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Circulating immune complexes in patients with necrotizing vasculitis
CLINICAL
IMMUNOLOGY
AND
Circulating
IMMUNOPATHOI.OGY
658-612 (1980)
Immune Complexes in Patients Necrotizing Vasculitisl
GARY M. KAMMER,' Departments
1.5,
of Medicine Medical
N.A.
SOTER,AND
and Dermatology. School. Boston.
P.H.
with
SCHUR
Robert B. Brigham Hospital, Massachusetts 02120
Hurvard
Received August 3. 1979 Sera from 29 patients with biopsy-proven idiopathic active cutaneous necrotizing venulitis (CNV) were examined for immune complexes by serum complement levels, Clq binding, and ADCC inhibition. For comparison these studies were also performed in patients with systemic lupus erythematosus (SLE). and rheumatoid arthritis (RA). Most of the patients with CNV, SLE, and RA have evidence of circulating immune complexes at a time during which they have active disease. Patients with CNV tend to have normal or slightly reduced serum complement levels, abnormal Clq binding assay levels. and abnormal ADCC inhibition. The complexes were mostly > 19 S in size. Density gradient analysis did not separate between the complexes in these three diseases. There was no association between ADCC inhibition and Clq binding or serum complement levels suggesting that these two assays detect different types of immune complexes. These observations suggest that multiple types of immune complexes exist in patients with different forms of vasculitis: those that appear to fix complement avidly and are cleared (low serum complement, low Clq binding); those that fix complement poorly in \?vo but are readily detected in vitro (high Clq binding): and those which bind to lymphocyte Fc receptors (ADCC inhibition). and appear not to fix complement.
INTRODUCTION
Numerous experimental animal studies have provided evidence strongly implicating immune complexes in the pathogenesis of necrotizing vasculitis (reviewed in Refs. 1 and 2). Moreover, extrapolating from the experimental studies in animals, it has been presumed that vasculitis in human disease is produced by the deposition of immune complexes in certain instances (3-8). The study of a welldefined patient population with one form of vasculitis, idiopathic cutaneous necrotizing venulitis (CNV) (9-l l), documented by examination of biopsy specimens. should permit a unique opportunity to characterize the circulating immune complexes in vasculitis. Therefore, 29 patients with idiopathic CNV documented by skin biopsy were studied at a time they had skin lesions by three serological assays for immune complexes: serum complement levels, which reflect in viva complement fixation by immune complexes (reviewed in 12, 13): a radioactive Clq binding assay which measures complexes still able to fix complement (14); and an antibody-dependent cellular cytotoxicity (ADCC) inhibition assay, which is complement independent, and reflects the ability of complexes to react with lymphocyte cell Fc receptors ’ This study was supported in part by USPHS Grants .4Mll414, AM05577. RRO05669, AIO0366, AIl0356. AM07031, by a Young Investigator Research Grant (AI-14292). and the New England Peabody Foundation, The Arthritis Foundation, and the Lupus Erythematosus Foundation. 2 Dr. Kammer was a postdoctoral fellow of the Arthritis Foundation. 658 0090-1229/80/040658-15$01.00/O Copyright 10 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
IMMUNE
COMPLEXES
IN
VASCULITIS
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(15). These studies in patients with CNV were compared to those in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Our results demonstrate the presence of heterogeneous circulating immune complexes of both complement-fixing and complement-independent types in most of the sera from patients with CNV. MATERIALS Patient
AND METHODS
Population
Two hundred twenty-four individuals were studied. Twenty-nine patients had idiopathic CNV documented by skin biopsy and unassociated with any other coexistent systemic disorder (9). Sera and skin biopsies were obtained simultaneously during a period when patients exhibited cutaneous lesions. Twenty-two of the patients had raised, erythematosus papules of various sizes that did not blanch with pressure (palpable purpura) and that occurred in crops that persisted for several days (9); episodes of recurrent urticaria occurred in seven patients (10). None of these patients had detectable hepatitis B antigenemia. Thirty-three patients had classical RA complicated by clinical features of vasculitis (17), including either digital infarcts, digital gangrene, leg ulcers, or peripheral neuropathy (mononeuritis multiplex). Twenty patients had definite or classical rheumatoid arthritis (RA) (16) with joint disease only and no clinical history or physical findings suggestive of vasculitis or other extraarticular manifestations of RA. Fortytwo. patients had active systemic lupus erythematosus (SLE), with either fevers not due to infection, arthritis or persistent arthralgia, anemia (hematocrit <30%), leukopenia ( <4000/mm3), a malar rash, and/or polyserositis (18). All SLE patients had four or more ARA criteria for SLE (19), and all had an ANA titer of > 1: 80 at the time they were studied. One hundred healthy hospital personnel served as normal controls. Serum Preparation
Venous blood was drawn into vacuum test tubes prewarmed to 37°C and allowed to clot at 37°C for 1 hr; the serum was separated at 37°C. Serum aliquots were immediately frozen at -70°C. Prior to use in experiments, frozen sera were thawed only once at 37°C. ADCC
Inhibition
Assay
This assay was performed as recently described (15). In summary 60 ~1 of 50 mM EDTA, pH 7.8, was mixed with 200 ~1 of test sera and incubated at 4°C for 15 min. Next, 50 ,ul of serum-EDTA was added to triplicate wells (U-shaped microtiter trays, Cook Laboratory Products, Alexandria, Va.) containing 100 ~1 of 2 x lo6 effector lymphocytes/ml, thoroughly mixed, and incubated for 60 min at 4°C. To remove residual sera following incubation, effector cells were centrifuged in the trays and thoroughly washed twice in the wells using a Caz+ -Mg*+-free culture medium. To replete Ca2+ and Mg2+ necessary for in vitro cytotoxicity, the effector cells were washed a third time in supplemented RPM1 1640. Finally, 75 ~1 of supplemented RPM1 1640, 100 ~1 of 51Cr-chicken red blood cells (51Cr-CRBC), and 25 ~1 of the appropriate anti-CRBC antiserum dilution were successively added to each well. Since this assay utilizes a single donor’s effector lymphocytes, the
660
KAMMER,
SOTER,
AND
SCHUR
standard final dilution of antiserum had been previously determined to be 2 x lo-“. Following centrifugation at 500 rpm for 5 min at 24”C, the microtiter trays were incubated at 37°C for 16 hr in a 5% CO,/95% air atmosphere with 100% humidity. The number of radioactive counts per minute in the supernatants was counted in a Packard Autogamma scintillation counter. The equations for calculation of the percentage ADCC inhibition3 have been given in detail elsewhere (20). Antibodies directed against both B and T lymphocytes have been demonstrated in the sera of some lupus patients (21). Since these antibodies might bind to the lymphocyte membrane via the Fc receptors or other membrane receptors, resulting in ADCC inhibition indistinguishable from that caused by immune complexlike reactants (hereafter referred to simply as immune complexes for the sake of brevity), the following studies were performed to determine whether antilymphocyte antibodies inhibit ADCC, as performed above. A rabbit antiserum prepared against human lymphocytes was incubated with the lymphocytes of 13 HLA-variable donors as target cells. These mixtures were then incubated with the standard effector lymphocytes. A mean percentage cytotoxicity of 58.3 (range = 33-95s) was induced, indicating that the antiserum possessed anti-lymphocyte antibodies. This antiserum was then ultracentrifuged at 100,OOOg for 90 min at 4°C (SW 50 rotor, Spinco Model L preparative centrifuge, Beckman Corp., LaJolla. Calif.) to remove aggregates. The upper one-third of the tube was collected. filtered (Millipore Corp., Bedford, Mass., 0.45 pm). and then incubated with the standard donor effector cells for 60 min at 4°C or 37°C. .These effector cells were then assessed for ADCC inhibition, as described above. Negligible inhibition (mean = 2.6 c 0.7%) of ADCC occurred after pretreatment of lymphocytes with the antiserum at 4°C; however, about 10% ADCC inhibition occured when effector cells had been pretreated with antiserum at 37°C. In addition, following partial blockade of surface Fc receptors with lOO-pg agg IgG/ml, cells were then reacted with antiserum for 60 min at either 4 or 37°C. No significant additional ADCC inhibition was observed compared to control effector cells pretreated with agg IgG only. In separate experiments, patients’ sera which had been shown to inhibit ADCC were chromatographed on DEAE. Fractions containing IgG inhibited ADCC while those containing IgM did not. Thus, these experiments suggest that antilymphocyte antibodies bind through another surface receptor and do not cause inhibition of ADCC. Clq Binding Assay (C/q BA) Clq was extracted from serum by the method of Yonemasu and Stroud (22), and was radiolabled with lz51 in the presence of lactoperoxidase. The Clq BA was carried out as described by Nydegger er u/. (23) and modified subsequently by Zubler et al. (14). An aliquot of the radiolabeled Clq was then incubated with both test and control sera to which EDTA had been added. Bound radiolabeled Clq was separated from free radiolabeled Clq by polyethylene gIyco1 precipitation. 3 Abbreviations used: ADCC inhibition, antibody-dependent cellular BA, 9-Clq binding activity; agg IgG, aggregated IgG; Ig, immunoglobulin.
cytotoxicity
inhibition;
Clq
IMMUNE
COMPLEXES
IN VASCULITIS
661
Standardization of the ADCC Inhibition Assay and Clq BA The isolation of Cohn fraction II, preparation of IgG aggregates (used herein as an in vitro model of immune complexes), and standardization of the ADCC inhibition assay have been previously described (15). The standard curve for the Clq BA was developed as follows: equal volumes of agg IgG in concentrations ranging from 20 mg/ml to 2 p.g/ml plus normal human serum (NHS) were mixed and assayed in the Clq BA. The estimated concentration of immune complexes in serum was expressed as equivalents of micrograms agg IgG per milliliter of serum by extrapolation from either the percentage Clq BA or percentage ADCC inhibition. The serum-agg IgG was prepared fresh with each experiment to standardize both assays. The standard curves in Fig. 1 give the percentage ADCC inhibition or Clq BA by different concentrations of agg IgG-serum. Sucrose Density Gradient Ultracentrifugation Into each cellulose nitrate tube a pellet of 0.3 ml of 70% sucrose in PBS -EDTA 15 mM (for samples later to be assayed by ADCC inhibition) or in barbital buffer, pH 8 (for fractions later to be assayed by Clq BA), is placed and then overlayed with 4.3 ml of sucrose in buffer, utilizing a density gradient former to produce an
PupAw IgG/mf FIG. 1. Standard curves of Clq BA and ADCC inhibition assay. Each point represents the mean 2 SEM of 25 experiments. The curves give the percent Clq BA or percentage inhibition of ADCC by different concentrations of agg IgG-NHS.
