSe
1,500
RA RA
Pa
32
320
Go
42
640
80
1,400
PC
64
5,120
250
1,400
GI
43
640
160
Sh
52
5,120
64
Hal
51
2,560
16
Hag
65
5,120
.
Ka
60
1,280
~~
RA RA
+
1,100 929
Cop
77
640
.. ..
8ac
66
5.120
256
+
RA RA
1,400
RA
1,600
RA
~
1,800
RA
t
1.530
RA
Is
30
640
256
t
t
2,200
SLE
St
40
640
320
+
+
1,400
SLE
HU
29
160
160
t
Ba
39
640
256
*
La
42
320
256
+
Ja
72
1,280
Do
60
...
so
50
640
Gr
48
640
co
53
.
.. . . ...
ZI
80
De
60
... ...
... ...
MO
65
Ma
60
640 ...
De
22
Gr
22
3,100
SLE
-
1.900
SLE
+
1,800
SLE
256
1,400
PSS
256
1,200
PSS
~
~~
1,000
Dermatomyosltls
1,400
Dermatomyosttls
1,230
Dermatomyosms
1,200
T-CLL
-
1,000
T-CLL
256 ...
+
450
Lymphoma
800
Lymphoma
...
...
~
900
Lymphoma
...
...
~
800
C-GVHD
83
SJDGREN SYNDROME
The common element among these disparate RNA species is their synthesis by RNA polymerase III.3o Another interesting feature of the SS-B antigen is that its location within the cell changes during different phases of the cell cycle.3’ During Go phase, the antigen is located in the nucleoplasm, but during S phase the localization becomes nucleolar. The significance of autoantibodies to SS-B or SS-A antigens in pathogenesis is unclear. Their detection may prove helpful in suggesting the diagnosis of SS, especially in patients where multisystem involvement may overshadow the sicca complaints.25 An association between the presence of anti-SS-A and vasculitis in primary SS patients has been reported by Alexander et aL3* The presence of extraglandular features also has been correlated with antibody to SS-B.24 Detection of other nonhistone proteins such as Sm and Scl-70” is also important, since it should suggest the presence of associated SLE or PSS, respectively. Organ-specific autoantibodies, such as antisalivary gland, are infrequent in primary SS patients.33 They have been reported in the majority of RA patients with secondary SS, suggesting that they may be an epiphenomena rather than playing a role in pathogenesis. In this regard, primary SS differs from organ specific autoimmune diseases such as thyroiditis, myasthenia gravis, and diabetes mellitus where antibodies to target organs can be found early in the disease process. Finally, RFs (antibodies to IgG) are consistently found in primary SS patients’ saliva, tears, and serum.34%3sThey may be of IgM, IgG, or IgA isotypes. Their role in pathogenesis is unclear. It has been suggested that their synthesis is normally induced to amplify other weak antibodyof antigen reactions36 or to aid in clearance immune complexes.37 The persistence of high titers of RF in primary SS patients may reflect continued antigenic stimulation, elevated immune complex level, or failure of the normal suppressive mechanisms to inhibit further RF synthesis. Ocular Features Patients generally complain of a burning and/ or foreign body sensation in their eyes, although a variety of our ocular complaints and signsI can be encountered (Table 5). Fewer than 50% of the
Table 5. Clinical Features
Clinml
Features
of KCS
SS Patients
Control Patients
(N = 64)
IN = 541
Symptoms Foreign-body sensation
81
4
Burning
80
4
Tiredness with or without diffi-
67
9
67
2
culty in opening the eyes Dry feeling with or without a poor response to physical or chemical irritants and emotions Redness
48
7
Difficulty in seeing
31
11 19
Itchiness
37
Aches
36
7
Soreness or pain
23
4
Photosensitivity and excess of
31
4
31
7
secretion which may appear to be watery, ropy, or as a film over the eye Signs Dilatation of the bulbar conjunctival vessels (usually interpalpebral) Mild pericorneal injection
36
0
Photophobia
47
13
Irregularity of the cornea1 image
63
5
White and frothy or Yellow and
22
0
27
0
11
0
tenacious discharge Dullness of the conjunctiva and/ or cornea Ptosis
NOTE. Data are percentage of patients with that clinical feature. Data taken from Whaley et al.”
patients report eye pain or photosensitivity. We ask the patients to grade the symptoms on a scale from 0 to 4: 0 for no symptoms, 1-t for mild irritation and burning, 2+ for redness and itching, 3+ for pain and photophobia that mildly limit function, and 4-t for severe pain and photophobia that dramatically limit functional activity. We encourage our patients to keep a diary of their symptoms on a weekly basis to identify the general tempo of their disease and recognize factors that may lead to exacerbation. The symptoms of sicca are nonspecific and are influenced by environmental irritants, medications (especially those containing anticholinergic or antihistamine activity), and emotional upset. However, it is important to identify these factors so that they may be altered to improve the patient’s functional status3’ Signs of KCS include the dilation of bulbar conjunctival vessels (especially interpalpebral),
a4
pericorneal injection, ptosis, and tenacious discharge that may form filamentary strands. Biomicroscopic examination often shows attached cornea1 filaments, abnormal amounts of precorneal debris, and superficial punctate keratitis5,‘2 as well as larger epithelial erosions. The diagnosis of KCS should be confirmed by instilling a 1% aqueous solution of rose bengal or fluorescein dye into each eye. The eyes are then examined and with the slit lamp to determine areas of punctate keratitis (Fig 2). Staining localized to areas previously in contact with the filter paper used in Schirmer test should be discounted. Keratitis, including punctate or filamentary changes, is not specific for SS; it can occur in patients with pemphigoid, sarcoidosis, trauma (such as contact lenses), or infections of bacterial, chlamydial (ie, trachoma), and viral etiology (Table 6). Neuropathy on a congenital basis (Reily-Day syndrome) or acquired basis (ie, damage to 5th cranial nerve) can lead to keratitis. In patients with known SS, a sudden flare of ocular symptoms should always raise suspicion of cornea1 erosion or infection. Oral Features
Virtually all patients describe a dry mouth necessitating increased fluid intake, particularly when eating dry foods such as crackers. In our series, 30% gave a history of parotid gland swelling which was unilateral and intermittent. Twenty percent had an enlarged parotid and/or submandibular glands on examination on at least one occasion. Bilateral parotid glands that
Fig 2. Schematic representation of KCS in a primary SS patient as visualized by rose-bengal staining. The left panel shows increased interpalpebral staining with fine punctate lesions. In the right panel, more severe imflammation is associated with coalescent erosions and filamentary strands on the cornea. These strands should be distinguished from mucoid strands that are often present in the bulbar conjunctiva but that may occasionally migrate to the cornea region during examination. These patients may also suffer cornea1 abrasions.
FOX
ET AL
Table 6. Causes of Keratitis and Salivary Gland Enlargement
Other Than SS
Keratitis
1. Mucus 2. 3.
membrane
Infections:
virus (adenovirus,
ria, or chlymdia 4.
Trauma tant
herpes,
vaccinia),
bacte-
tie, trachoma)
(ie. after
including
lights 5.
pemphrgord
Sarcoidosis
contact
chemrcal
lens) and envrronmental burns,
exposure
in-
to ultraviolet
or roentgenograms
Neuropathy
includmg
to 5th cranial
nerve)
neurotropic and familial
keratitis
he, damage
dysantonomia
(Reily-
Day syndrome) 6.
Hypovitaminosis
7.
Erythema
Salivary
gland
1.
Sarcoidosis. Bactenal
gonococci
tie, infectious
Tuberculosis,
and syphilis)
mononucleosis,
actinomycosis.
and vwal
mumps)
histoplasmosis,
tracho-
leprosy
4.
Iodide,
5.
Hyperlrpemrc
6.
Tumors mor).
syndrome)
amylordosrs
(including
infecttons
ma,
(Stevens-Johnson
enlargement
2.
3.
