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Light- and electron-microscopic findings of juvenile rheumatoid arthritis synovium: Comparison with normal juvenile synovium
Light- and Electron-microscopic Findings of Juvenile Rheumatoid Arthritis Synovium: Comparison with Normal Juvenile Synovium By C. Rosales Wynne-Roberts,
Carolyn
P
RESENTATION of juvenile rheumatoid arthritis (JRA) is diverse and often remarkably different from that in adults, and these differences have been summarized by several authors. l--3 Because of the wide range of clinical expression of JRA, there are many unanswered questions; Is JRA a disease different from that in adults or part of a wide spectrum of rheumatoid arthritis (RA) seen at any age? Is JRA, with its frequent acute onset simulating an infectious disease, due to an infectious agent or agents? Synovial tissues of patients with JRA have been studied by light microscopy4.5 and the findings described as indistinguishable from those of adult disease. Less information, however, is available about the ultrastructural abnormalities because reports of electron microscopy of synovia from patients with JRA are mostly scattered among those of adults.‘*’ Recently, Borrachero et a1.8 and Bierther and Schafer9 have reported electron-microscopic findings of synovia from two patients with Still disease and four patients with JRA. We present information derived from lightand electron-microscopic study of synovia from 15 patients with JRA. Ultrastructural evidence of any bacteria or any viral or bacterial product such as endotoxin1° or of mycoplasmal infection was carefully sought but not found. However, marked abnormalities were observed, and the findings are compared with those of seven normal juvenile synovia.” Comparisons with the electron-microscopic studies of JRA,s~g~‘z with cases of JRA found among those of the adult where the information can be separated and with the information available from the ultrastructural studies of adult synovia, are made. MATERIALS
AND
in Arthritis
and Rheumatism,
A. M. Turano, and M. Baron
from all patients were negative for tubercle bacillus and anaerobic and aerobic bacteria. The synovial specimens were obtained either during surgery or by closed biopsy using a Parker-Pearson needle. For surgery, a tourniquet pneumatic cuff was applied on some of the patients for hemostasis, and ah synovial specimens were obtained within 20 min of cuff inflation. The specimens were divided immediately for light or electron microscopy. Light-microscopy specimens were fixed in phosphatebuffered formaldehyde and embedded in paraffin. Fivemicron sections were stained with hematoxylin and eosin; additional sections were stained with Prussian blue iron stain.‘” Synovia for electron-microscopic study were fixed in 2% paraformaldehyde in phosphate buffer at pH 7.2 and 680 mOsm or in 2.5% glutaraldehyde in Verona1 buffer at pH 7.2 for 8-12 hr at 4°C on a turntable. After phosphate buffer or veronal buffer rinses, the tissues were postfixed in Millonig 1% osmic acidI or veronal-buffered 1% osmic acid, then dehydrated with a graded sequence of ethanols followed by propylene oxide and embedded in Epon-araldite or Maraglas epoxy resins. Thick (0.5-1.0 hrn) sections were stained with aqueous I % crystal violet, and ultrathin sections were cut on a Sorvall MT-2 ultramicrotome, mounted on Formvar carbon-coated copper grids, stained with Reynolds lead citrateI and aqueous 1% uranyl acetate solutions, and viewed at 60 kV in a Phillips EM-300 electron microscope. RESULTS
Macroscopically the synovia showed marked villous hypertrophy and ranged in appearance from white to pink. Microscopically, fibrin was seen on the surface of 13 of 15 synovia; most fibrin was noted in those specimens from JRA of more recent onset. In all synovia, fibrin was seen also in the lining and subintimal layers in small to moderate amounts. Synovial lining layers showed mild hyperplasia and marked hypertrophy (Fig. 1) and were sometimes four to six cells deep. On epoxy resin sections the lining cells were principally phagocytic. Vacuoles, dark granules, and phagocytosed materials
METHODS
Synovia were obtained from 15 patients with mono-, oligo-, or polyarthritis or Still disease who fulfilled Classification I criteria of the American Rheumatism Association for the diagnosis of JRA.13 The clinical and laboratory data of the patients studied are shown in Table 1. The antinuclear antibody and lupus erythematosus preparations were negative in all and are therefore not listed. Gammaglobulins were normal or slightly elevated. Synovial fluid cultures
Seminars
H. Anderson,
Vol. 7. No.
4
(Mav). 1978
From the Electron Microscopy Unit of the Veterans Administration Hospital, and the Children’s Hospital of Pittsburgh, Pittsburgh, Pa. Address for reprint requests: Carolyn H. Anderson, M.D.. Children’s Hospital of Pittsburgh, 125 DeSoto St., Pittsburgh, Pa. 15213. 0 1978 by Grune & Stratton. Inc. ISSN 0049-0172/78/0704-0004$02.00/0
287
2 mo
14yr
1.5yr
9/F
9/F
11/M
13/F
13/F
14’/r/M
16/F
16/F
8
9
10
11
12
13t
14
15
monoart~cular.
L. knee
L. knee
R. knee
R knee
L ankle
I-. knee
mtp
R. hallux
R. wrist
R. knee
L knee
R. knee
L. knee
Ft. knee
L knee
R. knee
surg. Site
test
ohgo. ollgoartwlar
“ectomy
SylK-
prostheses
of
removal
FUSlOlX
biopsy
Open
biopsy
Needle
vectomy
SY”0-
fusion
Triple
biopsy
Needle
w?ctcmly
SyrU-
“ectOmy
SYrlO-
biopsy
Needle
poly. polyartlcular.
0.5 yr
1 mo
1.5 yr
5.5 yr
1.5yr
biopsy
Needle
vectomy
SyllO-
vectomy
SyllCI-
biopsy
Needle
biopsy
Needle
biopsy
Needle
surg PrOC.
t ND, not done; SCAT, sheep cell agglutmatlon
*Mono.
7 Yr
8WF
7
1 mo
MOW2
8/F
6
4 vr
Still
6/M
5
3 mo
MCln0
6/M
4
3wk
Mono
6/F
3
3mo
MOflO
4/F
2
2mo
Mona
2 ‘/r/F
1
Duratlo”
Patlent
D,sHse
Age Iv)/ Sex
RA
URI
fluonde
2-3
Aspwm g/day
3 0 g/day
Aspmn
NOrW
wth
Multwltamms
3 6 g/day
Aspwin
current
for con-
Penicillm
None
1.8 g/day
Aspmn
None
over 12 yr
mfectmn
UWlav
NOllt?
rhlnms
Allergw
None
rhlnltls
Allergvz
NOIW
NOW
rhinltls
RA
1All patents
tulle
space
Narrow
joint
waldous
Bilateral
condyles
med. fem.
Bilat. notching
swelling
soft tl*S”e
Normal
osteoporosis
Juxtaarticular
demmeralozatmn
Dislocation,
jomt space
Increased
swelling
Soft tissue
swelling
soft ttssue
arrest lmes
growth
dascrepancy:
Leg length
Normal
swelling
Soft tissue
swelling
Soft tissue
x-rays
were whtte except for No
OnSet same
Mother
NO!%?
NOW
NOW
NOrll?
NOfW
COUSI” RA
Maternal
NOW
NOW
RA
Paternal
NOW
aunt
Maternal
3.6 g/day NOlIe
Allergx
RA NOW
grandmother
None
NOW
N0ll.Z
NOW
grandmother
Maternal
anemia NOlhZ
grandmother
Maternal
Family Hostory
deficiency
Other Disease
Aspirm
Vat. E iron
1.2 g/day
Aspirin
2.4 g/day
Aspirm
N0ll.Z
1.2 g/day
Aspirin
NOIll?