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KAMMER,
SOTER,
AND
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8-40% gradient. Four-tenths milliliter of experimental serum, agg IgG-NHS, or PBS-NHS were then overlayed onto each of three gradients and allowed to equilibrate at 4°C for l-2 hr. The gradients were then centrifuged at 37,000 rpm for 18 hr at 4°C. Twenty fractions were collected from the bottom of each tube. Samples to be assayed by ADCC inhibition were dialyzed against PBS-EDTA 15 mM, pH 7.8, at 4°C for 18-24 hr to remove residual sucrose; the remainder of the fractions were tested by the Clq BA. The concentrations of Ig and total protein in each fraction were determined as described below; IgG and IgM were utilized at 7 and 19 S markers, respectively. Other
Methods
Total serum hemolytic complement (CH50) was assayed by the method of Kent and Fife (24). Serum protein concentrations of complement proteins Clq, C4. C2. and C3, and immunoglobulins IgG and IgM were measured by radial immunodiffusion using monospecific antisera developed in this laboratory (25). Protein concentrations were determined by the Folin method (26). Antinuclear antibodies and precipitins to nuclear antigens and nucleic acids were detected by immunofluorescence and by counter immunoelectrophoresis (27). Statistical Methods
Statistical testing was performed on a Hewlett-Packard 9815 calculator. Results were expressed as the arithmetic mean F standard error of the mean (SEM), unless otherwise stated. Statistical significance was assessed by the Mann- Whitney test. Correlations were performed by the nonparametric Spearman’s rank correlation test (r,). RESULTS Complexes Quantitated by the Clq BA
The mean binding (2 2 SD) in sera from 100 normal individuals was 6.8 i 16.5 pg agg IgG/ml serum. As little as 1 pg agg IgG/ml could be measured by this system. Binding activities of greater than 23 pg agg IgG/ml were regarded as abnormally elevated, or positive. Fifteen of 29 sera from CNV patients had elevated binding activities. In addition, 8 of 20 sera from the uncomplicated RA group, 27 of 46 sera from the complicated RA group, and 20 of 42 sera from the SLE group were positive. The mean Clq BA of the CNV sera was 248 Fg agg IgG/ml; the mean Clq BA in uncomplicated RA was 103 pg agg IgG/ml; in the SLE group was 105 pg agg IgG/ml; and, in the complicated RA group was 929 kg agg IgG/ml (Table 1). The observed mean Clq BA was significantly higher in the CNV group than in normal controls (P < 0.0001). Complexes Quantitated by the ADCC tnhibition Assu~
The mean ADCC inhibition (5 2 SD) in sera from 100 normal individuals was 0.35 t 1.2 pg agg IgG/ml serum. As little as 0.1 pug agg IgG/ml could be quantitated. ADCC inhibition exceeding 1.5 pg agg IgG/ml was considered abnormally high, or positive. Twenty of 29 sera from the CNV group demonstrated elevated ADCC inhibition. Two of 20 sera from the uncomplicated RA group, 28 of 46 sera from the complicated RA group, and 34 of 42 sera from the SLE group had
RA
u Mean
-t 2 SD (range).
200+50 (150-250)
Healthy
controls
88 rt 77 (20- 192)
112 k 49 (20-244)
SLE
Complicated
251 2 75 (198-334)
RA
COMPLEMENT,
Uncomplicated
CHSO Wml)
OF SERUM
165 2 220 (O-342)
LEVELS
CNV
Disease
MEAN
343 2 200 (258 -745)
(288 -608)
144 2 248 (18-579)
245 r 250 (10-453)
484 + 248 (259-727)
308 + 419 (22-661)
c4 b.dml)
complement” c3 b.udml)
IN FOUR
(910-1980)
1340 zt 78
908 t 880 (310-2243)
10602960 (110-1780)
1539 r 386 (1240-2027)
1356 r 1550 (343 -3230)
TABLE I INHIBITION. AND Clq BINDING
416 k99
234 -c 300 (16-582)
207 2 459 (10-714)
477 + 158 (303 -584)
348 5 348 (16-576)
Clq (k.dml)
Serum
ADCC
PATIENT
(O-1.55)
0.35 + 1.2
6.5 + 26 (0.1-76)
8.3 k 25 (0.1-45)
1.4 + 8 (0.1-17)
1.5 k 25 (0. I-60)
ADCC* inhibition (@AHG/ml)
GROUPS
“SI-(-Jq”
(O-30)
6.8 2 16.5
105 t 506 (l-1550)
929 k 2667 (l-5600)
103 r 431 (l-870)
248 f 935 (l-1900)
binding (&AHG/ml)
AND CONTROLS
s 7 5
5 E
52
m CA
664
KAMMER,
SOTER,
AND
SCHUR
abnormally raised ADCC inhibition. The mean ADCC inhibition; of the CNV group (7.5 pg agg IgG/ml) was greater than that of normal controls (0.35 pg agg IgG/ml (P < 0.0001) (Table 1). While the mean ADCC inhibitions of each clinical group differed, overlap between individual values within each patient group as well as among groups was present. The levels of complexes detectable by the Clq BA were generally considerably higher than those detected by the ADCC inhibition assay. Often a 15- to loo-fold difference in concentrations between the two kinds of complexes was observed in individual sera. Serum Complement
Levels
The mean concentrations of serum CH50 and of Clq, C4, and C3 proteins in the CNV group and in other groups are given in Table 1. Mean levels in the CNV group were similar to those in the normal group, whereas the uncomplicated RA group tended to have elevated complement levels and the complicated RA group had significantly reduced levels of CH50 (P < 0.0001) and of C3 protein (P < 0.004). SLE sera had significantly reduced concentrations of CH50 (P < O.OOOl), Clq (P = 0.02), C4 (P < O.OOS), and C3 (P < 0.0001). Four sera from the CNV group had serum Clq levels ~100 &ml; of these, two had raised Clq BA. Compurison Levels
of Cly BA, ADCC
Inhibition
Assuy,
and Serum
Complement
A determination was made of the number of sera positive by both the Clq BA and ADCC inhibition assay, by a single assay, or in neither assay. The data are presented in Table 2. Most of the sera in the CNV, complicated RA, and SLE group had circulating immune reactants, and a large proportion of sera in these groups exhibited both kinds of circulating complexes (Table 2). The relationship between serum complement levels and serum concentrations of circulating complexes was then determined. In the CNV group a negative correlation of border-line statistical significance was demonstrated between the Clq BA and serum Clq levels (r s = -0.36, P < 0.05), and serum C4 levels (r, = -0.36, P < 0.05). There was no relationship between the Clq BA and the CHSO or C3 levels. In the complicated RA group a negative correlation was observed between the Clq BA TABLE THE
NUMBER
OF SERA IN CNV AND/OR ADCC
““I-Clq Disease
Poslpos
CNV Controls Uncomplicated Complicated SLE
RA RA
2
AND CONTROLS GIVING POSITIVE INHIBITION ASSAYS FOR IMMUNE
binding Pos/neg
assay/ADCC
AND/OR NEGA~WE COMPLEXES
inhibition
Neg/pos
10
5
10
Negineg ..-~~-~~~~4
0 23 17
8 4 3
2 5 17
10 1 5
Clq BINDING
Incidence circulating immune complexes CT) -- .._. _ . 86 50 97 88
IMMUNE
COMPLEXES
IN VASCULITIS
665
and serum CH50 levels (r, = -0.58, P < O.Ol), serum C4 levels (r, = -0.57, P < O.Ol), and serum C3 levels (r, = -0.50, P < 0.01). In the SLE group, a negative correlation of borderline significance was noted between Clq BA and serum CHSO levels (Y, = -0.32, P < 0.05), and serum Clq levels (r, = 0.37, P < 0.05). No relationship was demonstrated between this assay and serum C4 or C3 levels. The uncomplicated RA group showed no correlation whatsoever between the Clq BA and CH50 or any component levels. There were no apparent relationships between levels of complexes detected by the ADCC inhibition assay and levels of complexes detected by the Clq BA assay or serum complement levels. Sucrose Density Gradient Ultracentrifugation To determine whether circulating immune complexes in CNV exhibit differential sedimentation patterns from other sera, 21 sera positive by both assays from patients with CNV (9), SLE (4), and complicated RA (8) were subjected to density gradient ultracentrifugation in an 8-40% sucrose medium. The results of 6 of these 21 analyses are depicted in Fig. 2. CNV sera produced ADCC inhibition and Clq BA in both the 7- 19 S and >19 S regions (Fig. 2A and B), indicating that heterogeneous reactants of variable sizes circulate in these sera. Sera from the SLE (Fig. 2C and D) and complicated RA groups also yielded similar distributions of complexes (Fig. 2E and F). Normal sera did not inhibit in either assay. Aggregated IgG when added to normal serum and subjected to density gradient analysis showed that most ADCC inhibition was in the >19 S region, but some was in the 7- 19 S region; most of the Clq BA was in the > 19 S region. The absence of both ADCC inhibition and Clq BA in the 7 S region is expected as immune complexes are not found in this region.