A
multiforma
lead,
or copper states,
(usually
epithelial
ma, and mrxed
hypersensitwrty especially
unilateral) (adenoma. salivary
types
including
IV and V cysts
adenocarcinoma),
gland
(Warthrn
tu-
lympho-
tumors
remained enlarged for at least 12 months were seen in only seven patients: four with pseudolymphoma, two with recurrent bacterial parotitis superimposed on primary SS and only one with uncomplicated primary SS. These findings are comparable to those reported by Bloch et al and Whaley et al,‘,” where approximately 20% had a history of unilateral parotid enlargement, 10%’ had bilateral parotid enlargement, and 5% had submandibular gland swelling. Thus, the majority of primary SS patients do not have significant parotid or submandibular enlargement. Lymphocytic infiltration is present in biopsy specimens of the major salivary glands in SS patients even when swelling is not clinically apparent.j9 4’ Salivary gland enlargement can occur in diseases other than SS (Table 6). Diseases such as sarcoidosis, amyloidosis, infection (including bacterial, fungal, viral), and tumors must be considered. In previous years, reports of an unusual form of sarcoidosis called uveoparotid fever (Heerfordt disease) were common.4’ These patients had parotid enlargement and ocular symptoms but often lacked other features of sarcoidosis (hilar lymphadenopathy, diffuse lung infiltrates, skin sarcoid or hepatosplenomegaly). It is likely that some of the cases published as examples of Heerfordt disease were instances of
85
SJOGREN SYNDROME
SS4’ An SS-like syndrome with salivary gland involvement may occur after bone marrow transplantation.42 It has been proposed that this may be due to chronic stimulation of the immune system by allogeneic cells or to opportunistic viral infections.” A sudden increase in salivary gland pain or size should suggest the possibility of infection, which is more frequent in these patients due to inadequate salivary secretion and ductal lavage. The most frequent offending organisms are Staphylococcus, Streptococcus viridans, Streptococcus hemolyticus, and Pneumococcus which are normal flora of the oral cavity.4’ Cultures must be taken of the pus escaping from Stensen or Wharton duct. Salivary gland infections must be treated promptly to prevent formation of abscesses. A high frequency of stomatitis due to Candida albicans is also present in SS patients, particularly after receiving antibiotics for other illnesses.
Table 7. Extraglandular
Manifestations
Respiratory Chronic bronchitis secondary to dryness of upper and lower airway with mucus plugging Lymphocytic interstitial pneumonitis Pseudolymphoma with nodular infiltrates Pleural effusions Pulmonary hypertension Lymphoma Gastrointestinal Dysphagia associated with xerostomia Atrophic gastritis Liver disease including biliary cirrhosis and sclerosing cholangitis Skin Vaginal dryness Hyperglobulinemic purpura-nonthrombocytopenic Raynaud phenomena Vasculitis Renal Interstitial nephritis and renal tubular acidosis Glomerulonephritis-in
absence of antibodies to DNA
Endocrine, neurologic. and muscular ThYroiditis Peripheral neuropathy-symmetric
involvement of Extraglandular
Sites
Pulmonary. Involvement of exocrine glands in the upper respiratory tract leads to dryness of the nasal passages in approximately 50% of the patients (Table 7).13 In a comprehensive review, Hunninghake and Fauci43 emphasized the high incidence of pulmonary abnormalities in patients with SS, including pleurisy with and without effusion, interstitial fibrosis, dessication of tracheobroncheal mucous membrane, and lymphoid interstitial disease. Clinical studies of pulmonary function in primary SS patients have yielded conflicting results. Newball et a144 and Siegal et a14’ found evidence of mild to moderate obstructive airway disease in almost 50 of their primary SS patients. In another study, Oxholm et a146did not find obstructive changes in any of 43 patients with primary SS, but did note decreased diffusing capacity in patients with prior pneumonia or pleurisy. Pathologic changes in the lung of primary SS patients include atrophy of mucous glands, tenacious secretions, focal atelectases, recurrent infections, and increased frequency of bronchiectasis.43-45 These complications appear to result from infiltration of the bronchial glands by lymphocytes in a nodular or diffuse pattern.46A8 Although this article deals primarily with primary SS patients, it should be noted that non-
in Patients
With Primary SS
involvement of hands
and/or feet Mononeuritis multiplex Myalgias Hematologic Pseudolymphoma Angioblastic lymphadenopathy Lymphoma and myeloma Lymphopenia and neutropenia Aplastic anemia
smoking RA patients with secondary SS have a higher incidence of airway disease than nonsmoking RA patients lacking sicca sympon chest toms.49150 The presence of infiltrates roentgenogram in primary SS patients may indicate the emergence of lymphoma or infection in these patients. We have noted one primary SS patient with primary pulmonary hypertension, a condition that has not previously been reported with primary SS, but has been associated with PSS ” SLE 52and RA.53 Gktroiniestinal. Difficulty in swallowing is frequent in primary SS patients, due primarily to severe dryness of the esophagus as a result of lymphocytic infiltration and destruction of the submucosal glands.54 Abnormal esophageal motility (particularly in the upper one third of the esophagus) may also contribute to dysphagia in some primary SS patients.55 Gastric biopsy specimens show chronic atrophic gastritis and lym-
86
phocytic infiltrates.56 Patients with marked gastric involvement and hyposecretory states can be recognized by their elevated serum pepsinogen leveIs.57 Investigation of pancreatic function has shown impaired response to secretin and pancreozymin, suggesting subclinical pancreatic disease.5R,59 Clinical and/or biochemical evidence of liver disease is found in 5% to 10% of primary SS patients.60 Patients with positive antibody to smooth muscle or antibody to mitochondrial were found to have a higher prevalence of hepatomegaly, splenomegaly, and abnormal liver function tests.@‘* Liver biopsy specimens in these patients show nodular lymphoid infiltrates and cholangitis. These findings suggest that the pathogenetic process responsible for salivary gland destruction and damage to the exocrine hepatic apparatus may be similar. The high incidence of SS in patients with primary biliary cirrhosish”63 lends support to this concept. Drugs such as methyldopa, isoniazid, nitrofurantoin, oxyphenasatin, aspirin, and other nonsteroidal antiinflammatory drugs may contribute to liver dysfunction. Cutaneous, renal, and endocrine. Dryness of the vagina and consequent vulvar pruritis are common problems and often contribute to disturbing symptoms of dyspareunia. This has been attributed to chronic inflammation of the exocrine sweat glands.6,h4 Dependent nonthrombocytopenic purpura was present in about 10% of our patients and was associated with hyperglobulinemia.h5 Raynaud phenomena occurred in approximately 20% of our primary SS patients; these patients also have an increased frequency of telangiectasia although nail-fold changes, calcinosis or digital ulceration were rare. Renal tubular acidosis (especially type II) has been associated with SS; histologic examination of kidney shows interstitial infiltration of lymphocytes.66.h7 The lymphocytes appear to invade the renal parenchyma and immunologically damage the tubules. Similar infiltrates have been noted in patients receiving renal allografts.6x Another renal manifestation in some SS patients is immune complex, hypocomplementemic glomerulonephritis.69 These patients, in contrast to SLE, do not have antibodies to DNA in their serum.‘7,h9 Clinically apparent hypothyroidism has been
FOX ET AL
reported in 10% to 15% of SS patients6.“.” and occurred in a similar proportion of our patients. Antibody to thyroglobulin, antibody to thyroid microsomal, and thyroid-stimulating hormone levels may be elevated in 40% to 50% of patients”.” suggesting that subclinical thyroid damage may be relatively common. Thus endocrine as well as exocrine glandular cells may be targets for immune attack in primary SS. The known similarity in genetic background, HLADR3, and histologic similarities between Hashimoto thyroiditis and primary SS supports the hypothesis that a similar mechanisms may be operating in each disease. Insulin dependent diabetes mellitus’ and pernicious anemia” occur with similar frequency in primary SS to that noted in the general population. Hematologic abnormalities. Many primary SS patients have a normocytic. normochronic anemia,‘,’ with low serum iron and transferrin levels as in other chronic inflammatory diseases. Occasionally bone marrow aspiration shows decreased iron stores that probably result from chronic gastorintestinal blood loss associated with use of salicylates and nonsteroidal antiinflammatory drugs. Leukopenia (total WBC count ~~4,000 cells/ pL) was present in 30% of our patients; previous reports have noted leukopenia in 6% to 33%:. of their patients.‘.” The mechanism of this leukopenia remains unclear but may involve antibodies to leukocytes, splenic sequestration, or abnormal bone marrow maturation in certain patients.” Certain patients have marked lymphadenopathy and/or salivary gland swelling 5.6.‘o,7’~74 (Fig 3). Since their biopsies do not fulfill criteria for malignancy, these patients’ syndrome has been termed “pseudolymphoma.” Other forms of nonmalignant lymphoid proliferation in primary SS patients include thymomah and angioimmunoblastic lymphadenopathy.” In both pseudolymphoma and angioblastic lymphadenopathy, there appears to be a high frequency of progression to frank lymphoma.‘0~7h The relative risk of developing lymphoma for primary SS patients has been estimated to be approximately 40-fold higher than age-, sexmatched control subjects by investigators at the NIH.” However, Whaley et al” in Glasgow did not find such an increased prevalence. These discrepancies may be attributed to several fac-
SJOGREN SYNDROME
87
mucosa are more likely to form scar tissue and to encounter anatomic structures such as blood vessels and nerves. As shown in Fig 4, the lower lip is reflected and local anesthetic (0.5% marcaine with epinephrine as a vasoconstrictor), administered at least 1.5 cm posterior to the site where the biopsy will be taken (frame 1). With a scalpel, a l-cm incision was made through the mucosa (but not
Fig 3. Massive bilateral parotid gland enlargement in patient IA. with pseudolymphoma and SS. Clinical and histologic features of this patient and three additional pseudolymphoma are presented in reference 74.