MedlCatl0”S at Surgew
1.640
ND
ND
Neg
1:128
Neg.
ND
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
(SCAT)
1:128
Latex
13. who was nonwhite
41
47
28
7
37
22
20
31
7
10
59
56
20
12
40
ESR ,mm,hr, Wl”~r&-Z?
NO
NO
NO
NO
NO
NO
Y&S
NO
NO
NO
NO
NO
110
123
15.0
13.1
11 6
124
14.6
14.1
13.0
12.9
8.6
11.9
13.0
12.4
10.2
NO
NO
(91
Nodules
Hemoglobin
I2.000
6.500
7,800
5.300
7.900
8.200
9.800
8.700
11.300
6.000
9.000
4.900
8.000
6.DDO
8.600
wh1te count (cells/cu mm)
JUVENILE
SYNOVIUM.
JRA VS. NORMAL
289
Fig. 1. Synovial lining layer with large cells containing azurophilic granules and some vacuoles. S, surface; V, small venule. Crystal violet. x 980.
were contained within large cells with long processes and one or two nuclei. The subintimal fibroblasts were likewise mildly increased, although frequently their hyperplasia was masked by the presence of large numbers of inflammatory cells. Most cells shared in the hyperplastic response and were increased in numbers. They were especially numerous in two synovia. tnjlammatory Response The inflammatory cell response was generally marked. There were differences in degree among synovia and often from one area to another in any one synovium. The most striking finding was the heavy infiltration by plasma cells and lymphocytes. These cells were principally located around blood vessels of the subintimal layer, but often were widely scattered throughout the subintimal and lining layers. in which either plasma cells or Foci, lymphocytes predominated , were frequent. Plasma cells were large, lx-2 x the size of small lymphocytes, and binucleate plasma cells were frequently seen (Fig. 2). All tissues showed various numbers of large plump macrophages in the subintimal layer.
Fig. 2. Synovial villus. Eynovial lining layer on either side of the villus is relatively normal here. Plasma cells and lymphocytes dominate the perivascular inflammatory response. S, surface; arrows, binucleate plasma cells. Crystal violet. x 530.
Giant cells with peripheral oval nuclei were seen in three tissues. In the epoxy resin sections phagocytic giant cells (20-70 pm) were present in almost all tissues and were seen more clearly. They contained one to three nuclei and their cytoplasm was spread out, often ending in long cell processes. Dark granules and cytoplasmic vacuoles were present. Synovium from one patient contained many macrophages that had clearly ingested various materials including cells (Fig. 3). Monocytes were sometimes seen within the lumina of blood vessels, and migrating monocytes were observed in the walls of vessels, especially venules. Polymorphonuclear leukocytes of neutrophilic type formed a prominent part of the inflammatory infiltrate in one synovium and were occasionally seen scattered or in the lining layer in most other synovia. They were clearly not a prominent feature of the inflammatory responseof JRA.
WYNNE-ROBERTS
ET AL
Fig. 4. Synovial surface 6) and subintimal blood vessels (BV). Subintimal vessels show marked endothelial proliferation with obliteration of vessel lumina. Surrounding cellular response is made up mainly of macrophages. Crystal violet. x 530.
Fig. 3. Subintimal area of synovium. Small vein (V) is shown, and around it are various inflammatory cells. Lymphocytes, plasma cells and macrophages IfUll containing phagocytosed materials are shown. The vessel (BV) seen in its long axis contains cells, one of which may be a monocyte. Crystal violet. x 3900.
timal vessel endothelial cells, such that some lumina were obliterated (Fig. 4). Often the vessels were ringed by a great variety of inflammatory cells. Both normal and abnormal vessels could be seen in any one tissue.
Iron
Miscellaneous
Occasionally, the yellow pigment of intracellular iron was seen. This was generally in the subintimal or stromal areas, and the amount in any tissue was small. The Prussian blue stain showed occasional small pockets of iron located perivascularly and in the synovial lining layer.
Edema was seen in the synovial lining layer of two tissues. Fibrinoid necrosis was noted in three synovia, with palisading cells in one. Despite the hyperplasia of synovial cells at all levels, mitoses were uncommon and were seen in only three of the tissues. Mitoses of inflammatory cells were also extremely rare; although binucleate (telophase) plasma cells were frequently seen in all tissues, cells in metaphase were seldom found. Lastly, calcification was recognized in one subintimal locus of a single synovium.
Blood Vessels
Increased vascularity was common and was noted in most synovia. Blood vessels, especially venules and capillaries, were generally open. While arterioles usually appeared normal, capillaries and venules showed mild or moderate endothelial ceil hypertrophy and sometimes hyperplasia. In larger vessels endothelial hypertrophy appeared to be accompanied by more smooth muscle cells around the vessels. Furthermore, increased thickness of the adventitial sheath was often present. Two tissues showed marked proliferation of subin-
Other Findings
ELECTRON
MICROSCOPY
Synovial Lining Layer
One of the striking abnormalities noted by electron microscopy was loss of integrity of the synovial lining layer. Whereas the normal lining layer often had a definite edge facing the joint cavity, the inflammed synovium had no such
JUVENILE
SYNOVIUM:
291
JRA VS. NORMAL
synovial surface some cells appeared necrotic and apparently were separating from the synoival membrane. In this mesh of separating cells much fibrin was usually seen. All of the lining cells showed differences from normal. Lining Cells A cells were large, and their surface activity showed many micropinocytotic vesicles, filopodia, and long cell processes. Their cytoplasm contained many vacuoles and numerous pleomorphic lysosomes and phagolysosomes. Small round or elongated lysosomes were seen in the areas of well-developed Golgi apparatuses and contained dark-staining fine granular or homogeneous material. Medium-sized secondary lysosomes contained material of varied density; some of this was lipidlike. The large phagolysosomes, giant sized and often bizarre in shape, did not usually contain recognizable material. B cells were more nearly like those of normal synovia, except that they had more rough endoplasmic reticulum, often with dilated cisternae containing homogeneous material. Two synovia contained rough endoplasmic reticulum-bound material with electron-dense central and pale outer areas. Ribosomal stacking was frequent, and ribosomal rosettes were increased. Some B cells contained a few lysosomes. C cells appeared most like B cells, except that well-developed Golgi systems were observed in addition to the endoplasmic reticulum. The overlap between B and C cells was marked because both had filopodia and micropinocytotic vesicles as well as the other features listed below. Fig. 5. Montage of the synovial lining layer in JRA showing tightly packed pleomorphic cells. Necrotic lining cell (DLC) is separating from the surface (S). Lining cells are predominately phagocytic in type, with many primary and secondary lysosomes. Free myeloid membranes (arrows) and accumulations of fibrin (F) are shown. RBC, erythrocyte. x 3000.
margin. The lining layer (Fig. 5) was made up of A (phagocytic), B (synthetic), and C (intermediate) cells. I7 They were increased in numbers, and many were larger than their normal counterparts. The A cells were most numerous, followed by B and then C cells. A cells appeared to be most responsible for the lining cell hyperplasia. Fibrocytes were not observed.lL~‘s At the
Miscellaneous Features of All Cells of the Synovia Nuclei and nucleoli of the lining cells appeared normal. No nuclear inclusions were seen. Non-membrane-bound lipid was frequently observed in A, B, and C cells. Glycogen was noted in four synovia in the A cells and occasionally in the B cells. Myelinlike figures were most often seen in association with mitochondria, especially in cells of the lining layer; rarely they were seen in Golgi lamellae and associated with some phagolysosomes. Sometimes free or extracellular myeloid membranes were present. Mitochondria were more pleomorphic than in
292
normal tissue. Round or swollen mitochondria were noted as well as the myeloid figures seen associated with them. One tissue showed mitochondria of low ADP or type IV configuration.‘” The more unusual mitochondria were usually seen in cells of the synovial surfaces. Crystals were not seen in any tissues. Microtubules were occasionally observed, primarily in relationship to centrioles. Microfilaments were seen in lining cells in subintimal and stromal fibroblasts, and in endothelial and smooth muscle cells of blood vessels. Centrioles were seen in all cell types in all synovia. No mitoses were observed ultrastructurally (although they were seen by light microscopy). Occasional free red cells were seen in two synovia. No siderosomes or ferritin particles were identified in synovial lining cell fibrolasts or macrophages. Mast cells, normally seen in healthy synovial membranes, were plentiful. They appeared similar to normal mast cells except that they were not observed to be releasing their fine fibrillar and homogeneous materials as often as normal, and they contained more lipidlike material. In one instance, granules with scroll-like configurations were seen. In another synovium, subintimal B-like cells were seen in close proximity to mast cells, with B-cell processes extending around the mast cells.