10,000
r
$ io,ooo
1000
p
TnM
r g ‘Ooo tLnoc -‘*‘I-Ciq
1600
Tm
Binding
Activity 700 q
y;:
?!
i tn
50
t
OL
“‘b
2
I 4
I 6
F-lln 8
too
I\
2
4
6
8
400
2
4
6
8
40
FRACTION NUMBER FIG. 2. Plots of sucrose density gradient profiles of two sera from each disorder demonstrated to have both complement-fixing and complement-independent immune complexes. A, B: CNV; C, D: SLE; E, F: complicated RA. Note the absence of any distinctive gradient patterns which might characterize a specific disorder.
666
KAMMER,
MEAN
LEVELS
AND
RANGES
____CNV
Disease
7-19
SLE Complicated
AND
TABLE
3
SCHUR
OF: COMPLEXES
IN AN 8-40s
ADCC
SOTER,
FROM
SUCROSE
SERA
SEDIMENI
“‘[eclq BA” _--~~-~.s .>I9 s
inhibition”
s
~NG
GRADIENT
-a 19 s
?--I9
0.7 (0.1-1.2)
3.0 (0. I - 10.4)
54.0 (l-200)
82.0 (l-300)
2.4 (0.1 - 10.8)
‘.I (0.2-5.2)
21.0 rl--205)
(l-25)
204.0 (0.1~-1000)
118.0 (l-290)
148.0 (l--310,
RA (O.l-
3.1 12.3)
...~~
6.0
fl In pg AHGlml.
The mean ADCC inhibition and Clq BA in both regions (7- 19 S, >I9 S) were determined for each disease category for the 21 sera examined (Table 3). Similar mean levels were obtained in both regions by both assays. However, they tended to be higher in the >I9 S region in patients with complicated RA and those with CNV. Sera from patients with SLE tended to have somewhat higher mean levels in the 7- 19 S region. Sera from normals did not react in either assay. Yet even with these differences no distinctive gradient pattern for any of these disorders was identified. Whether the absence of a discrete gradient pattern reflects the limitations of the sucrose gradient technique or indicates that no such differences exist among these disease sera is unresolved. Relationship Patients
of Lelvels of Serum Complement
und Immune
Complrxes
in CNb
Of the patients with CNV, 12 had hypocomplementemia. Five of these 12 patients had complexes detectable by Clq BA; 9 of 12 had complexes detected by the ADCC inhibition assay (Table 4). In the 17 CNV patients with normocomplementemia, 10 had complexes detected by Clq BA and 11 by ADCC inhibition (Table 4). There was no difference in ADCC inhibition or Clq BA levels in patients with CNV presenting as palpable purpura or urticaria. DISCUSSION
Certain similarities between experimental models of vasculitis in animals and vasculitis in humans have been noted, including similar pathology, demonstration TABLE THE RELATION
OF SERUM
Complement level Normocomplementemia Hypocomplementemia
4
TOTAL HEMOLYTIC COMPLEMENT TO Posn IVE AND/OR FOR IMMUNE COMPLEXES IN PATIENTS WITH CNV
“‘I-Clq Posipos 5 5
binding Poslneg 5 0
test/ADCC
NEGATIVE
ASSAYS
inhibition Negipos 6 4
Neglneg 1 3
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of immunoglobulins and complement components at the site of tissue damage, and decreased levels of serum complement. Taken together these observations suggest that circulating immune complexes may participate in certain forms of vasculitis in humans. The present studies were instituted to search for circulating immune complexes in patients with necrotizing vasculitis. There are numerous methods for detecting immune complexes that are dependent on the different physical and biological properties of immune complexes. These include: molecular weight and valence of the antigen; molecular weight, class, subclass, and affinity of the antibody; molecular weight and ratio of antigen to antibody in the immune complex; ability of the antigen, antibody, and/or immune complex to activate the classical and/or alternative complement pathways; and ability of the complex to react with rheumatoid factors and/or with cell receptors. No single assay can detect all immune complexes. Most assays have been tested or standardized with respect to immune complexes of known composition, or to aggregated human y-globulin-which behave like immune complexes (28). In addition some assays will react with nonimmune complex material such as DNA, endotoxin, and low molecular weight 7 S substances (29). While in most situations these assays appear to detect material in patients’ sera that behaves like immune complexes, the identification of actual antigen-antibody complexes has rarely been achieved (30). In the present study three assays thought to reflect those properties of immune complexes that might be relevant to a study of vasculitis were used: serum complement levels which may be depressed in the serum of patients with vasculitis and are than thought to represent in vivo complement fixation by immune complexes (13); an assay that detects complexes that can fix complement (the Clq binding assay); and an assay that is independent of complement and that interacts with the Fc receptor of cells (the ADCC inhibition assay). Patients with CNV tend to have: normal or slightly reduced complement levels-if low, usually CHSO, Clq, and C4 levels are low and C3 levels are normal; abnormal Clq binding assays in one-half of patients; ADCC inhibition assays in most patients; and more complexes in the > 19 S region than in the 7- 19 S region. In previous reports regarding patients with vasculitis, the patient populations studied formed a rather heterogeneous group of vasculitides, including patients with CNV. Cutaneous necrotizing venulitis, which is defined by its characteristic pathology, may present as a polymorphous array of different skin lesions; however, the most diagnostic and consistently present skin lesion is palpable purpura. Less common manifestations include urticaria (lo), angioedema, and macular erythema. This variety of cutaneous lesions has led to the description of similar patients with hypocomplementemia and Clq precipitins under the terms systemic lupus erythematosus-like syndrome, erythema multiforme, and hypocomplementemic vasculitis urticaria syndrome (29, 31-36). The infrequent recognition of these diverse skin lesions as manifestations of underlying cutaneous necrotizing venulitis and the failure to define the type of vasculitis results from the fact that skin biopsies are infrequently performed in such patients. Therefore it is often difficult to compare the results in studies in which the type of vasculitis was not defined. In a study of 17 patients with “idiopathic cutaneous vasculitis” (12
668
KAMMER,
SOTER,
AND
SCHUR
were our CNV patients) by the WHO collaborative study group 18 assays were used to detect circulating complexes; from 41 to 76% of patients had circulating complexes by the nine assays that discriminated this group of patients from normals (37). In a study of 25 patients with “cutaneous vasculitis” 14 (56%) had complexes as assessed by the Raji cell radioimmunoassay (6). These results included 5 of 7 (71.4%) with rheumatoid arthritis and “vasculitis,” 6 of 10 (60%) with Sjogren’s syndrome and/or cryoglobulinemia, and 3 of 8 (37.5%) with “idiopathic vasculitis“ (3 of 7 sera provided by us had a positive assay). In a study of 56 patients with cutaneous vasculitis, which included 17 with idiopathic vasculitis. 18 with vasculitis and associated diseases, and 21 with other forms of vasculitis, 21 of 56 (37%) had circulating complexes as detected by either Clq binding or monoclonal rheumatoid factor assays (38). These positive results occurred mainly in those individuals with vasculitis plus associated diseases. Most of the binding materials were of high molecular weight. ~19 S. Complexes have been detected by reaction with monoclonal rheumatoid factor in 3 of 11 patients with systemic and cutaneous vasculitis but in 0 of 7 patients with cutaneous vasculitis (5). In a study of 53 patients with biopsy-proven cutaneous vasculitis. 8% had precipitins with ‘Clq, 37% had anticomplementary activity, 43% could inhibit EA rosette formation, and none had precipitins with rheumatoid factors (39). Two patients with vasculitis associated with HBSAg had a positive Staph A binding assay (40). Sera from two patients with polyartertis nodosa inhibited ADCC (41). A number of patients with various cutaneous manifestations of vasculitis have had intermediate (7- 19 S) and large molecular weight complexes (> 19 S) detected by Clq binding (29, 31-33, 35, 36. 42). cryoglobulins (11. 43, 44). serum hypocomplementemia, and the deposition of immune reactants t38,45-47) in the skin. Not all patients with vasculitis, including CNV, have hypocomplementemia (10). In summary, these studies are limited in many instances by the presence of several disorders, lack of knowledge of the state of disease activity, limited or no follow-up intervals. and the absence of documentation of vasculitis by examination of tissue biopsy specimens. Therefore the results in which biopsies were not obtained may represent either different forms of vasculitis, if indeed it was present. The patients in this study with RA complicated by features suggestive of vasculitis (17) manifested by either peripheral neuropathy, digital gangrene, nailfold infarcts, cutaneous ulcers, coronary artery involvement, and/or pericarditis were studied for comparison to those patients with idiopathic CNV. These patients tended to have: somewhat reduced serum complement levels, especially CHSO and C4: most patients had abnormal Clq binding and ADCC inhibition assays, often with very high levels-Clq binding levels tended to be higher than in patients with CNV; complexes tended to be more in the ) 19 S region than in the 7- 19 S region, particularly those causing ADCC inhibition. Rheumatoid factors did not appear to affect immune complex assays or levels. These observations in complicated RA are similar to the limited reports by others in patients with “rheumatoid vasculitis.” Onyewotu rt ul. (48) noted enhanced uptake of aggregated IgG by guinea pigs macrophages by all 22 sera studied from patients with RA and cutaneous vasculitis-the type of vasculitis
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IN
VASCULITIS
669