tors including the relatively small numbers of patients reported with lymphoma and ascertainment bias in patient referral patterns. However, these differences also may reflect the difficulty in distinguishing lymphoma from extensive infiltrates due to “benign” SS or pseudolymphoma.74 We are currently using specific monoclonal antibodies (described below) to help make this distinction. Finally, the differences may be related to treatment patterns since some patients may have been treated with cytotoxic agents or irradiation that may increase the frequency of lymphoma.“,” THE SALIVARY GLAND BIOPSY
Procedure for Obtaining Minor Salivary Gland Biopsies We obtain minor salivary biopsies from the intraoral mucosal surface of the lower lip just below the corner of the mouth (ie, the commissure). Biopsies from the midline of the oral
Fig 4. Procedure for obtaining minor salivary gland biopsy. Frame 1 shows the site where the biopsy will be taken after local anesthesia. In frame 2, the glands are everted through the skin by gentle pressure and separated from underlying muscle by careful disection. Frame 3 shows black silk sutures to close the incision.
88
to enter the submucosa), and the glands were everted by gentle pressure on the outer lip (frame 2). The scalpel undermines the mucosa and the protruding glands are removed by separation from the muscle with care to avoid the sensory nerves. Black silk sutures are used because absorbable sutures may loosen or be absorbed too rapidly. The sutures are removed in approximately one week. No significant morbidity has been noted (eg, sagging of lip); however, 10% to 20% may note hypoesthesia in the region of the biopsy. Because of potential hazard, biopsy of the major salivary gland is recommended only when there is a suggestion of intraparotid malignancy or serious diagnostic doubt. Changes in the minor glands usually mirror changes in the major glands” so that biopsy of the minor gland is the procedure of choice. To help determine which patients should have a minor salivary gland biopsy and to assess the major salivary gland’s function, a Lashley cup (Fig 5) is placed over the opening of Stensen duct by gentle pressure on the outer concentric ring. Salivary flow is stimulated by a sugarless lemon drop, and the rate of flow is determined. A flow rate of >3 mL/5 min is normal; most primary SS patients have a rate ~0.5 mL/5 min. Histologic Structure Histologically salivary glands appear as a number of lobules separated from each other by
FOX ET AL
connective tissue septa. The entire structure is surrounded by a connective tissue capsule. The ducts are arranged in a tree-like pattern (shown schematically in Fig 6). A acinus consists of a number of pyramidal cells grouped around a small central lumen. The light- and electronmicroscopic structure of these cells varies with their product. Production of saliva begins in the terminally located acini. Two major types of acinar cells can be distinguished: serous and mucous. On routine hematoxylin-eosin stained slides, serous cells have round nuclei and basophilic cytoplasm. Mucous cells have pale cytoplasm with nuclei compressed against the cell bases. Each acini is surrounded by a basement membrane. Between the acinar cell and the basement membrane is a myoepithelial cell that consists of a cell body and several long cytoplasmic processes that contain myofilaments similar to those found in smooth muscle ceils. The contraction of myoepithelial cells causes a sudden release of secretory granules from the acinar cells and propels acinar contents into the ducts. The most distal ducts are termed “striated” while the larger, more proximal ducts are termed “intercalated.” The human salivary gland system consists of paired major glands (ie, parotid, submandibular, and sublingual) as well as numerous minor salivary glands located in the buccal submucosa. These glands differ in their composition of mucous, serous, and seromucous acini (Table 8). The parotid glands contain predominantly serous glands and the minor salivary glands (obtained by routine labial biopsy) contain predominantly
(a) Fig 5. Lashley cup for measurement of parotid flow rate. The plastic cap consists of concentric rings. The outermost ring is attached to a suction cup and is used to anchor the cup over the opening of the parotid gland. After stimulation with a lemon drop, the flow enters through the inner concentric ring into the collecting tube.
8eroum flw
(b) mucous Wnd
(c) hWrcalmted ducts
(d) rtrlatod ducts
excretory act
Microanatomy of the salivary gland. The excreFig 6. tory ducts are located in the septal connective tissue linterlobular). All other components (a through d) are located within the parenchymatous lobules fintralobular).
89
SJOGREN SYNDROME
Table 8.
Characterization
of Major and Minor
Salivary Glands Characterwation Histologic
Submandibular
Minor Salivan,
Mucous
Mucous
Sections
Predominant of acinar Presence Flow
Parotid
rate
SWOUS
type cell
and
serous
of ducts
Prominent
(mL/5
3.5
and
SerOm”cO”S
Rare
Rare
3.0
(1.5)
min) Proteins
present
in saliva
from
each type
of gland
Amylase
High
Low
. . .
Lysozyme
Low
High
...
Glycoprotein
Cationic
Anionic’
. . .
Lactoferrin
High
High
. . .
Kallikrein
High
High
...
Low
High
. . .
IgG
Low
Low
IsA
High
High
Epidermal
growth
factor
Complement *Due
C3
to high content
Low of sialidated
Low
... ... ...
glycoproteins.