Inflammatory Cell Response Plasma cells were most frequently found around vessels (but never within their lumina), in close relationship to subintimal fibroblasts, macrophages, and sometimes A cells. All had typical prominent rough endoplasmic lamellae, and often dilated cisternae; they had the morphologic appearance of active protein synthesis. In most synovia some plasma cells contained Russell bodies. Free Russell bodies were not observed.20 These bodies were dark staining and homogenous and bound by rough endoplasmic reticulum. Paranuclear starlike well-developed Golgi lamellae and vesicles were usually present, especially in mature plasma cells. Mitochondria were plentiful and sometimes large; lipid drops and lysosomes were occasionally seen especially in young cells. Plasma cells appeared to have active surfaces with short filopodia. Binucleate plasma cells were frequently seen and showed all the cytoplasmic
WYNNE-ROBERTS
ET AL
elements recorded above. No Mott cells were observed.” Lymphocytes were the second most commonly seen inflammatory cells. They were found around and in blood vessel walls, in the synovial lining and subintimal areas, and sometimes closely associated with A or B cells. The lymphocytes appeared to migrate through the endothelial cells of the vessels, especially the venules and veins. The lymphocytes in the vessel walls had well-developed filopodia. These lymphocytes often contained many large mitochondria and rough endoplasmic reticulum. Variations in size and shape of nuclei were seen, and nucleoli were often prominent. Larger lymphocytes with prominent nucleoli and variegated nuclei and active cytoplasmic elements were seen. Sometimes these cells were not easy to distinguish from monocytes, but the lymphocytes’ nuclear appearance helped to differentiate the two cell types. Thus lymphocytes more often appeared atypical (Fig. 6) or showed early blastic transformation. Centrioles were present, but mitoses were not observed. Cells with features of both lymphocytes and plasma cells were seen. Such cells had irregular nuclei and well-developed rough endoplasmic reticulum lamellae. Macrophages made up a large segment of the infiltrating cell population and were especially numerous in synovium from patient No. 3. They most closely resembled A cells, with many lysosomes and filopodia. Like A cells, macrophages were very pleomorphic. They contained many primary lysosomes, some of which appeared to be budding off the Golgi lamellae. The lysosomes were small (60-100 nm), dark staining, and membrane bound. Such lysosomes were also seen in the periphery of the cytoplasm of the macrophages. Some macrophages also contained small tubular structures of 22 36 nm outer diameter in their peripheral cytoplasm (Fig. 7A). Mitochondria, both rough and smooth endoplasmic reticula, and highly developed Golgi apparatuses were frequently seen. Some macrophages had features of the monocyte seen in the lumina of blood vessels. The mode of egress from vessel lumen to the tissue was by passage between (as opposed to through) endothelial cells of the vessel wall. Macrophages frequently showed evidence of
JUVENILE
SYNOVIUM:
JRA VS. NORMAL
Fig. 6. Parts of five cells. Macrophage (Mac 1) contains a large pleomorphic phagolysosome (PhL) and a small secondary lysosome (L). Lymphocyte (LY) shows early transformation with an irregular nucleus (N) and large mitochondria (M). Long cell process (CP), probably from a macrophage, extends in intimate contact along the lower margin of the lymphocyte. Large macrophage (Mac 2) contains a dark pleomorphic phagolysosome. Small dark granules (arrows) can be seen within the margin of the phagocytosad material. Parts of two other macrophages (Mac 3. Mac 4) are shown. C. collagen fibers; V. vacuoles. x 13,660.
WYNNE-ROBERTS
ET AL
Fig. 7. Two macrophages, one of which is in close contact with a lymphocyte. At points of cell contact desmosomelike junctions occur (arrows). Lysosomes IL), vacuoles P/I, mitochondria (MI. nuclei (N) are shown, x 14.000. Inset: Small lymphocyte and a small portion of a macrophage lie in relation to each other. Section of macrophage contains short tubular structures tarrows) of 22-36-nm outer diameter, with an occasional larger structure of diameter up to 73 nm. Ribosomes of the lymphocyte are about 15 nm. for comparison. x 13,680.
phagocytosis and contained variegated phagolysosomes of medium and giant size (Fig. 6). The material within the phagosomes ranged from unrecognizable, sometimes homogenous material to obvious fibrin, platelets, and plasma cells. The recently ingested plasma cells appeared somewhat necrotic, with clumped nuclear chromatin; these cells were seen in various stages of degradation. The plasma cell breakdown appeared as fragmentation and disorganization of the cells’ contents, with myeloid figure formation and shrinkage of the cell. Of interest, lysosomes were not specifically located in the vicinity of the degrading cells; however, sometimes dark-staining granules were seen around the edge of an ingested cell, most likely representing fusion of primary lysosomes with the phagosome. In other the close juxtaposition of instances, macrophage and plasma cell or lymphocyte was remarkable (Fig. 7). Sometimes a desmosomelike junction was present between the contiguous cells. Both macrophages and A cells
appeared to be by far the most active phagocytic cells in the inflammed synovia. Neutrophils were located both around vessels and scattered in the synovial lining and subintimal areas. Some had ingested fibrin or other material but otherwise appeared normal. They rarely were degranulated. Neutrophils were not numerous; they were occasionally observed to be ingested primarily by A cells of the lining layer. Platelets were seen in all synovia in the lumina of vessels, sometimes plugging gaps in their walls, or just outside them. “Inclusions” Tubularlike structures were seen in three synovia in the cytoplasm of B cells of the lining layer (patients No. I, 3, 14) (Table 1). The mean diameter of these short wavy tubules was 23 nm (Fig. 8). Blood Vessels Each synovium abnormal vessels.
contained both normal Some, notably venules
and and
JUVENILE
I:ig. 8.
SYNOVIUM:
295
JRA VS. NORMAL
Part of a 8 cell containing fine wavy structures (arrows) of 23-nm diameter.
Rough endoplasmic
reticulum her 1. nu-
clefus(N), mitochondria (MI indicated. x 21,780.
‘ig. 9. IA) Part of an andothalial cell of a venule showing rod bodies (RI, which are Sean at different tangents of sasction. tochondria (MI. nucleus (N). lumen of vassal (Lu), basal lumina (BL), myeloid figure (My) shown. x 41,040. (8) Regular array nembranes within the rod (RI arranged longitudinally. Rough endoplasmic reticulum her) indicated. x 74,250. (C) App #arant ralopmant of new rod bodies (arrows) by lateral budding from mature rods. x 31,920.