was not stated. Theofilopoulos et al. (6) noted increased Raji cell assays in 5 of 7 sera from patients with rheumatoid vasculitis-f these 7 sera 4 were provided by us and were patients with RA and CNV. All of 7 patients observed by us with either “complicated RA” plus CNV or “uncomplicated RA” plus CNV had immune complexes by our assays. Sera from patients with complicated RA contain both intermediate (7- 19 S) and large (>19 S) complexes detected by reaction with bovine conglutinin (50). One serum showed enhanced uptake of aggregated IgG by guinea pig macrophages by large complexes (48). We have previously noted low complement levels in patients with complicated RA (49). The patients in this study with SLE tend to have: low levels of serum complement including CHSO, Clq, C4, and C3; about one-half of the patients had immune complexes detected by Clq binding, usually of moderate titer; most of the patients had complexes detected by ADCC inhibition, usually of moderate titer; complexes tended to be more in the 7-19 S region than in the >19 S region. The patients in this study with uncomplicated RA tend to have: normal to elevated complement levels; about one-half have complexes detected by Clq binding and few by ADCC inhibition-levels of these complexes were only slightly elevated. These studies demonstrate a marked heterogeneity of complexes, although there is a tendency for different patterns in different clinical entities. These observations also confirm other studies which found complexes in the serum of patients with RA and SLE of molecular size 7- 19 S and > 19 S that can bind complement or bind lymphocyte to receptors. The wide range of frequency of positivity by different assays in these disorders suggests either that these assays have varying sensitivities, or that they measure different complexes. This point was examined in the present study by simultaneous assessment of sera by three different assays. If one compares the results obtained by these three assays there is little, if any, correlation in the levels between each assay. The level of circulating complexes detected by Clq binding appears to be considerably higher than the level of complexes detected by ADCC inhibition. The presence of complexes in the ADCC inhibition assays did not correlate with the presence of complexes in the Clq binding assay levels nor with serum complement levels. There was some, but not striking, correlation between Clq binding assay levels and: serum complement (Clq, C4) levels in patients with CNV; CHSO, C4, and C3 levels in patients with complicated RA; and with CH50 and Clq levels in patients with SLE. Either a lack of, poor correlations, or some correlations between the levels detected by different assays have been noted by others. Tung et al. (51) noted a good correlation between levels of complexes detected by the Raji cell assay and the Clq solid phase assay in patients with various forms of glomerulonephritis. Gabriel and Agnello (52) noted some correlation (r = 0.36) between levels of complexes detected by a Clq inhibition assay and a monoclonal rheumatoid factor assay. They noted a close correlation with complement levels in serial studies of 6 SLE patients. Zubler et al. (53) noted only some (r = 0.5) correlation between Clq binding activity and synovial fluid C4 levels in patients with RA, but no correlation between Clq binding levels and serum CHSO, Clq, C4, or C3 levels in these patients. In later studies this group (54) noted a correlation between plasma or synovial
670
KAMMER,
SOTER,
AND
SCHUR
fluid C3d levels and Clq binding levels in patients with rheumatoid arthritis. Davis er al. (55) noted a correlation between an anticomplementary assay and a platelet aggregation assay in examining SLE sera. Rossen et ul. (56) noted no correlation between Clq binding levels of complexes and serum levels of C3 and Clq in patients with nephritis. These observations suggest that different assays detect and measure complexes with different properties. Therefore the fair (at best) correlation between assays suggests that these sera contain more than one type of complex. The lack of association between ADCC inhibition, complement levels, and Clq binding suggests that ADCC inhibition detects another type of (noncomplement fixing) complex, that has the ability to bind Fc receptors. This observation is consistent with our previous observation ( 15) that ADCC detects complexes that have not fixed C3. These data also suggest that there are considerable levels of non-complement fixing complexes in this disease. In our understanding of the pathogenesis of cutaneous necrotizing venulitis (CNV) one can speculate that those patients with CNV and low serum complement levels have complexes which activated complement in IP~,Wand caused local tissue damage. The histopathology of the skin lesions in hypocomplementemic CNV shows predominantly neutrophils, with some lymphocytes (9). These findings are consistent with animal models where complement-fixing immune complexes mediate tissue injury and result in lower serum complement levels. Nine of twelve patients had complexes detectable in ~ritro by Clq binding. This observation points to the limitation of this assay. The patients with CNV and normal serum complement levels could have accelerated complement metabolism with a compensatory hypersynthesis of complement, similar to what has been documented in patients with RA and normal complement levels (57). These complexes in patients with normocomplementemic CNV may be weak complement fixers, strong enough to bind Clq in Grro but not enough to lower serum complement levels in \Y\*o. The biological role of these complexes at the tissue level is not clear. On the other hand 11 of the 17 patients had complexes detected by ADCC inhibition, demonstrating that these sera have complexes able to interact with lymphocytes. The fact that normocomplementemic CNV patients have a predominantly lymphocytic infiltration about vascular skin lesions (9) suggests a possible relationship between the in Gtro and in rlir~ findings. The observation that only 1 patient with CNV did not have complexes detected by these three assays (complement levels, Clq binding, and ADCC inhibition), strongly suggests that these complexes do play some role in CNV: however, other factors may be involved. These data also suggest that sera with low complement levels had complexes which fixed a substantial amount of complement, that is more complement than was synthesized. The observations of low serum complement levels and low Clq binding levels in patients with SLE suggests that most of the circulating immune complexes in this disorder are avid complement fixers and have been cleared or deposited in tissues. If complexes are then detected in these sera, in vitro, it suggests that their ability to fix complement (viz Clq) is weak: for if the complexes were avid complement fixers they would have been cleared from the circulation. The observation in patients with complicated RA of only mild serum complement
IMMUNE
COMPLEXES
IN VASCULITIS
671
depression and high levels of Clq binding suggests that the complexes detected in fixers and not be involved in tissue injury, in this disorder. This hypothesis could therefore explain the observation of elevated Clq binding in the presence of normal complement levels-i.e., the only complexes still present in the circulation are those with weak complement (viz Clq) binding. The biological role in vivo of these circulating complexes that are able to bind complement or cells in vitro remains speculative. Other interpretations of the present and other data are also plausible, including a possible role for non-immune complex-mediated mechanisms in patients with vasculitis (58). One point seems apparent in these patients; there are many types of complexes which possess various properties and are detected by different assays.
vitro may be poor complement
ACKNOWLEDGMENTS We gratefully acknowledge the expert laboratory assistance of Mr. Chip Kava and Ms. Sally Brown, the measurement of Clq BA by Dr. David Glass, and the secretarial assistance of Mrs. Brenda Tracey.
REFERENCES 1. Cochrane, C. G., and Koffler, D., Adv. Immune/. 16, 186, 1973. 2. Haakenstad, A. O., and Mann& M., In “Autoimmunity, Genetic, Immunologic, Virologic, and Clinical Aspects” (N. Talal, Ed.), pp. 278-360, Academic Press, New York, 1977. 3. McDufie, F. C., Sams, W. N., Maldonado, J. E., Andreini, P. H., Conn, D. L., and Samayoa, E. A., Mayo Clin. Proc. 48, 340, 1973. 4. Luthra, H. S., McDulTie, F. C., Hunder, G. G., and Samayoa, E. A., J. Clin. Invesr. 56, 458, 1975. 5. Conn, D. L., McDuITie, F. C., Holley, K. E.,andSchroeter, A. L. Mayo Clin. Proc. 51,511,1976. 6. Theotilopoulos, A. N., Wilson, C. B., and Dixon, F. J. J. C/in. Invest. 57, 169, 1976. 7. Pussel, B. A., Scott, D. M., Lockwood, C. M., Pinching, A. J., and Peters, D. K., Lancet 2, 359, 1978. 8. Zubler, R. H., and Lambert, P. H., In “Recent Advances in Clinical Immunology” (R. A. Thompson, Ed.), pp. 125- 147, Churchill Livingstone, Edinburgh/London/New York, 1977. 9. Soter, N. A. Mihm, M. C., Gigli, I., Dvorak, H. F., and Austen, K. F., J. Invest. Dermatol. 66, 344, 1976. 10. Soter, N. A., N. Engl. J. Med. 296, 1440, 1977. 11. Soter, N. A., J. Invest. Dermatol. 67, 353, 1976. 12. Schur, P. H., Amer. J. Clin. Pathol. 68, 647, 1977. 13. Ruddy, S., Gigli, I., and Austen, K. F., N. Engl. J. Med. 287, 489, 545, 592, 642, 1972. 14. Zubler, R. H., Lange, C., Lambert, P. H., and Miescher, P. A., J. Zmmunol. 116, 232, 1976. 15. Kammer, G. M., and Schur, P. H., C/in. Immunol. Immunopathol. 10, 202, 1978. 16. Primer on the Rheumatic Diseases. J. Amer. Med. Ass. 224, 137, 1973. 17. Glass, D. N., Soter, N. A., and Schur, P. H., Arthritis Rheum. 19, 950, 1976. 18. Schur, P. H., and Sandson, J., N. Engl. J. Med. 278, 533, 1968. 19. Cohen, A. S., Reynolds, W. E.. Franklin, E. C., Kulka, J. P., Ropes, M. W., Shulman, L. E., and Wallace, S. L., Bull. Rheum. Dis. 21, 643, 1971. 20. Simchowitz, L., and Schur, P. H., Stand. J. Zmmunol. 5, 759, 1976. 21. Messner, R. P., In “Lymphocytes and their interactions” (R. L. Williams, Jr., Ed.), pp. 169- 180, Raven Press, New York, 1975. 22. Yonemasu, K., and Stroud, R. M., J. Immunol. 106, 304, 1971. 23. Nydegger, U. E., Lambert, P. H., Gerber, H., and Miescher, P. A., J. Clin. Invest. 54,297, 1974. 24. Kent, J. F., and Fife, E. H., Amer. J. Trap. Med. Hyg. 12, 103, 1963. 25. Schur, P. H., Connelly, A., and Jones, T. C., J. Immunol. 114, 270, 1975. 26. Lowry. 0. H., Rosebrough, N. R., and Farr, A. L., J. Biol. Chem. 193, 265, 1951. 27. Schur, P. H., DeAngelis, D., and Jackson, J. M., Clin. Exp. Immunol. 17, 209, 1974. 28. Arend. W. P., and Mannik, M., J. Exp. Med. 136, 514, 1972. 29. Agnello, V., Winchester, R. J., and Kunkel, H. G., Immunology 19, 909, 1970.