mucous glands. The submandibular and sublingual glands contain roughly equal proportions of mucous and serous glands. Most of the saliva is produced by the parotid and submandibular glands, approximately 15% to 20% is produced by the labial submucosal (ie, minor) salivary glands. The protein composition and flow rates of the major glands can be accurately determined since it is possible to collect fluid directly from their opening (ie, Stensen or Wharten duct). In contrast, the minor glands discharge their contents through multiple small ducts directly into the oral cavity and precise quantitation and characterization of these fluids is difficult. Significant differences in the protein content of the saliva from major glands have been noted (Table 8) including amylase, lysozyme, and cationic proteins. Kallikrein, lactoferrin, lactoperoxidase, and lipoproteins are found in the secretions of both major glands. Thus, in analyzing possible targets for the immune response in SS, it is important to remember the heterogeneity of different salivary (or lacrimal) glands. Many biologically active polypeptides are located in the submandibular gland, including epidermal growth factor, endodermal growth factor, neural growth factor, erythropoietin, renin, and somatostatin.56’78 There are relatively low levels of these peptides in the parotid or minor salivary glands. Also, lower levels of these
factors are present in the submandibular gland of women than men.79 It is possible that this difference in distribution of biologically active peptides may contribute to the female predominance of patients with SS. Immunoglobulin in saliva is predominantly due to secretion by plasma cells that surround the intralobular ducts. Many of these plasma cells originate in Peyer patches in the intestine and subsequently migrate to the salivary gland.*&** For example, intragastric feeding of particular antigens leads initially to appearance of “blast” cells in Peyer patches that then migrate through the thoracic duct and localize in salivary or mammary glands.83 This pattern of “homing” is strongly influenced by female sex hormones.84 Thus, the salivary gland is part of a common mucosal immune system that includes at least the salivary gland, bronchial glands, intestine, and cervix.83-s5 This localization pattern of lymphocytes may help explain the distribution of organ involvement in patients with primary SS. IgA in salivary secretions may also result from immunoglobulin synthesized at nonsalivary gland sites and secreted into the saliva.58 The secretion of serum IgA into saliva is facilitated by “secretory component,” a low molecularweight protein synthesized by serous acinar and intercalated ductal cells.*’ This molecule serves as a specific receptor that binds polymeric IgA in the serum and participates in its endocytosis and vesicular transport to the ductal lumen. The mechanisms for transport of IgA into saliva are thought to be similar to those described for the mammary gland and biliary system.*’
THE SALIVARY GLAND IN SS
Light and Electron Microscopy In SS, focal lymphocytic infiltrations are initially observed in a periductal distribution of the minor salivary gland. The majority of lobules are involved and infiltrates are more prominent in the central portion of the lobule than the periphery. Well-defined germinal centers are infrequent in minor salivary gland biopsy specimens but occasionally present in parotid and submandibular infiltrates. Normal parenchymal structures such as acini and ducts may be extensively replaced by these infiltrates. “Activated” lymphocytes with large, vesicular nuclei and promi-
90
nent nucleoli are frequently present and may be difficult to distinguish from the lymphocytes in lymphoma.9~“~72-74 In the early stages of the SS lesion, the acini and ductal cells undergo degenerative changes, and there is hyperplasia of the myoepithelial cells. The ducts often become filled with inspissated material and become dilated. Since the stroma of the salivary gland remains, the lobular architecture is preserved, which differentiates SS from lymphoma of the salivary gland. In Fig 7, frame A shows a low-power photomicrograph of a minor salivary-gland biopsy specimen with acini surrounded by lymphocytes. At higher power, some acini are relatively intact (frame B), but occasional lymphocytes are present within the acinar structure (arrow). Frames C and D show other acinar and ductal structure with increasing numbers of infiltrating lymphocytes. These photographs suggest an active role of
FOX
ET AL
lymphocytes in the destruction of salivary gland acini and ducts. The histopathologic changes that occur in the minor salivary glands are similar to those in the major glands. However “myoepithelial islands” are rarely present in minor salivary gland biopsies, although are found in 40% of the major salivary gland biopsies in SS.Xh These islands are thought to arise from proliferation of ductal myoepithelial cells that obliterate the lumen, forming a mass of cells that becomes surrounded by lymphocytes. Our results using monoclonal antibodies support this hypothesis since myoepithelial islands do not react with antibodies to lymphocytes*’ but do react with tubular and myoepithelial cells antibodies.” Morgan and Castleman found that myoepithelial islands helped distinguish SS from malignant lymphoma.x’ At the electron microscopic level, most acinar
Photomicrograph of a minor salivary gland biopsy. Frame A shows e low-power view while frames B through D Fig 7. show different acinar and ductal structures with infiltrating lymphocytes (shown by arrows).
SJOGREN
91
SYNDROME
cells are collapsed and the amount of secretory granules are markedly decreased in glands from patients with primary SS.9&92 Mitochondria are decreased in number and the remaining ones were swollen. Nuclei of the acinar cells appeared relatively unremarkable except for prominent nucleoli. Tubuloreticular structures may be present and may represent a nonspecific response to epithelial cell injury.93,94 No electron dense deposits at the basement membrane of the ducts or blood vessels were noted in minor salivary gland, major salivary gland, or lung biopsy specimens.9’m93 These results suggest that immune complexes may not play a direct role in the destruction of salivary gland, and the presence of lymphocytic infiltrates (as shown in Fig 7) suggests that cellular mechanisms are more important. Grading Systems for Labial Salivary Gland Biopsies In 1968, Chrisholm and Mason” reported a grading scale to indicate the relative number of mononuclear cells infiltrating minor salivary gland tissue (Table 9). A “focus” was described as an aggregate of at least 50 mononuclear cells (lymphocytes, plasma cells, or histocytes). For the diagnosis of primary SS, a grade IV biopsy Table 9. Grading Systems for Minor Salivary Gland Biopsies Chisholm
and Masong
Grade
Lymphocytes/4
0
mm2
(ie, greater than or equal to two foci/4 mm2) was required. However, Chisholm and Mason required only a grade III biopsy for the diagnosis of secondary SS plus RA. However, since a grade III biopsy occurs in up to 20% of “normal” elderly individuals,95v96 we require a grade IV biopsy before classifying our patients as secondary SS. In 1974, Greenspan et a196 revised the Chisholm and Mason scale by presenting the actual number of foci/4 mm* (Table 10). The highest possible score was 12, where confluent lymphocytic infiltrates were noted. Although other parameters (number of pyrinophilic cells, number of mast cells, degree of acinar depletion, fatty infiltration and fibrosis) were described, they were not incorporated into the grading system. A third classification system was reported by Tarpley et a198who used five classes to assess the chronic inflammatory cell infiltration and acinar destruction. Ductal and acinar destruction were graded on a 1 to 4 scale independent of the lymphocytic infiltration scale but were judged not to be of additional value. The grading system used by Chisholm and Mason is sensitive at the low end of the scale; four of the five grades (grades 0 through 3) denote infiltrates of less than two foci and thus are sensitive in evaluating biopsies that are negative for SS. However, this scale is a poor discriminator at the high end of the scale where anywhere from 2 to 12 foci/4 mm* are lumped into a single category. The addition of the focus score
None Slight
Moderate
3
1 Focus
4
r2
Greenspan Focus
infiltrate
score
2
Cell-Surface Antigens
1 focus)
Leu Antibody
Foci Mature
Lymphocytes/4
T cells
mm2
No foci
T cell subset
1 Focus
T helper
2 Foci
including
lymphocytic
infiltrate
aI’*
including
T suppressor B cells
Normal
2
More
Plasma
than
Diffuse partial Diffuse 4
0KT3
Leu 3
0KT4
Leu 2
OKT8
IgD
0KB2
Leu cells
1 or 2 foci
3
OKTl
Leu 4
cells
Criteria
0
acinar
12
round
HLA-DR
cell infiltration
acinar
destruction
infiltration
with
destruction
with
(la-antigen)
0KB7
Cytoplasmic kappa,
2 foci
OKT Antibody
Leu 1
cells
T cell subset
Confluent
Class
(but
et a?’