WYNNE-ROBERTS
296
capillaries, seemed to have the most changes. Endothelial cell hypertrophy and hyperplasia were sometimes present. The endothelial cells themselves appeared more active than normal. Large nuclei, well-developed rough endoplasmic reticulum, free ribosomes, and Golgi stacks were present in increased amounts. Mitochondria were plentiful, lysosomes were increased, and occasionally lipid drops were present. Rod bodiesz2 (Fig. 9), seen mainly in venules, were often numerous, especially on the luminal side of the vessel. Seen in longitudinal section they contained regular membranes oriented in the long axis of the rods (Fig. 9B). In cross section the rods contained approximately 8-12 welldefined membrane bound vesicles of 12 nm outer diameter. Rods occasionally showed lateral budding, which was best seen in longitudinal section (Fig. 9C). Endothelial cell microvillus projections into the vessel lumina were plentiful. Micropinocytotic activity was marked on both luminal and basal aspects of the vessels. Smooth muscle cells were hypertrophied and appeared actively synthetic. Increase in vessel wall thickness was principally due to increase in the ad-
Fig.
10.
membrane.
Capillary x 5600.
with
thickened
adventitial
sheath.
Two
ET AL
ventitial sheath, particularly of basal laminae and collagen (Fig. 10). These features were most often seen in veins and venules and capillaries of all synovia, even in those of most recent onset of juvenile rheumatoid synovitis (Table 1). Intercellular Matrix In addition to easily recognized collagen, fibrin often was seen between cells, in vacuoles of A cells, and ingested by neutrophils in the synovial lining and subintimal layers. The fibrin was seldom seen with banding of regular periodicity but generally was of the fine fibrillar kind, with slight beading. There was a considerable increase in intercellular amorphous and fine fibrillar materials. These materials were distinct from fibrin. Effete cells as well as debriding surface cells were occasionally found in all synovia. DISCUSSION
We have croscopic patients (4 of JRA
lymphocytes
studied the light- and electron-miappearances of synovia from 15 males, 11 females), with four forms (8 with monarticular, 1 with
are just
outside
the vessel
wall.
My,
free
myeloid
JUVENILE
SYNOVIUM.
JRA VS. NORMAL
oligoarticular, 4 with polyarticular arthritis, and 2 with Still disease), of age range 2.5516 yr. The findings were compared with synovia from seven patients (two female, five male), of ages 6-20 yr, with no synovial disease. I ’ The age and sex patterns of the two groups were not precisely matched. Females showed a preponderance over males by 2.5:1 in our JRA group; this experience is similar to that of others.‘-” Initially five of our patients (Table 1) did not fulfill the criteria for JRA, but they subsequently did so. Diagnois of JRA required the persistence of arthritis for 3 moor more and the elimination of other causes of arthritis. I3 Comparison of Synovial Membrane Morphology From JRA Within the Group of I2 Patients and With That of Adult RA The light-microscopic histology of the synovia from the different types of expression of JRA within our group were compared, after coding the tissues as unknown, and we were unable to discern any differences. Neither the clinical severity nor the duration of disease significantly affected the histology. The same procedure was adopted for comparison with a number of slides from patients with adult RA or with osteoarthritis; it was not possible to distinguish consistently thejuvenile from the adult form of RA. The greatest variations found in the synovial and inflammatory responses were within the tissues themselves, between one area and another, rather than from among different patients, Focal synovitis was a marked feature. This experience was similar to that recorded by Bywaters and Ansell.” Comparison With Reports of JRA Synovial Ultrastructure Borrachero et al.,s in their study of synovia from two children with Still disease, noted hyperplasia of the synovial lining layer and a marked plasma cell and lymphocytic response. Bierther and Schafer9 studied four children with JRA and recorded endothelial cell damage of capillaries and postcapillary vessels, a neutrophil response, and increase in mast cells. They did not comment on plasma cell, lymphocyte, or macrophage infiltration. The duration of disease in their four patients was not stated, but the predominance of neutrophils in the inflammatory response contrasted with our
297
findings of neutrophils being a lesser part of the response. Clawson et al.‘* reported an inflammatory and synovial lining cell response similar to ours. Vascular changes with endothelial swelling, necrosis, and scarring were seen. We have not observed necrosis or scarring in our cases. Schumacher and Kitridou’ studied synovitis of recent onset and included three patients with JRA. They noted that plasma cells and lymphoid cells were rare in early JRA or RA, but no particular differences between the two groups were recorded. In our series we had only one patient whose onset of disease was less than 1 mo prior to synovial biopsy (No. 3, Table 1). In this instance, we observed a chronic inflammatory response with marked macrophage activity, many plasma cells, and lymphocytes. In our other four patients with JRA of up to 2 mo duration plasma cell response was likewise established. Assessment of the Synovial and Inflammatory Responses in the Synovitis of 15 Patients With JRA and Comparison With 7 Control Synovia The inflammatory abnormalities seen in juvenile rheumatoid synovitis may be divided into two parts, those involving the cells indigenous to the synovium and those relating to the immigrant cell response. The morphologic response in JRA synovium is compared diagrammatically with normal synovial structure (Fig. 11). The striking differences between the inflammed and normal synovia were seen in all areas of the joint lining. The well-defined surface of the normal synovial lining layer was lost, being replaced by fibrin, sloughing cells, and neutrophils. The effete phagocytic cells, including neutrophils, most likely release lysosomal enzymes into the joint cavity that in turn could contribute to cartilage damage.23 The hypertrophy and mild hyperplasia of the lining cells suggested increased activities in response in part to unknown stimuli and in part to their marked phagocytic activity. Furthermore, their altered appearance with increased lipid and glycogen indicated changes in metabolism compared with normal. Increased anaerobic glycolysis and lactate production have been reportedz4 and enhanced oxygen uptake recorded in inflammed synovia.2” However, these observations are not unique to synovia;
298
WYNNE-ROBERTS
ET AL
Joint Space
Joint Space
Venule
Fig. 11. (A) Diagrammatic concept of the structure of normal juvenile synovium. Fibrocvte IFc), phagocytic cell (A), synthetic cell (B), intermediate cell (CL amorphous material (AM). granulated mast cell, fibroblast (FL collagen (CL capillary (Cap). and a venule are shown. Interrupted lines from the vessel lumina to the synovial surface represent the sources and general direction of movement of fluid to which lining cells add hyaluronate, yielding highly viscous synovial fluid (SF). (B) Diagrammatic concept of the synovial and inflammatory responses in JRA. Synovial response consists of increase in numbers of phagocytic surface cells (A) and disappearance of fibrocytes (Fc). Synthetic cell (B). intermediate cell (CL debriding lining cell fdlc), lysosomes (Ly), fibroblast (F), collagen (C), and mast cells are shown. There is an increase in amorphous materials (AM). The immigrant inflammatory response is represented by fibrin (Fib), polymorphonuclear neutrophils (PMNL macrophages (ML lymphocytes (L). plasma cells (P), platelets (PI), and transformed lymphocytes (TL). Blood vessels show endothelial hyperplasia and thickened adventitial sheathes. Synovial fluid (SF) is formed as in normal synovium, except in increased amounts; to it are added some of the products of inflammation and increased amounts of hvaluronete
increased glycolysis has been noted in other inflammatory responses.‘6 The preponderance of A cells with many lysosomes in JRA contrasted with the B-cell predominance of the healthy tissues. This conversion of the synovial lining cells from mostly smaller synthetic-appearing ones to large active and predominantly phagocytic cells was remarkable. The possible origins of the increased numbers of A cells in the absence of increased mitoses may be speculated on. The fibrocytes”,‘* observed in normal synovia were no longer found; perhaps in the inflammatory state these cells may be the ones that differentiate into A cells. Alternatively, A cells may arise from C cells. Thirdly, some are possibly immigrant macrophages that in the lining layer become indistinguishable from indigenous A cells.