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30. Almedia, J. D., and Waterson, A. P., Lancer 2, 983, 1%9. 31. Agnello, V., KofBer, D.. Eisenberg, J. W.. Winchester, R. J., and Kunkel, H. G., J. Exp. Med. 134, 228s 1971. 32. Agnello, V.. Ruddy. S., Winchester. R. J., Christian, C. L., and Kunkel. H. G., Birth Defects Orig. Arf. Ser. 11, 312, 1975. 33. Oishi, M., Takano, M., Miyachi, K., Ichikawa, Y.. and Homma, M., Inr. Arch. Allergy Appl. Immunol. 50, 463, 1976. 34. Agnello. V., Medicine 57, 1, 1978. 35. Marder, R. J., Burch, F. X., Schmid, F. R.. Zeiss, C. R.. and Gewurz. H., .I. Immunol. 121, 613, 1978. 36. Agnello, V., Arthritis Rheum. Suppl. 21, S146, 1978. 37. Lambert. P. H., Dixon, F. J., Zubler, R. H., Agnello. V. Cambiaso, C., Casali, P., Clarke, J., Cowdery, J. S., McDuffte, F. C., Hay, I. C. M., MacLennan, P., Masson, P., Muller-Eberhard, H. J., Penttinen, K., Smith. M., Tappeiner, G.. Theolilopoulos, A. N., and Verrouse, P.. /. Ct’in. Lab. Immunol. 1, 1. 1978. 38. Mackel, S., Tappeiner, G., Brumtield, H., and Jordon. R. E., Clin. Res. M, 3OOA, 1978. 39. Andrews. B. S., Cains, G., McIntosh, J., Petts, V., and Penny, R., J. C/in. Lab. Zmmunol. 1, 3 11. 1979. 40. McDougal, J. S., Redecha, P. B., Inman, R. D., and Christian, C. L.,J. Clin. Invest. 63,27. 1979. 41. Feldman, J. L., Becker, M. J., Moutsopoulos, H., Fye. H., Blackman, M., Epstein, W. V., and Talal, N.. J. Clin. Invest. 58, 137, 1976. 42. Asghar, S. S., Faber, W. R., and Cormane, R. H.. J. Invest. Dermatol. 64, 113, 1975. 43. Meltzer, M., Franklin, E. C., Elias, K., McCluskey, R. T., and Cooper, N., Amer. J. Med. 40, 837, 1966. 44. Cream, J. J., Quarf. J. Med. 45, 255, 1976. 45. Miescher, P. A., Paronetto, F., and KofBer, D.. In “Immunopathology IV International Symposium” (P. Grabar and P. Miescher, Eds.), pp. 446-456, Grune & Stratton, New York, 1%5. 46. Schroeter, A. L., Copeman, P. W. M.. Jordon, R. E., Sams. W. M., Jr.. and Winkelmann. R. K., Arch. Dermatol. 104, 254, 1971. 47. Sams. W. M., Jr., Claman, H. N., Kohler, P. F., McIntosh, R. W., Small, P., and Mass, M. F.,J. Invest. Dermarol. 64, 441, 1975. 48. Gnyewotu, I. I., Johnson, P. M., Johnson, G. D., and Holborow, E. J., C/in. ~xp. Immunol. 19, 267, 1975. 49. France, A. E., and Schur, P. H., Arthritis Rheum. 14, 231, 1971. 50. Eisenberg, R. A. Theofdopoulos, A. N., and Dixon, F. J., J. Immunol. 118, 1428, 1977. 51. Tung, K. S. K., Woodroffe, A. J.. Ahlin, T. D., Williams, R. C., Jr., and Wilson, C. B., J. C[in. Invesf. 62, 61, 1978. 52. Gabriel, A., Jr., and Agnello, V., J. C/in. Invest. 58, 990, 1977. 53. Zubler, R. H., Nydegger, U., Perrin, L. H., Fehr, K., McCormick, J., Lambert, P. H., and Miescher. P. A., .I. C’lin. Invest. 57, 1308, 1976. 54. Nydegger, U. E., Zubler, R. H., Gabay, R., Jolait, G., Karagevrekis, C. H. Lambert, P. H., and Miescher, P. A., J. C&n. Invest. 59, 862, 1977. 55. Davis, P., Cumming, R. H., and Verrier-Jones, J., C/in Exp. Immunol. 28, 226, 1977. 56. Rossen, R. D., Reisberg, M. A., Singer, D. B., Schloeder, F. X., Suki, W. N., Hill, L. L., and Eknoyzn, G., Kidney Int. 10, 256, 1976. 57. Ruddy, S., Carpenter, C. B., Chin, K. W., Knostman, J. N., Soter, N. A., Gotze, O., MullerEberhard, H. J., and Austen, K. F., Medicine 53, 165, 1975. 58. Glass. D., Soter, N. A., Gibson, D., Carpenter, C. B., and Schur, P. H., Arthritis Rheum. 19,945, 1976.
IMMUNOLOGY
AND
Circulating
IMMUNOPATHOI.OGY
658-612 (1980)
Immune Complexes in Patients Necrotizing Vasculitisl
GARY M. KAMMER,' Departments
1.5,
of Medicine Medical
N.A.
SOTER,AND
and Dermatology. School. Boston.
P.H.
with
SCHUR
Robert B. Brigham Hospital, Massachusetts 02120
Hurvard
Received August 3. 1979 Sera from 29 patients with biopsy-proven idiopathic active cutaneous necrotizing venulitis (CNV) were examined for immune complexes by serum complement levels, Clq binding, and ADCC inhibition. For comparison these studies were also performed in patients with systemic lupus erythematosus (SLE). and rheumatoid arthritis (RA). Most of the patients with CNV, SLE, and RA have evidence of circulating immune complexes at a time during which they have active disease. Patients with CNV tend to have normal or slightly reduced serum complement levels, abnormal Clq binding assay levels. and abnormal ADCC inhibition. The complexes were mostly > 19 S in size. Density gradient analysis did not separate between the complexes in these three diseases. There was no association between ADCC inhibition and Clq binding or serum complement levels suggesting that these two assays detect different types of immune complexes. These observations suggest that multiple types of immune complexes exist in patients with different forms of vasculitis: those that appear to fix complement avidly and are cleared (low serum complement, low Clq binding); those that fix complement poorly in \?vo but are readily detected in vitro (high Clq binding): and those which bind to lymphocyte Fc receptors (ADCC inhibition). and appear not to fix complement.
INTRODUCTION
Numerous experimental animal studies have provided evidence strongly implicating immune complexes in the pathogenesis of necrotizing vasculitis (reviewed in Refs. 1 and 2). Moreover, extrapolating from the experimental studies in animals, it has been presumed that vasculitis in human disease is produced by the deposition of immune complexes in certain instances (3-8). The study of a welldefined patient population with one form of vasculitis, idiopathic cutaneous necrotizing venulitis (CNV) (9-l l), documented by examination of biopsy specimens. should permit a unique opportunity to characterize the circulating immune complexes in vasculitis. Therefore, 29 patients with idiopathic CNV documented by skin biopsy were studied at a time they had skin lesions by three serological assays for immune complexes: serum complement levels, which reflect in viva complement fixation by immune complexes (reviewed in 12, 13): a radioactive Clq binding assay which measures complexes still able to fix complement (14); and an antibody-dependent cellular cytotoxicity (ADCC) inhibition assay, which is complement independent, and reflects the ability of complexes to react with lymphocyte cell Fc receptors ’ This study was supported in part by USPHS Grants .4Mll414, AM05577. RRO05669, AIO0366, AIl0356. AM07031, by a Young Investigator Research Grant (AI-14292). and the New England Peabody Foundation, The Arthritis Foundation, and the Lupus Erythematosus Foundation. 2 Dr. Kammer was a postdoctoral fellow of the Arthritis Foundation. 658 0090-1229/80/040658-15$01.00/O Copyright 10 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
IMMUNE
COMPLEXES
IN
VASCULITIS
659
(15). These studies in patients with CNV were compared to those in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Our results demonstrate the presence of heterogeneous circulating immune complexes of both complement-fixing and complement-independent types in most of the sera from patients with CNV. MATERIALS Patient
AND METHODS
Population
Two hundred twenty-four individuals were studied. Twenty-nine patients had idiopathic CNV documented by skin biopsy and unassociated with any other coexistent systemic disorder (9). Sera and skin biopsies were obtained simultaneously during a period when patients exhibited cutaneous lesions. Twenty-two of the patients had raised, erythematosus papules of various sizes that did not blanch with pressure (palpable purpura) and that occurred in crops that persisted for several days (9); episodes of recurrent urticaria occurred in seven patients (10). None of these patients had detectable hepatitis B antigenemia. Thirty-three patients had classical RA complicated by clinical features of vasculitis (17), including either digital infarcts, digital gangrene, leg ulcers, or peripheral neuropathy (mononeuritis multiplex). Twenty patients had definite or classical rheumatoid arthritis (RA) (16) with joint disease only and no clinical history or physical findings suggestive of vasculitis or other extraarticular manifestations of RA. Fortytwo. patients had active systemic lupus erythematosus (SLE), with either fevers not due to infection, arthritis or persistent arthralgia, anemia (hematocrit <30%), leukopenia ( <4000/mm3), a malar rash, and/or polyserositis (18). All SLE patients had four or more ARA criteria for SLE (19), and all had an ANA titer of > 1: 80 at the time they were studied. One hundred healthy hospital personnel served as normal controls. Serum Preparation
Venous blood was drawn into vacuum test tubes prewarmed to 37°C and allowed to clot at 37°C for 1 hr; the serum was separated at 37°C. Serum aliquots were immediately frozen at -70°C. Prior to use in experiments, frozen sera were thawed only once at 37°C. ADCC