0
Tarple:Tkt
Table 10. Monoclonal Antibodies to Lymphocyte
infiltrate
2
lambda
L243
Okla- 1
Monocytes
Leu M3
OKM-
Natural
Leu 7
killer cells
complete
Leu Immature
T cells
11
Leu 6
0KT6
1
FOX ET AL
92
by Greenspan et a19’ increases the sensitivity of grade 4 by creating 11 subgrades (ie, focus scores 2 through 12). The Tarpley scale is intermediate between the other two systems since it provides three categories for biopsy specimens with two or more foci. IMMUNOLOGIC
EVOLUTION OF PERIPHERAL
BLOOD AND SALIVARY GLAND LYMPHOCYTES
Cell-Surface Antibodies
Antigens Dejined by Monoclonal
Based on studies in animal systems, it was recognized that immune responses depend on the interaction of different subsets of mononuclear cells: T cells, B cells, and monocytes.94 T ceils represent a class of cells that originate in bone marrow but migrate to the thymus to differentiate into functionally mature cells. B cells represent a different population that arises in the bone marrow that differentiate to become antibodyforming cells. Since this differentiation step occurred in the “bursa of Fabricius” in birds, such cells were termed B cells. In general, T cells provide regulatory function such as T helper cells that instruct B cells to make antibody or T suppressor cells that inhibit further antibody production. Some T cells regulate other T cells; T inducer cells are necessary for the production of additional T helper or T suppressor cells. Another subpopulation of T cells, termed cytotoxic lymphocytes, can lyse certain virally infected or neoplastic target cells; the differentiation of these cells is also controlled by T inducer cells. Additional subsets of mononuclear cells play important roles in fighting bacterial and viral infection: natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) cells each provide a first line of defense against potential pathogens. Finally, monocytes and macrophages are important in processing foreign antigens and presenting them in an immunogenic form to T cells or B cells. The interaction between these cell types depends on direct cellcell interaction but also involves the release of soluble factors.” Each of these interactions (as sites of factor production) represents a potential site where abnormality can lead to either autoimmunity or immunodeficiency. To further understand these regulatory systems, attempts have been made to identify characteristic cell-surface markers that would allow
recognition and separation of distinct mononuclear subsets. About ten years ago, initial studies showed that T cells could be identified by their ability to interact with sheep erythrocytes (ie, to form rosettes), B cells by their cell-surface immunoglobulin, and monocytes by their phagocytic-adherence properties. Histochemical stains for a-napthyl acid esterase (for most T cells) and nonspecific esterase (for monocytes) also were helpful. Recent attention has been directed to the use of monoclonal antibodies to detect particular subsets. Monoclonal antibodies are produced by immunizing mice with human lymphocytes; this leads to production of mouse B cells making antibodies to human lymphocytes antigens. These murine B cells are “immortalized” by fusion with a murine myeloma; the resulting “hybridoma” cell line secretes a single antibody (ie, a myeloma protein) that reacts with a single human cell surface antigen and thus can provide an unlimited amount of standardized reference reagent. Examples of monoclonal antibodies to human lymphocytes are shown schematically in Table 10 and in Fig 8. We list the commercially available preparations that are most widely used: the “Leu” series’“” from Becton-Dickinson (Mt. View, Calif) and the “OKT” series’“’ from Ortho Diagnostics (Raritan. NJ). The entire population of T cells can be enumerated with Leu 5; this consists of mature T cells (reactive with Leu I or Leu 4) or immature (T cells, Leu 6). Mature T
RECOGNIZED
SUBSETS OF PBL BY MONOCLONAL ANTIBODIES T-cells
. .. -0KT4
Non T-cell
SClp4 OKT3+O,KM’+lg-1 --+-OKT8+1 (Leu 3) bu
2)
-v
I ; l-la, I
Fig 8. Schematic representation of lymphocyte subsets. Total T cells are defined by the presence of receptor for sheep erythrocytes (detected by Leo 51. Mature T cells are defined by antibody SC1 (similar to Leu 1) or 0KT3. Reciprocal T cell subsets react with 0KT4 (same as Leo 3) and OKTS (same as Leu 2).
SJOGREN SYNDROME
cells express either Leu 3 (T helper/inducer cells) or Leu 2 (T suppressor/cytotoxic cells) (Fig 8). Although there are significant excep~~ons,102~104 there is generally good correlation between Leu 3 or Leu 2 phenotype and functional properties. Additional monoclonal antibodies identify NK cells (Leu 7, Leu 1 l)‘05-‘07 monocytes (Leu M3) and B cells. As more is learned about lymphocyte function, additional monoclonal antibodies will be developed to better define lymphocyte subsets. However, at the present time, the available markers permit an adequate assessment of whether lymphocytes in blood or tissue infiltrates are polyclonal or monoclonal. Characterization of Blood Lymphocytes in Primary SS Welo and others23 found that the number of Leu 3 T cells/pL (the helper/inducer subset) was relatively normal. While the number of Leu 2’ T cells (suppressor/cytotoxic subset) was significantly decreased in many primary SS patient’s blood, the mechanisms responsible for this decreased number of Leu 2 cells remain unclear. We did not find autoantibodies in primary SS patients directed against “normal” T cells, ‘Oxin contrast to some SLE patients where autoantibodies to suppressor cells have been reported.“’ Immunohistologic Characterization of Salivary Gland Lymphocytes To characterize salivary gland biopsy specimens, frozen tissue sections are fixed in acetone and specific monoclonal antibodies are applied. After rinsing, horseradish peroxidase conjugated goat antibody to mouse IgG is added, followed by the enzyme substrate 3’3’diaminobenzidine. This series of reactions leads to the deposition of an insoluble brown product (3’3’diaminobenzidine) at the site where the original antibody was bound. In some instances, additional amplification of the original antibody-binding signal is obtained by using biotin conjugated-goat antibody to mouse Ig and avidin-conjugated horseradish peroxidase.‘08,“0 An example is shown in Fig 9 (frame A) where the antibody to T cells, Leu 4, was used. When a control myeloma protein with no known antihuman activity (MOPC 21) was used, no staining
93
was evident. The majority of T cells belong to the Leu 3a subset (that includes T helper/inducer cells; frame C). Some Leu 2a+ T cells (T cytotoxic/suppressor cells) were also noted (frame D). B cells (detected by antibody to IgD) were also present in the gland (frame B). Of particular interest, a B cell subset defined by antibody B532 was present in salivary gland and lymph nodes A high degree of staining but not in blood.““” with antibody to HLA DR (anti-Ia) was present, including an interstitial pattern as well as in an intracytoplasmic distribution in some cells. When T cells were prepared from suspensions of salivary gland lymphocytes, approximately 50% of the T cells were Ia + . In contrast, < 10% of T cells are Ia+ in normal peripheral blood lymphocytes (PBL) or primary SS PBL. Together, these results emphasize that both T cells and B cells at the site of inflammation differ significantly from those in the same patient’s blood. In addition to minor salivary gland biopsies in primary SS, parotid gland (in six patients) and submandibular gland (in three patients) biopsy specimens were characterized. The majority of lymphocytes were again T cells of the Leu 3a subset although some Leu 2a cells were present. 74~‘08 Small clusters of germinal center like B cells (detected by antibody B532) were present but large lymphoid follicles were infrequent. Thus the lymphoid infiltrates in minor salivary gland biopsies appear representative of the process affecting the major salivary glands. To confirm these results and to obtain quantitation of lymphocyte subsets, suspensions of salivary gland were stained with monoclonal antibodies and analyzed cytofluorimetrically to demonstrate that >75% were Leu 3a+ and ~25% were Leu 2a+~llo.lll In several primary SS patients, we were able to study muscle biopsy specimens and transbronchial lung biopsy specimens. We found that infiltrates were generally present in the perivascular region and contained predominantly T cells similar to those in the salivary gland biopsy specimens described above. Monoclonal antibodies have proved most useful in distinguishing pseudolymphoma from lymphoma in biopsies of salivary glands and lymph node. In four patients with pseudolymphoma,74 we found a diffuse infiltration of T cells that were predominantly Leu 3a’. In contrast, most non-
94
FOX ET AL
Fig 9. Microanatomy of the salivary gland. The excretory ducts are located in the septal connective tissue (interlobular). All other components (A through D) are located within the parenchymatous lobules (intralobular).