The tremendous increase in lysosomes and phagocytic activity seen in both A cells and immigrant macrophages was probably partly induced by the presence of increased numbers of effete cells, fibrin, and other intercellular materials such as immune complexes. This process would be in keeping with the mechanism of removal of unwanted biologic materials described by Axline and Cohn.“’ B cells were larger than normal and showed much more endoplasmic reticulum with dilated cisternae. Possibly these cells were secreting more hyaluronate and other mucopolysaccharides into the adjacent matrix and the synovial effusion accompanying the synovitis.” Two rheumatoid tissues contained much electrondense amorphous material similar to that recorded in tissue-cultured fibroblasts derived
JUVENILE
SYNOVIUM:
JRA VS. NORMAL
from a patient with JRA who had a high hyaluronate synthesis rate and similar to material noted by others. 2g The significance of the unusual appearance of the rough endoplasmic reticulum contents is unknown but may be due partly to the effects of tissue processing. Of particular interest were the blood vessel changes, especially endothelial proliferation, evidence of increased cytoplasmic activities and adventitial sheath hypertrophy. These represented changes due to stimulation of the blood vessels that apparently take part in the overall hyperactivity of the synovium. Focal necrosis of part of a vessel wall or of whole vessels, with or without thrombosis, was not seen in our sampling. Despite the perivascular location of the invasive inflammatory response and of evidence of migration of inflammatory cells through the vessel walls, morphologic evidence of damage to the vessels, that is, a vasculitis, was not present. Vasculitis has been considered in adult RA by Goldie and co-workers,““~3’ who noted abnormalities by light microscopy. No firm conclusions were drawn by these authors about the role of a vasculopathy in RA. However, Marin et al.“” found vasculitis as a primary lesion; certainly in more malignant forms of RA, vasculitis may be a very prominent feature. The immigrant inflammatory cell response consisted of neutrophils, macrophages, and lymphocytes. The role of the neutrophil appeared to be that of scavenger in and on the surface of the synovial lining layer, whereas the macrophage phagocytized cells and debris both in the lining and subintimal areas. The frequent intimate relationship of macrophage and plasma cell or lymphocyte raised the question of passage of informational or other material between the intimate cells. The role of the macrophage in immunity has been widely explored and it has been shown that macrophages can ingest some organisms and can excrete antigens derived from the ingested organisms.33 The monocyte-macrophage response in JRA is a prominent feature of the inflammatory response. Perhaps this response has an important role to play in processing and modifying an antigen. The continual production of antigenic stimuli is suggested by the chronicity of the active inflammatory response. We may speculate on whether or not the
299
phagocytic behavior of lining A cells (matching that of macrophages) is accompanied by an ability on the part of A cells to “process” some infectious agents and produce antigen in a way similar to that documented for macrophages.33 Such an adaptation could be an important part of the synovitis of JRA. Our impression of marked increase of lysosomes in the inflammed synovial tissue was based only on morphologic observation because we have not specifically measured lysosomal increase using an enzyme marker. Our observations are similar to those who have studied the synovitis of adult RA. 34*35Primary lysosomes were seldom seen in the vicinity of phagosomes; the small dense granules lining membranebound material suggested that fusion of primary lysosomes with phagosomes occurred with the formation of secondary phagolysosomes. Autophagic vacuoles, residual bodies, and extracellular free membranes were all seen, indicating that the sequence of events of digestion of materials documented by deDuve and Whattiaux had taken place.“” Several authors have specifically studied the plasma cell component of the inflammatory response.2”~97~38 Curtiss noted in a review”!’ that plasma cell response does not occur early in the synovitis of RA but is established after 4-5 mo of active disease. We saw a well-established response of plasma cells and lymphocytes in our early cases of JRA (Table I). This is in keeping with the early findings of immunologically induced arthritis in rabbits.40 Furthermore, it has been shown that IgM and then IgG antibodies occur in response to a specific antigenic stimulus within 14 days from primary immunization by the antigen. 41 Therefore any primary antigenic stimulus (or stimuli) in RA or JA should attract antibody-forming cells, and antibodies might be expected to be produced within 3 wk of the onset of disease. Some have commented on the presence of Russell bodies, which have been shown to contain gamma globulins, within plasma cells.*“~~” We, too, have observed Russell bodies, but never large nor found outside cells.“’ In the tissues of our study, as in those of adult RA, all plasma cells appeared morphologically to be very active in protein synthesis, as indicated by well-developed ergastoplasm and Golgi systems. While we observed frequent close rela-
300
tionship of plasma cells to either A cells or macrophages and ingestion of plasma cells by phagocytes, only one other author has recorded plasma cell phagocytosis by histiocytes,J2 but others have noted the close juxtaposition of the two cell types.20,43 We may speculate on why plasma cells were ingested. ‘It may simply be to remove old or effete cells, because the life span of some plasma cells is relatively short,44 about 1 wk, or perhaps selected plasma cells are being removed from the inflammatory scene by phagocytosis because their products are injurious. Binucleate (telophase) plasma cells were noted frequently in the synovitides, which suggested that replication of plasma cells occurred. Binucleate and multinucleate plasma cells have been reported in other inflammatory situations.44 Our findings in JRA agree with those of others4” that plasma cells dominate the inflammatory responseof RA. Lymphocytes, like plasma cells, were observed in close juxtaposition with phagocytic cells; however, ingested lymphocytes were not detected. Three explanations can be considered for this latter observation. First, most lymphocytes survive longer than plasma cells; second, perhaps many were transformed into plasma cells; third, some lymphocytes may reenter the circu1ation.l” We noted that lymphocytes once outside the circulation often appeared atypical and that some were similar to those described as transformed.“X We did not observe mitoses among the lymphocyte series, although in vitro they have been reported to occur 667 days after phytohemagglutinin stimulation.“’ That the small lymphocyte was most likely the source of plasma cells was supported by our never visualizing intraluminal plasma cells and by our seeing all intermediate stages of transformation between lymphocyte and plasma cell. This behavior of many lymphocytes therefore placed them in the B (bone marrow) dependent category of the immune defense were not distinguished system. 48 T lymphocytes from untransformed B lymphocytes. The question of an infectious agent or agents as the possible cause(s) of RA has been raised by many; and we have raised it with respect to the etiology of JRA. Neumark and Farkas have consistently reported nuclear particles that they considered to be viral.4g In 1973 Neumark et
WYNNE-ROBERTS
ET AL
al.“” noted cytoplasmic viral particles in 42 rheumatoid patients and not in 17 nonrheumatoid controls. We have carefully studied their work and reviewed our synovia for similar particles but have not seen them. Neumark et al. stated specifically that they were able to distinguish the particles of size 120-220 nm from small lysosomes. We have seen membrane-bound dark-staining particles predominantly in phagocytes. They appeared to be derived from Golgi systems, and therefore we thought them most likely primary lysosomes. Morphologically these lysosomes appeared quite similar to elementary bodies observed by Schumacher and Kitridou7 in the cytoplasm of a perivascular cell from a patient with transient synovitis. Clearly, it is extremely difficult to distinguish single viruses or certain mycoplasmal organisms from normal cell organelles in some instances, in contrast to viral particles that form lattices. Rod bodies have previously been considered abnormalZZ and possibly an infective agent but most likely a normal component of at least some small blood vessels.“’ Rod bodies were more plentiful than normal, and it was interesting to note that they appeared to replicate by budding. Their functional significance is not known. Tubularlike structures were seen by Helder et al.,“2 and we saw similar structures in three synovia. Certainly these structures were reminiscent of some viral configurations. They were unlike the lacey tubular structures seen in the endothelial cells of blood vessels of patients with systemic lupus erthematosus or polymyositis.“’ Interestingly, the morphologic appearance of the tubular structures seen by us had staining characteristics suggesting they comprised primarily membrane material. The tubules of about 30-nm outer diameter frequently seen in the peripheral cytoplasm of macrophages were longer than the wavy 23-nm structures seen in synovial lining cells and may have been an abnormal component of the macrophage because we have found no documentation of these tubuIes in other situations. The tubules just discussed appeared different from the microtubules of 1526-nm diameter seen as a normal cytoplasmic component of all cells. The presence of iron in rheumatoid synovia
JUVENILE
SYNOVIUM:
JRA VS. NORMAL
301
has been extensively reported by Muirden in his studies of chronic anemia of RA. Two of our patients were anemic, and four of the group had active arthritis for more than 4 yr. Prussian blue staining for iron was present by light microscopy, but neither siderosomes nor ferritin granules were seen ultrastructurally in any cells, This negative result represents a problem
of sampling, since sequestration occurred in those with JRA.