Inhibition
Assay
This assay was performed as recently described (15). In summary 60 ~1 of 50 mM EDTA, pH 7.8, was mixed with 200 ~1 of test sera and incubated at 4°C for 15 min. Next, 50 ,ul of serum-EDTA was added to triplicate wells (U-shaped microtiter trays, Cook Laboratory Products, Alexandria, Va.) containing 100 ~1 of 2 x lo6 effector lymphocytes/ml, thoroughly mixed, and incubated for 60 min at 4°C. To remove residual sera following incubation, effector cells were centrifuged in the trays and thoroughly washed twice in the wells using a Caz+ -Mg*+-free culture medium. To replete Ca2+ and Mg2+ necessary for in vitro cytotoxicity, the effector cells were washed a third time in supplemented RPM1 1640. Finally, 75 ~1 of supplemented RPM1 1640, 100 ~1 of 51Cr-chicken red blood cells (51Cr-CRBC), and 25 ~1 of the appropriate anti-CRBC antiserum dilution were successively added to each well. Since this assay utilizes a single donor’s effector lymphocytes, the
660
KAMMER,
SOTER,
AND
SCHUR
standard final dilution of antiserum had been previously determined to be 2 x lo-“. Following centrifugation at 500 rpm for 5 min at 24”C, the microtiter trays were incubated at 37°C for 16 hr in a 5% CO,/95% air atmosphere with 100% humidity. The number of radioactive counts per minute in the supernatants was counted in a Packard Autogamma scintillation counter. The equations for calculation of the percentage ADCC inhibition3 have been given in detail elsewhere (20). Antibodies directed against both B and T lymphocytes have been demonstrated in the sera of some lupus patients (21). Since these antibodies might bind to the lymphocyte membrane via the Fc receptors or other membrane receptors, resulting in ADCC inhibition indistinguishable from that caused by immune complexlike reactants (hereafter referred to simply as immune complexes for the sake of brevity), the following studies were performed to determine whether antilymphocyte antibodies inhibit ADCC, as performed above. A rabbit antiserum prepared against human lymphocytes was incubated with the lymphocytes of 13 HLA-variable donors as target cells. These mixtures were then incubated with the standard effector lymphocytes. A mean percentage cytotoxicity of 58.3 (range = 33-95s) was induced, indicating that the antiserum possessed anti-lymphocyte antibodies. This antiserum was then ultracentrifuged at 100,OOOg for 90 min at 4°C (SW 50 rotor, Spinco Model L preparative centrifuge, Beckman Corp., LaJolla. Calif.) to remove aggregates. The upper one-third of the tube was collected. filtered (Millipore Corp., Bedford, Mass., 0.45 pm). and then incubated with the standard donor effector cells for 60 min at 4°C or 37°C. .These effector cells were then assessed for ADCC inhibition, as described above. Negligible inhibition (mean = 2.6 c 0.7%) of ADCC occurred after pretreatment of lymphocytes with the antiserum at 4°C; however, about 10% ADCC inhibition occured when effector cells had been pretreated with antiserum at 37°C. In addition, following partial blockade of surface Fc receptors with lOO-pg agg IgG/ml, cells were then reacted with antiserum for 60 min at either 4 or 37°C. No significant additional ADCC inhibition was observed compared to control effector cells pretreated with agg IgG only. In separate experiments, patients’ sera which had been shown to inhibit ADCC were chromatographed on DEAE. Fractions containing IgG inhibited ADCC while those containing IgM did not. Thus, these experiments suggest that antilymphocyte antibodies bind through another surface receptor and do not cause inhibition of ADCC. Clq Binding Assay (C/q BA) Clq was extracted from serum by the method of Yonemasu and Stroud (22), and was radiolabled with lz51 in the presence of lactoperoxidase. The Clq BA was carried out as described by Nydegger er u/. (23) and modified subsequently by Zubler et al. (14). An aliquot of the radiolabeled Clq was then incubated with both test and control sera to which EDTA had been added. Bound radiolabeled Clq was separated from free radiolabeled Clq by polyethylene gIyco1 precipitation. 3 Abbreviations used: ADCC inhibition, antibody-dependent cellular BA, 9-Clq binding activity; agg IgG, aggregated IgG; Ig, immunoglobulin.
cytotoxicity
inhibition;
Clq
IMMUNE
COMPLEXES
IN VASCULITIS
661
Standardization of the ADCC Inhibition Assay and Clq BA The isolation of Cohn fraction II, preparation of IgG aggregates (used herein as an in vitro model of immune complexes), and standardization of the ADCC inhibition assay have been previously described (15). The standard curve for the Clq BA was developed as follows: equal volumes of agg IgG in concentrations ranging from 20 mg/ml to 2 p.g/ml plus normal human serum (NHS) were mixed and assayed in the Clq BA. The estimated concentration of immune complexes in serum was expressed as equivalents of micrograms agg IgG per milliliter of serum by extrapolation from either the percentage Clq BA or percentage ADCC inhibition. The serum-agg IgG was prepared fresh with each experiment to standardize both assays. The standard curves in Fig. 1 give the percentage ADCC inhibition or Clq BA by different concentrations of agg IgG-serum. Sucrose Density Gradient Ultracentrifugation Into each cellulose nitrate tube a pellet of 0.3 ml of 70% sucrose in PBS -EDTA 15 mM (for samples later to be assayed by ADCC inhibition) or in barbital buffer, pH 8 (for fractions later to be assayed by Clq BA), is placed and then overlayed with 4.3 ml of sucrose in buffer, utilizing a density gradient former to produce an
PupAw IgG/mf FIG. 1. Standard curves of Clq BA and ADCC inhibition assay. Each point represents the mean 2 SEM of 25 experiments. The curves give the percent Clq BA or percentage inhibition of ADCC by different concentrations of agg IgG-NHS.
662
KAMMER,
SOTER,
AND
SCHUR
8-40% gradient. Four-tenths milliliter of experimental serum, agg IgG-NHS, or PBS-NHS were then overlayed onto each of three gradients and allowed to equilibrate at 4°C for l-2 hr. The gradients were then centrifuged at 37,000 rpm for 18 hr at 4°C. Twenty fractions were collected from the bottom of each tube. Samples to be assayed by ADCC inhibition were dialyzed against PBS-EDTA 15 mM, pH 7.8, at 4°C for 18-24 hr to remove residual sucrose; the remainder of the fractions were tested by the Clq BA. The concentrations of Ig and total protein in each fraction were determined as described below; IgG and IgM were utilized at 7 and 19 S markers, respectively. Other
Methods
Total serum hemolytic complement (CH50) was assayed by the method of Kent and Fife (24). Serum protein concentrations of complement proteins Clq, C4. C2. and C3, and immunoglobulins IgG and IgM were measured by radial immunodiffusion using monospecific antisera developed in this laboratory (25). Protein concentrations were determined by the Folin method (26). Antinuclear antibodies and precipitins to nuclear antigens and nucleic acids were detected by immunofluorescence and by counter immunoelectrophoresis (27). Statistical Methods
Statistical testing was performed on a Hewlett-Packard 9815 calculator. Results were expressed as the arithmetic mean F standard error of the mean (SEM), unless otherwise stated. Statistical significance was assessed by the Mann- Whitney test. Correlations were performed by the nonparametric Spearman’s rank correlation test (r,). RESULTS Complexes Quantitated by the Clq BA
The mean binding (2 2 SD) in sera from 100 normal individuals was 6.8 i 16.5 pg agg IgG/ml serum. As little as 1 pg agg IgG/ml could be measured by this system. Binding activities of greater than 23 pg agg IgG/ml were regarded as abnormally elevated, or positive. Fifteen of 29 sera from CNV patients had elevated binding activities. In addition, 8 of 20 sera from the uncomplicated RA group, 27 of 46 sera from the complicated RA group, and 20 of 42 sera from the SLE group were positive. The mean Clq BA of the CNV sera was 248 Fg agg IgG/ml; the mean Clq BA in uncomplicated RA was 103 pg agg IgG/ml; in the SLE group was 105 pg agg IgG/ml; and, in the complicated RA group was 929 kg agg IgG/ml (Table 1). The observed mean Clq BA was significantly higher in the CNV group than in normal controls (P < 0.0001). Complexes Quantitated by the ADCC tnhibition Assu~
The mean ADCC inhibition (5 2 SD) in sera from 100 normal individuals was 0.35 t 1.2 pg agg IgG/ml serum. As little as 0.1 pug agg IgG/ml could be quantitated. ADCC inhibition exceeding 1.5 pg agg IgG/ml was considered abnormally high, or positive. Twenty of 29 sera from the CNV group demonstrated elevated ADCC inhibition. Two of 20 sera from the uncomplicated RA group, 28 of 46 sera from the complicated RA group, and 34 of 42 sera from the SLE group had
RA
u Mean
-t 2 SD (range).