Hodgkin lymphomas are characterized by B cell infiltrates.” Although T cell lymphomas rarely occur in adults, these patients experience rapidly enlarging lymph nodes and tissue lesions accompanied by a poor prognosis. In contrast, the pseudolymphomatous lesions may remain stable for years,74 and the karyotype of lymphocytes from blood and salivary glands of pseudolymphoma was normal, in contrast to patients with non-Hodgkin lymphoma.74 These results suggest that the cellular content of the pseudolymphoma lesions is similar to that found in the “benign” lesions. The factors stimulating this extensive proliferation of lymphocytes remain to be determined. The progression from pseudolymphoma (similar to benign lesions with polyclonal T cells and B cells) to lymphoma (monoclonal B cells with either kappa or lambda) has been reported.“* Also, it might be expected that some biopsies will show an intermediate stage during which a predominant B cell emerges.“2m”5
Our finding of predominantly T cell infiltrates in the minor salivary gland biopsy specimens of SS patients are in contrast to several previous studies. Greenspan et a19’ reported a preponderance of plasma cells based on pyrinophilic staining. Although this histochemical stain was thought specific for plasma cells, recent studies demonstrated pyrinophilic staining of metabolically active T cells due to their increased RNA and production ‘I6 in cases of T cell lymphoma’” immunoblastic lymphadenopathy.“’ Konttinen et ,]“9,‘20 have used staining for acid alphanapthyl acetate esterase since peripheral blood T cells give a “dot-like” pattern of staining while B cells are negative. They found that the majority of salivary gland lymphocytes lacked the dot-like staining pattern and concluded that these were B cells. Data now show that mitogen activated T cells lose their alpha-napthyl acetate esterase activity.‘2’m’23 Thus, presence of pyrinophilic cells and alpha-napthyl acetate esterase-negative cells
95
SJOGREN SYNDROME
in salivary gland may result from in vivo activation of T cells, a supposition that is consistent with our finding of activated T cells reactive with antibodies OKTlO and antibody to Ia. Other methods to enumerate T cells and B cells in the salivary gland have included specific antiserum to immunoglobulin and rosetting assays with complement-coated erythrocytes.‘24*‘25 These studies have yielded conflicting results. Using heteroserum to thymocytes Talal et a1’24found a predominance of T cells only in very large foci while we found a majority of T cells regardless of focus size. This difference from our study may result from different antibodies used as detecting reagents. Also our patient selection was totally primary SS while the other studies were predominantly secondary SS plus RA. In our experience, the enumeration of B cells in tissue sections using monoclonal antibodies is difficult. Antibodies to IgG, IgA, IgM, kappa, or lambda are suitable for staining plasma cells but the large amount of interstitial immunoglobulin prevent the enumeration of surface Ig+ B cells. Relatively few B cells were noted with antibody to IgD, a marker present on the majority of peripheral blood B cells. However, IgD cannot be used as a measure of total B cells since it is known that germinal center B cells in lymph node lack IgD.99 However, germinal center B cells can be detected by monoclonal antibody B532llO.lli; therefore, we have used the combination of IgD and B532 antibodies to enumerate the B cell subpopulations. It remains possible that additional B cell subpopulations lacking these markers are present in primary SS biopsy specimens and studies are currently in progress using newly developed monoclonal antibodies to nonimmunoglobulin antigens on B cells including the complement C3 receptors. Using these methods 10% to 25% of lymphoid cells in primary SS salivary gland biopsies have B cell markers.74 This includes 5% to 10% of cells bearing cytoplasmic IgG or IgA and having a plasmacytoid appearance.“‘*‘26 Also, we have noted a higher proportion of B cells in secondary SS biopsies than in primary SS biopsies and that these secondary SS B cells are more likely to be located in germinal center-like structures (authors’ unpublished observations). These differences in the immunohistologic architecture between primary and secondary SS may
represent different mechanisms of pathogenesis or may reflect prior treatment in the secondary SS patients.
In Vitro Functional Studies of Lymphocyte Function PBL from primary SS patients can be stimulated by mitogens to secrete immunoglobulin in vitro (Table 11). Production of IgG and IgM was comparable to that of PBL from normal subof these T cells (E’ = sheep jects.‘08 Treatment erythrocyte receptor-positive cells) with monoclonal antibody 0KT4 plus complement abolished IgM and IgG synthesis whereas antibody 0KT4 alone or complement alone was without with antibody OKT8 plus effect.74”08 Treatment complement did not alter the amount of Ig synthesized or lead to the production of RF. Thus, T helper activity in the blood of primary SS patients is included in the OKT4 subset; this is similar to results obtained with normal blood lymphocytes. Similar to PBL, the salivary gland lymphocyte (SGL) could be induced to secrete immunoglobulin in vitro (Table 11). However, the SGL produced RF while the PBL did not. Lysis of the T cells with antibody 0KT4 plus complement blocked this production. This suggests that tissue lymphocytes are the major source of RF production and that this synthesis remains under normal immunoregulation by T cells. Also the OKT4’ cells were noted to possess T inducer activity necessary for the generation of T cytotoxic cells.74 T suppressor and T cytotoxic activ-
Table 11. Production of Antibody In Vitro by SGL and PBL of Primary SS Patients IgM*
IgG’
BFt
SGL
310 ? 80 25 + 5 60 + 4
Total SGL
E+ EE+ (OKT4
+ C’) + E-
E+ IOKT8
+ C’) + E-
69 + 7
670
2 110
27+
10
110 + 15 cl0
85 k 25
60 + 25
76 2 21
295
k SO
416
? 104
371
+ 161
700
+ 186
PBL Total PBL
19 k 7
342
13
E-
27 + 5
39k
10
cl0
38 + 7
46 + 7
E+ (OKT4
+ C’) + E-
E+ (OKTE + C’) + E‘ng antibody 743
E+
t
315
* 152
698
+ SO for six normal PBL, IgM = 297
188.
tRF = IgM RF (ng k SO).
+ 179
+ 122 and IgG =
96
ity generated from primary SS PBL and SGL were included in the OKT8’ subset. Since 0KT4’ T-cells from normal PBL have recently been shown to possess NK and ADCC activities under certain circumstances,‘02~‘04 we examined SGL for these activities. Little NK activity (ie, lysis of “Cr-K562 cells) or ADCC function (ie, lysis of 5’Cr Chang cells coated with antiserum to Chang cells) was noted.74 The NK and ADCC levels in these patients blood was at the lower limit of normal range for this age group; the SGL’s activity was even lower than their respective PBL. In summary, our results suggest that the OKT4 and OKT8 T-cell subsets of PBL and SGL of primary SS patients possess functions similar to those in normal blood or tonsillar lymph node. However, the presence of “activation” antigens on the SGL (defined by monoclonal antibodies as described above) suggested that the initial stages of lymphocyte activation have already occurred in vivo for SGL. Further studies are in progress to determine the response of these PBL and SGL to soluble factors derived from lymphocytes. The ability to augment or inhibit these T cell responses may provide additional avenues for future therapy. Most previous studies on the functional properties of PBL from primary SS patients have indicated similar immunoregulatory properties as found in normal subjects. No increase in spontaneous IgG production or anti-TNP plaque forming cells was present;‘,‘*’ these findings contrasted with SLE patients where a marked increased in such activated B cells were presNormal conconavalin A suppressor cell ent. ‘27m’29 function was noted* and ADCC levels were within normal limits in one study13’ although low in a subsequent report.13’ Miyasaka et al’32.‘33 recently reported that some primary SS patients have a deficient response in autologous and allogeneic mixed lymphocyte reaction; this might reflect abnormality in T cell-macrophage interactions. In assessing functional studies of primary SS patients, it may be important to compare these patients to disease-free control subjects that bear histocompatibility antigens HLA-DR3.‘34 Ambinder et al’35 showed that such normal control subjects have increased IgG secretion in vitro as compared to HLA-DR3-negative normal sub-
FOX ET AL
jects. Defects in clearance of immune complexes or IgG-sensitized erythrocytes have been noted in HLA-DR3 positive individuals with SS’36 but also in HLA-DR3 normal control subjects.‘37.‘38 SPECULATIONS
ON PATHOGENESIS
In a general sense, the presence of autoimmune disease implies the presence of T-B cell and T-T cell interactions that allow responses against host tissues. This is shown schematically in Fig 10, where an extrinsic injury probably leads to release of tissue antigens. T helper and B cells are activated to produce autoantibodies; such T cells and B cells may be induced directly within the salivary gland or may migrate (ie, “home”) to this site after their encountering antigen in some of other lymphoid regions. In addition to antigenspecific responses, many antigen nonspecific responses may also result as a consequence of the release of soluble factors that enhance immunologic responses. Under normal circumstances, this “autoreaction” would be limited by suppressor cells. Therefore, the presence of autoantibodies implies either insufficient T-suppressor responses or failure of other cells to respond to these signals. The crucial questions to be answered are whether this represents an exaggerated form of normal immune response or whether there exists intrinsic defects in the T or B cells responsible for the disordered immunoregulation. The initial inciting lesion in SS remains unknown. Leading candidates include Epstein-
Extrinsic Injury
I
I
---Lhost cell disruption with release of autoantigens
1
7 generate T-suppressor
I generate l-helper B-cells
I
I
I
I
I
I
oolvclonal B-cell ktivation (RF, ANA)
I
s:A,
I
T-cells infiltrate salivary gland
I I
-
I
-- failure of T-suppressor cells tb'modulate
I
--__
I Tissue injury
I
I 1_
Fig tion
I
__
Release of autoantigens
10.