of iron clearly
ACKNOWLEDGMENT
Dr. J. Cassidy of the Rackham
Arthritis Research Unit, of Michigan, Ann Arbor, kindly reviewed the manuscript. Dr. W. Green and Dr. H. Swensen of the Orthopedic Department of Presbyterian University Hospital, University of Pittsburgh, kindly obtained some of the synovia for our study. University
REFERENCES 1. Calabro JJ, Katz RM, Maltz BA: A critical reappraisal of juvenile rheumatoid arthritis. Clin Orthop 74:101-119,1971 2. Schaller J, Wedgwood RJ: Juvenile rheumatoid arthritis: A review. Pediatrics 50:94&953, 1972 3. Calabro JJ, Holgerson WB, Sonpal GM, et al: Juvenile rheumatoid arthritis: A genera1 review and report of 100 patients observed for 15 years. Semin Arthritis Rheum 51257-298, 1976 4. Gardner DL: Pathology of Rheumatoid Arthritis. Baltimore Williams & Wilkins, 1972, p 173 5. Bywaters EGL, Ansell BM: Monoarticular arthritis in children. Ann Rheum Dis 24:116-122, 1965 6. Norton WL, Ziff M: Electron microscopic observations on the rheumatoid synovial membrane. Arthritis Rheum 9:589-610, 1966 7. Schumacher HR, Kitridou RC: Synovitis of recent onset: A clinicopathologic study during the first month of disease. Arthritis Rheum 15:465-485, 1972 8. Borrachero J, Valle A, Diaz-Flores L, et al: Estudio con microscopio optic0 y electronico de la membrana sinovial en la enfermedad de Still. Rev Esp Rheum Enferm Osteoartic 13:527-532, 1970 9. Bierther M, Schafer U: Elektronenmikroskopische Untersuchung des Synovialgewebes bei der juvenilen rheumatoiden Arthritis. Z Rheumatol 33:43-53, 1974 IO. Garcia MM, Charlton KM, McKay KA: Characterization of endotoxin from Fusobacterium necrophorum. Infect Immun 11:371~379,1975 II. Wynne-Roberts CR, Anderson C: Lightand electron-microscopic studies of normal juvenile human synovium. Semin Arthritis Rheum (this issue) 7:279%286, 1978 12. Clawson CC, Lounberg J, Good RA: Synovial microvasculature in juvenile rheumatoid arthritis. Abstracts of the 25th Annual EMSA Meeting, pp 174-175, 1967 (unpublished) 13. Brewer EJ Jr, Bass JC, Cassidy JT, et al: Criteria for the classification of juvenile rheumatoid arthritis. Bull Rheum Dis 23:712-719, 1972 14. Luna LG (ed): Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology (ed 3). Washington, D.C., U.S. GPO, 1968, p 179 15. Millonig G: Advantages of a phosphate buffer for 0~0, solutions in fixation. J Appl Physiol32:1637, 1961 16. Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain for electron microscopy. J Cell Biol 17:208, 1963 17. Barland P, Novikoff’ AB, Hamerman D: Electron mi-
croscopy of the human synovial membrane. J Cell Biol 14:207-220, 1962 18. Porter KR: Morphogenesis of connective tissue, in Stephens CAL Jr, Stanfield AB (cds): Cellular Concepts in Rheumatoid Arthritis. Springfield, Ill., Thomas, 1965, pp 6-36 19. Tandler B, Hoppel CL: Mitochondria. New York, Academic, 1972, p 18 20. Orlovskaya GV, Muldiyarov PY, Kazakova IS: Synovial plasma cells in rheumatoid arthritis: Electron microscope and immunofluorescence studies. Ann Rheum Dis 29:524532, 1970 21. Bessis M: Living Blood Cells and Their Ultrastructure. New York, Springer, 1973, pp 533-535 22. Highton TC, Caughey DE, Rayns DG: A new inclusion body in rheumatoid synovia. Ann Rheum Dis 25:149-155, 1966 23. Weissmann G: Lysosomal mechanisms of tissue injury in arthritis. New Engl J Med 286:141-146, 1972 24. Stossel TP: Phagocytosis. New Engl J Med 290:717-723.774-780,833X338, 1974 25. Thomas DP, Dingle JT: Studies on human synovial membrane in vitro: The metabolism of normal and rheumatoid synovia and the effect of hydrocortisone. Biochem J 68:231-238, 1958 26. Beck WS: The control Chem 232:25 l-270, 1958
of leukocyte
glycolysis.
J Biol
27. Axline SG, Cohn ZA: In vitro induction of lysosomal enzymes by phagocytosis. J Exp Med I3 I : l239- 1260, 1970 28. Wynne-Roberts CR, Castor CW: Ultrastructural comparison of rheumatoid and non-rheumatoid synovial fibroblasts grown in tissue culture. Arthritis Rheum 15:65-83, 1972 29. Ghadially FN, Roy S: Ultrastructure membrane in rheumatoid arthritis. Ann 261426-443, 1967
of synovial Rheum Dis
30. BrBnemark PI, Ekholm R, Goldie I: To the question of angiopathy in rheumatoid arthritis. An electron microscopic study. Acta Orthop Stand 40: 153-175, 1969 31. Goldie I: The synovial microvascular derangement in rheumatoid arthritis and osteoarthritis. Acta Orthop Stand 40:75 l-764, 1970 32. Marin D, Negoercu M, Stoia I, et al: The tnorphology of the synovial tissue and articular fluid cells in rheumatoid polyarthritis-Studied with the optical and electronic microscope. Acta Rheum Stand 15:126-134, I969 33. Uranue ER, Cerottini JC: The function of
WYNNE-ROBERTS
macrophages in the immune response. Semin Hematol 7:225-248, 1970 34. Barland P, Novikoff AB, Hamerman D, et al: Lysosomes in the synovial membrane in rheumatoid arthritis: A mechanism for cartilage erosion. Trans Assoc Am Physicians 72:2399247, 1964 35. Barland P, Novikoff AB, Hamerman D: Fine structure and cytochemistry of the rheumatoid synovial membrane, with special reference to lysosomes. Am J Pathol44:853 866, 1964 36. deDuve C, Wattiaux R: Functions of lysosomes. Annu Rev Physiol2:435-492, 1966 37. Neumark T, Farkas K: Ultrastructural aspects of lymphoreticular cells in rheumatoid synovium. Ann Rheum Dis 32:524 530, 1973 38. Kobayashi I, Ziff M: Electron microscopic studies of lymphoid cells in the rheumatoid synovial membrane. Arthritis Rheum 16:471 486, 1973 39. Curtiss PH Jr: Changes produced in the synovial membrane and synovial fluid by disease. J Bone Joint Surg 46A:873 888, 1964 40. Dumonde DC, Glynn LE: The production of arthritis in rabbits by an immunological reaction to fibrin. J Exph Pathol43:373 383, 1962 41. Humphrey JH, White RG: Immunology for Students of Medicine (ed 3). Philadelphia, Davis, 1970, p I25 42. Bierther M, Streit W: Die Synovialis bei chronischer Polyarthritis. Dtsch Med Wochenschr 97:453 458, 1972 43. Inoue H: A light and electron microscopic study of the distribution of gold sodium thiomalate in the rheumatoid synovial membranes. Acta Med Okayama 22:293 317, 1968
ET AL
44. Bessis M: Living Blood Cells and Their Ultrastructure. New York, Springer, 1973, pp 5 19 541 45. Hirohata K, Kobayashi I: Fine structures of the synovial tissues in rheumatoid arthritis. Kobe J Med Sci IO:195 225, 1964 46. Bessis M: Living Blood Cells and Their Ultrastructure. New York, Springer, 1973, pp415 419 47. Nowell PC: Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res 20:462, 1960 48. Humphrey JH, White RG: Immunology for Students of Medicine (ed 3). Philadelphia, Davis, 1970, p 325 49. Neumark T, Farkas K: Nuclear synovium. Ann Rheum Dis 29:653-659.