200+50 (150-250)
Healthy
controls
88 rt 77 (20- 192)
112 k 49 (20-244)
SLE
Complicated
251 2 75 (198-334)
RA
COMPLEMENT,
Uncomplicated
CHSO Wml)
OF SERUM
165 2 220 (O-342)
LEVELS
CNV
Disease
MEAN
343 2 200 (258 -745)
(288 -608)
144 2 248 (18-579)
245 r 250 (10-453)
484 + 248 (259-727)
308 + 419 (22-661)
c4 b.dml)
complement” c3 b.udml)
IN FOUR
(910-1980)
1340 zt 78
908 t 880 (310-2243)
10602960 (110-1780)
1539 r 386 (1240-2027)
1356 r 1550 (343 -3230)
TABLE I INHIBITION. AND Clq BINDING
416 k99
234 -c 300 (16-582)
207 2 459 (10-714)
477 + 158 (303 -584)
348 5 348 (16-576)
Clq (k.dml)
Serum
ADCC
PATIENT
(O-1.55)
0.35 + 1.2
6.5 + 26 (0.1-76)
8.3 k 25 (0.1-45)
1.4 + 8 (0.1-17)
1.5 k 25 (0. I-60)
ADCC* inhibition (@AHG/ml)
GROUPS
“SI-(-Jq”
(O-30)
6.8 2 16.5
105 t 506 (l-1550)
929 k 2667 (l-5600)
103 r 431 (l-870)
248 f 935 (l-1900)
binding (&AHG/ml)
AND CONTROLS
s 7 5
5 E
52
m CA
664
KAMMER,
SOTER,
AND
SCHUR
abnormally raised ADCC inhibition. The mean ADCC inhibition; of the CNV group (7.5 pg agg IgG/ml) was greater than that of normal controls (0.35 pg agg IgG/ml (P < 0.0001) (Table 1). While the mean ADCC inhibitions of each clinical group differed, overlap between individual values within each patient group as well as among groups was present. The levels of complexes detectable by the Clq BA were generally considerably higher than those detected by the ADCC inhibition assay. Often a 15- to loo-fold difference in concentrations between the two kinds of complexes was observed in individual sera. Serum Complement
Levels
The mean concentrations of serum CH50 and of Clq, C4, and C3 proteins in the CNV group and in other groups are given in Table 1. Mean levels in the CNV group were similar to those in the normal group, whereas the uncomplicated RA group tended to have elevated complement levels and the complicated RA group had significantly reduced levels of CH50 (P < 0.0001) and of C3 protein (P < 0.004). SLE sera had significantly reduced concentrations of CH50 (P < O.OOOl), Clq (P = 0.02), C4 (P < O.OOS), and C3 (P < 0.0001). Four sera from the CNV group had serum Clq levels ~100 &ml; of these, two had raised Clq BA. Compurison Levels
of Cly BA, ADCC
Inhibition
Assuy,
and Serum
Complement
A determination was made of the number of sera positive by both the Clq BA and ADCC inhibition assay, by a single assay, or in neither assay. The data are presented in Table 2. Most of the sera in the CNV, complicated RA, and SLE group had circulating immune reactants, and a large proportion of sera in these groups exhibited both kinds of circulating complexes (Table 2). The relationship between serum complement levels and serum concentrations of circulating complexes was then determined. In the CNV group a negative correlation of border-line statistical significance was demonstrated between the Clq BA and serum Clq levels (r s = -0.36, P < 0.05), and serum C4 levels (r, = -0.36, P < 0.05). There was no relationship between the Clq BA and the CHSO or C3 levels. In the complicated RA group a negative correlation was observed between the Clq BA TABLE THE
NUMBER
OF SERA IN CNV AND/OR ADCC
““I-Clq Disease
Poslpos
CNV Controls Uncomplicated Complicated SLE
RA RA
2
AND CONTROLS GIVING POSITIVE INHIBITION ASSAYS FOR IMMUNE
binding Pos/neg
assay/ADCC
AND/OR NEGA~WE COMPLEXES
inhibition
Neg/pos
10
5
10
Negineg ..-~~-~~~~4
0 23 17
8 4 3
2 5 17
10 1 5
Clq BINDING
Incidence circulating immune complexes CT) -- .._. _ . 86 50 97 88
IMMUNE
COMPLEXES
IN VASCULITIS
665
and serum CH50 levels (r, = -0.58, P < O.Ol), serum C4 levels (r, = -0.57, P < O.Ol), and serum C3 levels (r, = -0.50, P < 0.01). In the SLE group, a negative correlation of borderline significance was noted between Clq BA and serum CHSO levels (Y, = -0.32, P < 0.05), and serum Clq levels (r, = 0.37, P < 0.05). No relationship was demonstrated between this assay and serum C4 or C3 levels. The uncomplicated RA group showed no correlation whatsoever between the Clq BA and CH50 or any component levels. There were no apparent relationships between levels of complexes detected by the ADCC inhibition assay and levels of complexes detected by the Clq BA assay or serum complement levels. Sucrose Density Gradient Ultracentrifugation To determine whether circulating immune complexes in CNV exhibit differential sedimentation patterns from other sera, 21 sera positive by both assays from patients with CNV (9), SLE (4), and complicated RA (8) were subjected to density gradient ultracentrifugation in an 8-40% sucrose medium. The results of 6 of these 21 analyses are depicted in Fig. 2. CNV sera produced ADCC inhibition and Clq BA in both the 7- 19 S and >19 S regions (Fig. 2A and B), indicating that heterogeneous reactants of variable sizes circulate in these sera. Sera from the SLE (Fig. 2C and D) and complicated RA groups also yielded similar distributions of complexes (Fig. 2E and F). Normal sera did not inhibit in either assay. Aggregated IgG when added to normal serum and subjected to density gradient analysis showed that most ADCC inhibition was in the >19 S region, but some was in the 7- 19 S region; most of the Clq BA was in the > 19 S region. The absence of both ADCC inhibition and Clq BA in the 7 S region is expected as immune complexes are not found in this region.
10,000
r
$ io,ooo
1000
p
TnM
r g ‘Ooo tLnoc -‘*‘I-Ciq
1600
Tm
Binding
Activity 700 q
y;:
?!
i tn
50
t
OL
“‘b
2
I 4
I 6
F-lln 8
too
I\
2
4
6
8
400
2
4
6
8
40
FRACTION NUMBER FIG. 2. Plots of sucrose density gradient profiles of two sera from each disorder demonstrated to have both complement-fixing and complement-independent immune complexes. A, B: CNV; C, D: SLE; E, F: complicated RA. Note the absence of any distinctive gradient patterns which might characterize a specific disorder.
666
KAMMER,
MEAN
LEVELS
AND
RANGES
____CNV
Disease
7-19
SLE Complicated
AND
TABLE
3
SCHUR
OF: COMPLEXES
IN AN 8-40s
ADCC
SOTER,
FROM
SUCROSE
SERA
SEDIMENI
“‘[eclq BA” _--~~-~.s .>I9 s
inhibition”
s
~NG
GRADIENT
-a 19 s
?--I9
0.7 (0.1-1.2)
3.0 (0. I - 10.4)
54.0 (l-200)
82.0 (l-300)
2.4 (0.1 - 10.8)
‘.I (0.2-5.2)
21.0 rl--205)
(l-25)
204.0 (0.1~-1000)
118.0 (l-290)
148.0 (l--310,
RA (O.l-
3.1 12.3)
...~~
6.0
fl In pg AHGlml.
The mean ADCC inhibition and Clq BA in both regions (7- 19 S, >I9 S) were determined for each disease category for the 21 sera examined (Table 3). Similar mean levels were obtained in both regions by both assays. However, they tended to be higher in the >I9 S region in patients with complicated RA and those with CNV. Sera from patients with SLE tended to have somewhat higher mean levels in the 7- 19 S region. Sera from normals did not react in either assay. Yet even with these differences no distinctive gradient pattern for any of these disorders was identified. Whether the absence of a discrete gradient pattern reflects the limitations of the sucrose gradient technique or indicates that no such differences exist among these disease sera is unresolved. Relationship Patients
of Lelvels of Serum Complement
und Immune
Complrxes
in CNb
Of the patients with CNV, 12 had hypocomplementemia. Five of these 12 patients had complexes detectable by Clq BA; 9 of 12 had complexes detected by the ADCC inhibition assay (Table 4). In the 17 CNV patients with normocomplementemia, 10 had complexes detected by Clq BA and 11 by ADCC inhibition (Table 4). There was no difference in ADCC inhibition or Clq BA levels in patients with CNV presenting as palpable purpura or urticaria. DISCUSSION
Certain similarities between experimental models of vasculitis in animals and vasculitis in humans have been noted, including similar pathology, demonstration TABLE THE RELATION
OF SERUM
Complement level Normocomplementemia Hypocomplementemia
4
TOTAL HEMOLYTIC COMPLEMENT TO Posn IVE AND/OR FOR IMMUNE COMPLEXES IN PATIENTS WITH CNV
“‘I-Clq Posipos 5 5
binding Poslneg 5 0
test/ADCC
NEGATIVE
ASSAYS
inhibition Negipos 6 4
Neglneg 1 3
IMMUNE
COMPLEXES
IN
VASCULITIS
667
of immunoglobulins and complement components at the site of tissue damage, and decreased levels of serum complement. Taken together these observations suggest that circulating immune complexes may participate in certain forms of vasculitis in humans. The present studies were instituted to search for circulating immune complexes in patients with necrotizing vasculitis. There are numerous methods for detecting immune complexes that are dependent on the different physical and biological properties of immune complexes. These include: molecular weight and valence of the antigen; molecular weight, class, subclass, and affinity of the antibody; molecular weight and ratio of antigen to antibody in the immune complex; ability of the antigen, antibody, and/or immune complex to activate the classical and/or alternative complement pathways; and ability of the complex to react with rheumatoid factors and/or with cell receptors. No single assay can detect all immune complexes. Most assays have been tested or standardized with respect to immune complexes of known composition, or to aggregated human y-globulin-which behave like immune complexes (28). In addition some assays will react with nonimmune complex material such as DNA, endotoxin, and low molecular weight 7 S substances (29). While in most situations these assays appear to detect material in patients’ sera that behaves like immune complexes, the identification of actual antigen-antibody complexes has rarely been achieved (30). In the present study three assays thought to reflect those properties of immune complexes that might be relevant to a study of vasculitis were used: serum complement levels which may be depressed in the serum of patients with vasculitis and are than thought to represent in vivo complement fixation by immune complexes (13); an assay that detects complexes that can fix complement (the Clq binding assay); and an assay that is independent of complement and that interacts with the Fc receptor of cells (the ADCC inhibition assay). Patients with CNV tend to have: normal or slightly reduced complement levels-if low, usually CHSO, Clq, and C4 levels are low and C3 levels are normal; abnormal Clq binding assays in one-half of patients; ADCC inhibition assays in most patients; and more complexes in the > 19 S region than in the 7- 19 S region. In previous reports regarding patients with vasculitis, the patient populations studied formed a rather heterogeneous group of vasculitides, including patients with CNV. Cutaneous necrotizing venulitis, which is defined by its characteristic pathology, may present as a polymorphous array of different skin lesions; however, the most diagnostic and consistently present skin lesion is palpable purpura. Less common manifestations include urticaria (lo), angioedema, and macular erythema. This variety of cutaneous lesions has led to the description of similar patients with hypocomplementemia and Clq precipitins under the terms systemic lupus erythematosus-like syndrome, erythema multiforme, and hypocomplementemic vasculitis urticaria syndrome (29, 31-36). The infrequent recognition of these diverse skin lesions as manifestations of underlying cutaneous necrotizing venulitis and the failure to define the type of vasculitis results from the fact that skin biopsies are infrequently performed in such patients. Therefore it is often difficult to compare the results in studies in which the type of vasculitis was not defined. In a study of 17 patients with “idiopathic cutaneous vasculitis” (12
668
KAMMER,
SOTER,
AND
SCHUR