Schematic
in SS.
representation
for
immunoregula-
97
SJOGREN SYNDROME
Barr virus (EBV) and cytomegalovirus. These viruses are thought to reside in the salivary EBV can be glands normally.‘39*‘40 Infectious cultured from the opening of Stensen duct in 20% of normal subjects and in >50% of patients receiving corticosteroids or immunosuppresand sives.‘4’ Thus, these viruses are ubiquitous already present at the relevant target organ.‘4o9’42 Further, EBV can stimulate polyclonal antibody production and autoantibody synthesis such as RF.‘43 Recently, Yang et a1299’44reported that antibody SS-B found in many SS patients was directed against a ribonuclear protein that selectively associate with EBV-encoded RNA. In nasopharyngeal carcinoma, a disease that is known to be associated with EBV transformation of epithelial cells,‘45-‘47 an intense lymphocytic response occurs that has been termed a “lymand these lesions resemble phoepithelioma”,‘48 the changes found in primary SS. Taken together, these findings suggest a role for EBV initiating SS, but direct evidence for the presence of viral genomes and gene products still must be established. Since HLA-DR 3 is more prevalent in primary SS, genetic factors may be important. Evidence for three possible mechanisms for the role of histocompatibility antigens and disease have been presented. These genes may predispose patients to an exaggerated or deficient response to certain infections such as EBV.‘49-‘5’ The gene products of the HLA-DR gene (termed Iaantigens for “immune associated” proteins with molecular weight 34,000 and 28,000) may serve as a “target” for autoimmune lymphocytes. This is presumed to be the mechanism in the salivary gland involvement in mice with chronic graftversus-host disease.ls2 Finally, HLA-DR3 has been associated with defective clearance of immune complexes by the reticuloendothelial system in liver and spleen.‘37m’38 Possibly allowing immune complexes to localize at other target sites such as salivary gland where they could stimulate immune responses. After the “initial lesion” in a genetically susceptible individual, lymphocytic infiltration of T cells and B cells occurs. These cells are predominantly T-helper cells, and they induce local active immunoglobulin synthesis.‘53*‘54 These immunoglobulins are broken down to form Fc-
fragments that further stimulate antibody production’5s*‘56 or form immune complexes that activate complement, which in turn can further activate lymphocyte function.‘57*‘58 It is therefore possible that chronic stimulation of B cells within lymph nodes or other tissues may result from exposure to antigen(s), T cell derived soluble factors, and fragments derived from immune complexes and complement. The original initiating antigen need no longer be present by the time that disease is clinically apparent. Since the MRL/l mouse shows tissue lesions similar to human SS (ie, salivary glands infiltrated by Lyt l+, 2- T cells, hyperglobulinemia, and RF),‘59*‘62 it is important to note that these animals have marked abnormalities in production of their T cell derived lymphokines; they have diminished levels of interleukin 2 production but high levels of factors that drive B cell growth and differentiation.‘60.‘6’ The actual mechanism of destruction of the salivary and lacrimal gland also remains unknown. The cells infiltrating the acini and tubules are probably responsible for cellmediated destruction. Although ADCC mechanisms remain possible, antibodies to salivary duct are infrequent in primary SS patients.60 The actual antigens being detected may be a viral antigen or perhaps a host antigen crossreactive with a viral gene product.‘63 TREATMENT OF PATIENTS WITH SS
The treatment of SS is aimed at symptomatic relief and limiting the damaging local effects of chronic xerophthalmia and xerostomia. They may have specific problems such as thyroiditis, nephritis, gastritis, or pneumonitis. In addition many have symptoms suggestive of a chronic pain syndrome that includes myalgias, arthralgias, and depression. The goals of therapy must be to develop programs to address organ specific as well as organ nonspecific manifestations of this disease. The Dry Mouth Clinical management of the dry mouth is a difficult problem. In addition to rampant dental caries and chronic pain, patients have troublesome intraoral soft tissue problems that include infections due to Candida and difficulty with dentures due to dryness. Before any treatment
98
FOX
program is started, it is important to identify contributing factors such as mouth breathing, heavy smoking, stress, depression, and drugs that have anticholinergic side effects. The most frequently implicated drugs are the phenothiazines, tricyclic antidepressants, antispasmodics, antiparkinsonian, and decongestant medications. Home remedies and nonprescription medications may possess anticholinergic side effects even though the patients may not recognize these agents as “drugs.” Many of the treatments (or approaches to treatment) suggested below have initially been used by radiotherapists for patients with prior radiation of the head and neck, and we have found similar treatments helpful in our patients. Most patients discover that it is helpful to carry water with them, and to avoid dry foods, alcohol, tobacco, and mouth breathing. Dental prophylaxis by their dentists is supplemented by frequent use of a toothpick or Water Pik device. Sugarless chewing gum and sugarless lemon drops are helpful in some cases. A variety of saliva substitutes are available (Table 12). These differ in their flavoring agents and preservatives. Salivart spray has the theoretical advantage that it contains no preservatives since these agents may be responsible for topical irritation in some patients. After administration of these sprays, parotid flow rates are increased for seven to eight minutes in both normal subjects and SS patients.‘64m’66 However, the patient’s sense of Table
12.
Commercial Tears
Preparation Artificial
Preparations
of Artificial
and Saliva
Manufacturer
PR?SerVstlVe
saliva
Saliment
Ferring
Parahydroxybenzoate
Xerolube
Scherer
Paraben
Saliva
Roxane
Paraben
Westoort
None
Substitute
Salivart Artificial
tears
Liquifilm Liquifllm Tears
Forte
Plus
Allergan
Chlorbutanol
Allergan
Thimerosol
Allergan
Chlorbutanol
Hypotears
cooper
Benzalkonium
chloride
Tears
Alcon
Benzalkonium
chloride
Adsorbotears
Alcon
Thimerosol
Mum
Muro
Benzalkonium
Duolube
Muro
None
Duratears
Alcon
Methylparaben
Lacrilube
Allergan
Chlorbutanol
Lacrlserts
Merke
None
Ocular
Naturale
Tears ointments
chloride
ET AL
xerostomia may be decreased for up to several hours. Treatment with a 0.4% stannous fluoride has been suggested to enhance dental remineralization of damaged tooth surfaces. Previous studies have reported increased salivary flow rates after administration of parasympathomimetic agents such as piocarpine or neostigmine as either a mouthwash or as a systemic medication. Others have used potassium iodide solutions (SSKI) for this purpose. Varying degrees of success have been noted. Our experience and that reported at the NIH” indicates that these medications may help some patients with relatively early or mild xerostomia but not patients with severe xerostomia. Treatment
of Dry Eves
The treatment of dry eyes is not only symptomatic but also designed to prevent scarring secondary to KCS or infection. The administration of artificial tears containing methylcellulose (Table 12) gives considerable relief to most patients, but disabling symptoms remain present in some cases. Failure of artificial tears has been attributed in some cases to the preservatives in the artificial tears (such as benzalkonium chloride, chlorbutanol, or thimerosal) that provoke further topical irritation.“’ We routinely ask patients to try several different preparations sequentially to identify those that seem most tolerable. Lacrilube or Duolube ointment is prescribed for use at bedtime. In patients who have mild dry eyes, Lacriserts have been helpful, but our patients with marked xerophthalmia have frequently noted increased ocular irritation on inserting the pellets or visual blurring leading them to discontinue this medication. In all patients, symptoms tend to wax and wane, particularly in association with the seasons when dry winds are prevalent. When patients can identify exacerbating problems increased frequency of artificial tear application should be started before symptoms develop in the hope of preventing objective eye findings. The use of humidifiers at night, wrap around glasses, and even goggles during flares often is helpful. Sudden worsening of ocular symptoms should always suggest possible ocular infection. In patients with associated diseases such as RA, nodular corneai lesions and vasculitic lesions also must be considered.