bodies in rheumatoid 1970
50. Neumark T, Hellos I, Farkas K: Virus-like particles in rheumatoid synovium. Stand J Rheum 2:21 28, 1973 51. Weibel ER, components in arterial 1964
Palade GE: New cytoplasmic endothelia. J Cell Biol 23:lOl 112.
52. Helder AW. Feltkamp-Vroom TM, Nienhuis RLF: Electron and light microscopical observations and serological findings in rheumatoid arthritis. Ann Rheum Dis 32:515 523, 1973 53. Hashimoto K, Chandler RW: Paramyxovirus-like inclusions in systemic lupus erythematosus, electron microscopic and cell culture studies. Acta Derm (Stockh) 521263 277, 1972 54. Muirden KD: The anaemia of rheumatoid arthritis: The significance of iron deposits in the synovial membrane. Aust Ann Med 2:97 104, 1970
Carolyn
P
RESENTATION of juvenile rheumatoid arthritis (JRA) is diverse and often remarkably different from that in adults, and these differences have been summarized by several authors. l--3 Because of the wide range of clinical expression of JRA, there are many unanswered questions; Is JRA a disease different from that in adults or part of a wide spectrum of rheumatoid arthritis (RA) seen at any age? Is JRA, with its frequent acute onset simulating an infectious disease, due to an infectious agent or agents? Synovial tissues of patients with JRA have been studied by light microscopy4.5 and the findings described as indistinguishable from those of adult disease. Less information, however, is available about the ultrastructural abnormalities because reports of electron microscopy of synovia from patients with JRA are mostly scattered among those of adults.‘*’ Recently, Borrachero et a1.8 and Bierther and Schafer9 have reported electron-microscopic findings of synovia from two patients with Still disease and four patients with JRA. We present information derived from lightand electron-microscopic study of synovia from 15 patients with JRA. Ultrastructural evidence of any bacteria or any viral or bacterial product such as endotoxin1° or of mycoplasmal infection was carefully sought but not found. However, marked abnormalities were observed, and the findings are compared with those of seven normal juvenile synovia.” Comparisons with the electron-microscopic studies of JRA,s~g~‘z with cases of JRA found among those of the adult where the information can be separated and with the information available from the ultrastructural studies of adult synovia, are made. MATERIALS
AND
in Arthritis
and Rheumatism,
A. M. Turano, and M. Baron
from all patients were negative for tubercle bacillus and anaerobic and aerobic bacteria. The synovial specimens were obtained either during surgery or by closed biopsy using a Parker-Pearson needle. For surgery, a tourniquet pneumatic cuff was applied on some of the patients for hemostasis, and ah synovial specimens were obtained within 20 min of cuff inflation. The specimens were divided immediately for light or electron microscopy. Light-microscopy specimens were fixed in phosphatebuffered formaldehyde and embedded in paraffin. Fivemicron sections were stained with hematoxylin and eosin; additional sections were stained with Prussian blue iron stain.‘” Synovia for electron-microscopic study were fixed in 2% paraformaldehyde in phosphate buffer at pH 7.2 and 680 mOsm or in 2.5% glutaraldehyde in Verona1 buffer at pH 7.2 for 8-12 hr at 4°C on a turntable. After phosphate buffer or veronal buffer rinses, the tissues were postfixed in Millonig 1% osmic acidI or veronal-buffered 1% osmic acid, then dehydrated with a graded sequence of ethanols followed by propylene oxide and embedded in Epon-araldite or Maraglas epoxy resins. Thick (0.5-1.0 hrn) sections were stained with aqueous I % crystal violet, and ultrathin sections were cut on a Sorvall MT-2 ultramicrotome, mounted on Formvar carbon-coated copper grids, stained with Reynolds lead citrateI and aqueous 1% uranyl acetate solutions, and viewed at 60 kV in a Phillips EM-300 electron microscope. RESULTS
Macroscopically the synovia showed marked villous hypertrophy and ranged in appearance from white to pink. Microscopically, fibrin was seen on the surface of 13 of 15 synovia; most fibrin was noted in those specimens from JRA of more recent onset. In all synovia, fibrin was seen also in the lining and subintimal layers in small to moderate amounts. Synovial lining layers showed mild hyperplasia and marked hypertrophy (Fig. 1) and were sometimes four to six cells deep. On epoxy resin sections the lining cells were principally phagocytic. Vacuoles, dark granules, and phagocytosed materials
METHODS
Synovia were obtained from 15 patients with mono-, oligo-, or polyarthritis or Still disease who fulfilled Classification I criteria of the American Rheumatism Association for the diagnosis of JRA.13 The clinical and laboratory data of the patients studied are shown in Table 1. The antinuclear antibody and lupus erythematosus preparations were negative in all and are therefore not listed. Gammaglobulins were normal or slightly elevated. Synovial fluid cultures
Seminars
H. Anderson,
Vol. 7. No.
4
(Mav). 1978
From the Electron Microscopy Unit of the Veterans Administration Hospital, and the Children’s Hospital of Pittsburgh, Pittsburgh, Pa. Address for reprint requests: Carolyn H. Anderson, M.D.. Children’s Hospital of Pittsburgh, 125 DeSoto St., Pittsburgh, Pa. 15213. 0 1978 by Grune & Stratton. Inc. ISSN 0049-0172/78/0704-0004$02.00/0
287
2 mo
14yr
1.5yr
9/F
9/F
11/M
13/F
13/F
14’/r/M
16/F
16/F
8
9
10
11
12
13t
14
15
monoart~cular.
L. knee
L. knee
R. knee
R knee
L ankle
I-. knee
mtp
R. hallux
R. wrist
R. knee
L knee
R. knee
L. knee
Ft. knee
L knee
R. knee
surg. Site
test
ohgo. ollgoartwlar
“ectomy
SylK-
prostheses
of
removal
FUSlOlX
biopsy
Open
biopsy
Needle
vectomy
SY”0-
fusion
Triple
biopsy
Needle
w?ctcmly
SyrU-
“ectOmy
SYrlO-
biopsy
Needle
poly. polyartlcular.
0.5 yr
1 mo
1.5 yr
5.5 yr
1.5yr
biopsy
Needle
vectomy
SyllO-
vectomy
SyllCI-
biopsy
Needle
biopsy
Needle
biopsy
Needle
surg PrOC.
t ND, not done; SCAT, sheep cell agglutmatlon
*Mono.