were our CNV patients) by the WHO collaborative study group 18 assays were used to detect circulating complexes; from 41 to 76% of patients had circulating complexes by the nine assays that discriminated this group of patients from normals (37). In a study of 25 patients with “cutaneous vasculitis” 14 (56%) had complexes as assessed by the Raji cell radioimmunoassay (6). These results included 5 of 7 (71.4%) with rheumatoid arthritis and “vasculitis,” 6 of 10 (60%) with Sjogren’s syndrome and/or cryoglobulinemia, and 3 of 8 (37.5%) with “idiopathic vasculitis“ (3 of 7 sera provided by us had a positive assay). In a study of 56 patients with cutaneous vasculitis, which included 17 with idiopathic vasculitis. 18 with vasculitis and associated diseases, and 21 with other forms of vasculitis, 21 of 56 (37%) had circulating complexes as detected by either Clq binding or monoclonal rheumatoid factor assays (38). These positive results occurred mainly in those individuals with vasculitis plus associated diseases. Most of the binding materials were of high molecular weight. ~19 S. Complexes have been detected by reaction with monoclonal rheumatoid factor in 3 of 11 patients with systemic and cutaneous vasculitis but in 0 of 7 patients with cutaneous vasculitis (5). In a study of 53 patients with biopsy-proven cutaneous vasculitis. 8% had precipitins with ‘Clq, 37% had anticomplementary activity, 43% could inhibit EA rosette formation, and none had precipitins with rheumatoid factors (39). Two patients with vasculitis associated with HBSAg had a positive Staph A binding assay (40). Sera from two patients with polyartertis nodosa inhibited ADCC (41). A number of patients with various cutaneous manifestations of vasculitis have had intermediate (7- 19 S) and large molecular weight complexes (> 19 S) detected by Clq binding (29, 31-33, 35, 36. 42). cryoglobulins (11. 43, 44). serum hypocomplementemia, and the deposition of immune reactants t38,45-47) in the skin. Not all patients with vasculitis, including CNV, have hypocomplementemia (10). In summary, these studies are limited in many instances by the presence of several disorders, lack of knowledge of the state of disease activity, limited or no follow-up intervals. and the absence of documentation of vasculitis by examination of tissue biopsy specimens. Therefore the results in which biopsies were not obtained may represent either different forms of vasculitis, if indeed it was present. The patients in this study with RA complicated by features suggestive of vasculitis (17) manifested by either peripheral neuropathy, digital gangrene, nailfold infarcts, cutaneous ulcers, coronary artery involvement, and/or pericarditis were studied for comparison to those patients with idiopathic CNV. These patients tended to have: somewhat reduced serum complement levels, especially CHSO and C4: most patients had abnormal Clq binding and ADCC inhibition assays, often with very high levels-Clq binding levels tended to be higher than in patients with CNV; complexes tended to be more in the ) 19 S region than in the 7- 19 S region, particularly those causing ADCC inhibition. Rheumatoid factors did not appear to affect immune complex assays or levels. These observations in complicated RA are similar to the limited reports by others in patients with “rheumatoid vasculitis.” Onyewotu rt ul. (48) noted enhanced uptake of aggregated IgG by guinea pigs macrophages by all 22 sera studied from patients with RA and cutaneous vasculitis-the type of vasculitis
IMMUNE
COMPLEXES
IN
VASCULITIS
669
was not stated. Theofilopoulos et al. (6) noted increased Raji cell assays in 5 of 7 sera from patients with rheumatoid vasculitis-f these 7 sera 4 were provided by us and were patients with RA and CNV. All of 7 patients observed by us with either “complicated RA” plus CNV or “uncomplicated RA” plus CNV had immune complexes by our assays. Sera from patients with complicated RA contain both intermediate (7- 19 S) and large (>19 S) complexes detected by reaction with bovine conglutinin (50). One serum showed enhanced uptake of aggregated IgG by guinea pig macrophages by large complexes (48). We have previously noted low complement levels in patients with complicated RA (49). The patients in this study with SLE tend to have: low levels of serum complement including CHSO, Clq, C4, and C3; about one-half of the patients had immune complexes detected by Clq binding, usually of moderate titer; most of the patients had complexes detected by ADCC inhibition, usually of moderate titer; complexes tended to be more in the 7-19 S region than in the >19 S region. The patients in this study with uncomplicated RA tend to have: normal to elevated complement levels; about one-half have complexes detected by Clq binding and few by ADCC inhibition-levels of these complexes were only slightly elevated. These studies demonstrate a marked heterogeneity of complexes, although there is a tendency for different patterns in different clinical entities. These observations also confirm other studies which found complexes in the serum of patients with RA and SLE of molecular size 7- 19 S and > 19 S that can bind complement or bind lymphocyte to receptors. The wide range of frequency of positivity by different assays in these disorders suggests either that these assays have varying sensitivities, or that they measure different complexes. This point was examined in the present study by simultaneous assessment of sera by three different assays. If one compares the results obtained by these three assays there is little, if any, correlation in the levels between each assay. The level of circulating complexes detected by Clq binding appears to be considerably higher than the level of complexes detected by ADCC inhibition. The presence of complexes in the ADCC inhibition assays did not correlate with the presence of complexes in the Clq binding assay levels nor with serum complement levels. There was some, but not striking, correlation between Clq binding assay levels and: serum complement (Clq, C4) levels in patients with CNV; CHSO, C4, and C3 levels in patients with complicated RA; and with CH50 and Clq levels in patients with SLE. Either a lack of, poor correlations, or some correlations between the levels detected by different assays have been noted by others. Tung et al. (51) noted a good correlation between levels of complexes detected by the Raji cell assay and the Clq solid phase assay in patients with various forms of glomerulonephritis. Gabriel and Agnello (52) noted some correlation (r = 0.36) between levels of complexes detected by a Clq inhibition assay and a monoclonal rheumatoid factor assay. They noted a close correlation with complement levels in serial studies of 6 SLE patients. Zubler et al. (53) noted only some (r = 0.5) correlation between Clq binding activity and synovial fluid C4 levels in patients with RA, but no correlation between Clq binding levels and serum CHSO, Clq, C4, or C3 levels in these patients. In later studies this group (54) noted a correlation between plasma or synovial
670
KAMMER,
SOTER,
AND
SCHUR
fluid C3d levels and Clq binding levels in patients with rheumatoid arthritis. Davis er al. (55) noted a correlation between an anticomplementary assay and a platelet aggregation assay in examining SLE sera. Rossen et ul. (56) noted no correlation between Clq binding levels of complexes and serum levels of C3 and Clq in patients with nephritis. These observations suggest that different assays detect and measure complexes with different properties. Therefore the fair (at best) correlation between assays suggests that these sera contain more than one type of complex. The lack of association between ADCC inhibition, complement levels, and Clq binding suggests that ADCC inhibition detects another type of (noncomplement fixing) complex, that has the ability to bind Fc receptors. This observation is consistent with our previous observation ( 15) that ADCC detects complexes that have not fixed C3. These data also suggest that there are considerable levels of non-complement fixing complexes in this disease. In our understanding of the pathogenesis of cutaneous necrotizing venulitis (CNV) one can speculate that those patients with CNV and low serum complement levels have complexes which activated complement in IP~,Wand caused local tissue damage. The histopathology of the skin lesions in hypocomplementemic CNV shows predominantly neutrophils, with some lymphocytes (9). These findings are consistent with animal models where complement-fixing immune complexes mediate tissue injury and result in lower serum complement levels. Nine of twelve patients had complexes detectable in ~ritro by Clq binding. This observation points to the limitation of this assay. The patients with CNV and normal serum complement levels could have accelerated complement metabolism with a compensatory hypersynthesis of complement, similar to what has been documented in patients with RA and normal complement levels (57). These complexes in patients with normocomplementemic CNV may be weak complement fixers, strong enough to bind Clq in Grro but not enough to lower serum complement levels in \Y\*o. The biological role of these complexes at the tissue level is not clear. On the other hand 11 of the 17 patients had complexes detected by ADCC inhibition, demonstrating that these sera have complexes able to interact with lymphocytes. The fact that normocomplementemic CNV patients have a predominantly lymphocytic infiltration about vascular skin lesions (9) suggests a possible relationship between the in Gtro and in rlir~ findings. The observation that only 1 patient with CNV did not have complexes detected by these three assays (complement levels, Clq binding, and ADCC inhibition), strongly suggests that these complexes do play some role in CNV: however, other factors may be involved. These data also suggest that sera with low complement levels had complexes which fixed a substantial amount of complement, that is more complement than was synthesized. The observations of low serum complement levels and low Clq binding levels in patients with SLE suggests that most of the circulating immune complexes in this disorder are avid complement fixers and have been cleared or deposited in tissues. If complexes are then detected in these sera, in vitro, it suggests that their ability to fix complement (viz Clq) is weak: for if the complexes were avid complement fixers they would have been cleared from the circulation. The observation in patients with complicated RA of only mild serum complement
IMMUNE
COMPLEXES
IN VASCULITIS
671
depression and high levels of Clq binding suggests that the complexes detected in fixers and not be involved in tissue injury, in this disorder. This hypothesis could therefore explain the observation of elevated Clq binding in the presence of normal complement levels-i.e., the only complexes still present in the circulation are those with weak complement (viz Clq) binding. The biological role in vivo of these circulating complexes that are able to bind complement or cells in vitro remains speculative. Other interpretations of the present and other data are also plausible, including a possible role for non-immune complex-mediated mechanisms in patients with vasculitis (58). One point seems apparent in these patients; there are many types of complexes which possess various properties and are detected by different assays.
vitro may be poor complement
ACKNOWLEDGMENTS We gratefully acknowledge the expert laboratory assistance of Mr. Chip Kava and Ms. Sally Brown, the measurement of Clq BA by Dr. David Glass, and the secretarial assistance of Mrs. Brenda Tracey.
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