99
SJOGREN SYNDROME
European studies have suggested that bromhexine, a synthetic alkaloid derivative originally used as a mucolytic agent in cough remedies, may have beneficial effects in increasing lacrimal and salivary gland function.‘68*‘69 Doses of at least 48 mg/d were required since no benefits were reported at lower doses. This medication is not yet available in the United States. In patients at our clinic who have been refractory to commercially available artificial tears and ointments, we have recently begun a study of using artificial tears prepared from the patient’s own serum. The serum is diluted with two volumes of sterile normal saline and aliquoted into 1 mL vials (in the absence of added preservative). These vials are then stored at -20 “C, and the patient removes one vial per day to use as a source of artificial tears. In 15 consecutive KCS patients we observed improvement in symptoms and in rose bengal and/or fluorescein staining. None of the patients had complete resolution of symptoms but each described a generally soothing effect of the autologous serum tears.“’ Patients could easily distinguish between these eye drops and normal saline or their commercially available tears. However, double-blind prospective studies must be conducted before this approach can be advocated for general use. Finally, in patients in whom response to commercially available (or serum) tears remains too transient, consideration can be given to closure of the puncta and canaliculi by surgery or cautery. This opening is surrounded by dense, relatively avascular connective tissue and can be occluded by electrocautery as an outpatient. Theoretically, soft contact lenses might help prevent the tear film by preventing evaporation. However, great care must be taken to maintain frequent lubrication with a low-viscosity artificial tear preparation or severe abrasions may result. Rarely, a partial tarsorrhaphy (sewing the lateral portion of the eyelids together) may be required. Dryness of the Nasal Passage and Sinusitis Many patients complain of nasal dryness and symptoms of sinusitis with postnasal drip. Our initial approach is to provide increased moisture to this region by use of normal saline sprays (Ocean Spray) and humidifiers at night. In some cases, nasal irrigation with normal saline proves helpful. This can be easily performed by the
patient using an irrigation syringe or a Water Pik device (set for the lowest volume-pressure delivery level). The diagnosis of sinusitis is confirmed by sinus roentgenogram with air-fluid levels and CBC with leukocytosis. In these cases, it may be necessary to establish drainage, obtain definitive cultures, and treat with antibiotics. Role of Diet Patients frequently ask about the role of diet either in causing their disease or in their treatment. No definite answers are known but environmental agents (perhaps food antigens) may play a role. One of the best examples of diet-related autoimmune disease is celiac sprue, where autoimmune reaction against gliadin (a wheatderived product) plays an important role.“’ These patients have a high frequency of histocompatibility antigen HLA-DR3, similar to primary SS patients. Another example is dermatitis herpetaformis patients who possess iodide sensitivity and HLA-DR3.‘72 Therefore, some undetected antigen in food may play an analogous role in primary SS. If this analogy is correct, then a high titer of antibodies to the particular food antigens should be detectable. However, caution must be exercised in looking for “anti-food” antibodies since antibodies to milk protein have been found in normal individuals’73 as well as those with apparent food allergies. Recent interest has centered on the possible role of linoleic acids since these fatty acids are precursors of prostaglandins and/or leukotrienes that are important in the inflammatory response. One preliminary report suggests a deficiency of prostaglandin E, in SS that was treated with dietary supplement of fatty acids.‘74*‘75 Vitamin A deficiency has also been suggested since patients with this deficiency may have xerophthalmia.“‘j However, these reports must be considered preliminary. Many of our patients have tried these treatments and various elimination diets without benefit. In our clinic, we have suggested that patients take a supplemental multivitamin of their choice. We suggested yogurt or buttermilk added to diet since some patients have noted beneficial responses and had an apparently lower incidence of oral candida. Based on reports that zinc was helpful in reducing stomatitis in patients after
FOX ET AL
head and neck irradiation’76 we tried zinc sulfate (220 mg/d) without significant improvement in most cases. In summary, double-blind studies on large numbers of primary SS patients will be required before the role of dietary factors can be assessed. Approach to Treatment of the Myalgias. Arthralgias. and Fatigue in SS Patients Many patients complain of profound myalgias. In most cases, normal (or only slight elevation) of muscle enzymes such as aldolase are present. Several patients had electromyographic studies with mild myopathic changes and their muscle biopsy specimens showed perivascular lymphocyte accumulations (mostly T cells) without frank changes of muscle necrosis. Similar perivascular changes have been noted in muscle biopsy specimens in RA or SLE that were performed because of myalgias. However, the significance of these biopsies remains unclear until results are available on biopsies from primary SS patients who lack myalgias. Frequently, the patient has already been told that their symptoms are due to depression. Although this may in part be true, it often leads to anger and loss of the therapeutic physician-patient relationship. When antidepressants are used, medications with minimal anticholinergic side effects (Amoxapine, Norpramin, Trazadone) are preferable since amitrypthine (Elavil) may cause significant ocular and mucous membrane drying that exacerbates underlying sicca symptoms. The Role of Corticosteroids and Immunosuppressive Agents In general, we try to avoid these medications except in cases where visceral disease (pneumonitis, nephritis, CNS symptoms) requires their use. Since our patients do not frequently have clinically significant nephritis or CNS disease, the major use has been for pneumonitis or polyserositis refractory to nonsteroidal agents. The role of immunosuppressive and/or cytotoxic drugs in the development of lymphoma has been extensively discussed.‘0,“,‘7.74.‘77 Some groups believe that these treatments predispose to development of lymphoma.” Other groups assume that only the most severe patients are treated with cytotoxics and these patients were more likely to develop lymphoma because of
severe disease.” Because of the increased incidence of lymphoma in animals treated with cyclophosphamide and in other patients receiving this drug, we try to avoid its use. In many cases, patients have been inactive for many months and have marked “deconditioning” of their cardiovascular and musculoskeletal system. After reassurance that muscle necrosis has not occurred, we emphasize physical therapy and gradually increasing exercise programs. Lowdose salicylates or nonsteroidal drugs are frequently helpful. Surgical Approach to the Salivary Gland In certain patients, biopsy of the major salivary glands may be required to rule out lymphoma. The basic approach is a superficial parotid lobectomy or removal of the entire submandibular gland. The frequent occurrence of scarring and cyst formation due to the disease process may make identification of the facial nerve difficult. In other patients, parotid infections or abscess may require surgical drainage. In this type of case, a facial flap may be raised and then a tunnel created parallel to the branches of the facial nerve to break the septate connections that exist within the gland and to allow adequate drainage. Ultrasound may aid in identifying abscesses within the gland preoperatively. SUMMARY
In this review, we have concentrated on the immunohistologic features of primary SS. The precise relationship between primary and secondary SS and SLE or RA is unclear in many patients, particularly early in the disease. Nevertheless, recognition of sicca components is important in patient management. The extraglandular features found in primary SS were reviewed to emphasize the reported histologic changes. Routine studies on tissue biopsy specimens can be extended by using monoclonal antibodies that detect particular subsets of mononuclear cells. Studies of lymphocyte function show in primary SS patients that salivary glands contain predominantly T-helper/inducer cells. Based on these results, speculations in the pathogenesis of primary SS are presented that incorporate current concepts of soluble factor (ie, lymphokine) function. Finally, we outline the mul-
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tidisciplinary approach to therapy followed by our divisions of rheumatology, opthalmology, otolaryngology, and oral medicine. ACKNOWLEDGMENT We thank the members of the division of rheumatology (Drs K. Hench, J. Vaughan, E. Tan, C. Robinson, J. Curd, and F. Kozin), otolaryngology (Drs D. Bell and R. Fantozzi), and ophthalmology (Drs J. Michelson and R. Chan) for allowing us to study their patients. We appreciate the technical assistance of D. Harlow, R. Burhrow, S. Brewster, and F.
Kral in performing these experiments and preparing the manuscript. Special thanks to Carol Peebles, who evaluated autoantibody profiles on some of our patients, and to Drs Tom Adamson, Scott Carsten, and Carol Young who helped perform immunohistochemical studies during their rheumatology fellowships. Generous gifts of monoclonal antibodies were made available by Drs N. Warner (Becton-Dickinson, Mt View, Calif) and G. Goldstein (Ortho Diagnostic, Raritan, NJ). We appreciate the helpful discussion with Drs S. Fong, C. Tsoukas, D. Carson, E. Morgan, and A. Theofilopoulos on the mechanisms and immunoregulation. Finally, we thank our primary SS patients for participating in our studies.
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