7 Yr
8WF
7
1 mo
MOW2
8/F
6
4 vr
Still
6/M
5
3 mo
MCln0
6/M
4
3wk
Mono
6/F
3
3mo
MOflO
4/F
2
2mo
Mona
2 ‘/r/F
1
Duratlo”
Patlent
D,sHse
Age Iv)/ Sex
RA
URI
fluonde
2-3
Aspwm g/day
3 0 g/day
Aspmn
NOrW
wth
Multwltamms
3 6 g/day
Aspwin
current
for con-
Penicillm
None
1.8 g/day
Aspmn
None
over 12 yr
mfectmn
UWlav
NOllt?
rhlnms
Allergw
None
rhlnltls
Allergvz
NOIW
NOW
rhinltls
RA
1All patents
tulle
space
Narrow
joint
waldous
Bilateral
condyles
med. fem.
Bilat. notching
swelling
soft tl*S”e
Normal
osteoporosis
Juxtaarticular
demmeralozatmn
Dislocation,
jomt space
Increased
swelling
Soft tissue
swelling
soft ttssue
arrest lmes
growth
dascrepancy:
Leg length
Normal
swelling
Soft tissue
swelling
Soft tissue
x-rays
were whtte except for No
OnSet same
Mother
NO!%?
NOW
NOW
NOrll?
NOfW
COUSI” RA
Maternal
NOW
NOW
RA
Paternal
NOW
aunt
Maternal
3.6 g/day NOlIe
Allergx
RA NOW
grandmother
None
NOW
N0ll.Z
NOW
grandmother
Maternal
anemia NOlhZ
grandmother
Maternal
Family Hostory
deficiency
Other Disease
Aspirm
Vat. E iron
1.2 g/day
Aspirin
2.4 g/day
Aspirm
N0ll.Z
1.2 g/day
Aspirin
NOIll?
MedlCatl0”S at Surgew
1.640
ND
ND
Neg
1:128
Neg.
ND
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
(SCAT)
1:128
Latex
13. who was nonwhite
41
47
28
7
37
22
20
31
7
10
59
56
20
12
40
ESR ,mm,hr, Wl”~r&-Z?
NO
NO
NO
NO
NO
NO
Y&S
NO
NO
NO
NO
NO
110
123
15.0
13.1
11 6
124
14.6
14.1
13.0
12.9
8.6
11.9
13.0
12.4
10.2
NO
NO
(91
Nodules
Hemoglobin
I2.000
6.500
7,800
5.300
7.900
8.200
9.800
8.700
11.300
6.000
9.000
4.900
8.000
6.DDO
8.600
wh1te count (cells/cu mm)
JUVENILE
SYNOVIUM.
JRA VS. NORMAL
289
Fig. 1. Synovial lining layer with large cells containing azurophilic granules and some vacuoles. S, surface; V, small venule. Crystal violet. x 980.
were contained within large cells with long processes and one or two nuclei. The subintimal fibroblasts were likewise mildly increased, although frequently their hyperplasia was masked by the presence of large numbers of inflammatory cells. Most cells shared in the hyperplastic response and were increased in numbers. They were especially numerous in two synovia. tnjlammatory Response The inflammatory cell response was generally marked. There were differences in degree among synovia and often from one area to another in any one synovium. The most striking finding was the heavy infiltration by plasma cells and lymphocytes. These cells were principally located around blood vessels of the subintimal layer, but often were widely scattered throughout the subintimal and lining layers. in which either plasma cells or Foci, lymphocytes predominated , were frequent. Plasma cells were large, lx-2 x the size of small lymphocytes, and binucleate plasma cells were frequently seen (Fig. 2). All tissues showed various numbers of large plump macrophages in the subintimal layer.
Fig. 2. Synovial villus. Eynovial lining layer on either side of the villus is relatively normal here. Plasma cells and lymphocytes dominate the perivascular inflammatory response. S, surface; arrows, binucleate plasma cells. Crystal violet. x 530.
Giant cells with peripheral oval nuclei were seen in three tissues. In the epoxy resin sections phagocytic giant cells (20-70 pm) were present in almost all tissues and were seen more clearly. They contained one to three nuclei and their cytoplasm was spread out, often ending in long cell processes. Dark granules and cytoplasmic vacuoles were present. Synovium from one patient contained many macrophages that had clearly ingested various materials including cells (Fig. 3). Monocytes were sometimes seen within the lumina of blood vessels, and migrating monocytes were observed in the walls of vessels, especially venules. Polymorphonuclear leukocytes of neutrophilic type formed a prominent part of the inflammatory infiltrate in one synovium and were occasionally seen scattered or in the lining layer in most other synovia. They were clearly not a prominent feature of the inflammatory responseof JRA.
WYNNE-ROBERTS
ET AL
Fig. 4. Synovial surface 6) and subintimal blood vessels (BV). Subintimal vessels show marked endothelial proliferation with obliteration of vessel lumina. Surrounding cellular response is made up mainly of macrophages. Crystal violet. x 530.
Fig. 3. Subintimal area of synovium. Small vein (V) is shown, and around it are various inflammatory cells. Lymphocytes, plasma cells and macrophages IfUll containing phagocytosed materials are shown. The vessel (BV) seen in its long axis contains cells, one of which may be a monocyte. Crystal violet. x 3900.
timal vessel endothelial cells, such that some lumina were obliterated (Fig. 4). Often the vessels were ringed by a great variety of inflammatory cells. Both normal and abnormal vessels could be seen in any one tissue.
Iron
Miscellaneous
Occasionally, the yellow pigment of intracellular iron was seen. This was generally in the subintimal or stromal areas, and the amount in any tissue was small. The Prussian blue stain showed occasional small pockets of iron located perivascularly and in the synovial lining layer.
Edema was seen in the synovial lining layer of two tissues. Fibrinoid necrosis was noted in three synovia, with palisading cells in one. Despite the hyperplasia of synovial cells at all levels, mitoses were uncommon and were seen in only three of the tissues. Mitoses of inflammatory cells were also extremely rare; although binucleate (telophase) plasma cells were frequently seen in all tissues, cells in metaphase were seldom found. Lastly, calcification was recognized in one subintimal locus of a single synovium.
Blood Vessels
Increased vascularity was common and was noted in most synovia. Blood vessels, especially venules and capillaries, were generally open. While arterioles usually appeared normal, capillaries and venules showed mild or moderate endothelial ceil hypertrophy and sometimes hyperplasia. In larger vessels endothelial hypertrophy appeared to be accompanied by more smooth muscle cells around the vessels. Furthermore, increased thickness of the adventitial sheath was often present. Two tissues showed marked proliferation of subin-
Other Findings
ELECTRON
MICROSCOPY
Synovial Lining Layer
One of the striking abnormalities noted by electron microscopy was loss of integrity of the synovial lining layer. Whereas the normal lining layer often had a definite edge facing the joint cavity, the inflammed synovium had no such
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synovial surface some cells appeared necrotic and apparently were separating from the synoival membrane. In this mesh of separating cells much fibrin was usually seen. All of the lining cells showed differences from normal. Lining Cells A cells were large, and their surface activity showed many micropinocytotic vesicles, filopodia, and long cell processes. Their cytoplasm contained many vacuoles and numerous pleomorphic lysosomes and phagolysosomes. Small round or elongated lysosomes were seen in the areas of well-developed Golgi apparatuses and contained dark-staining fine granular or homogeneous material. Medium-sized secondary lysosomes contained material of varied density; some of this was lipidlike. The large phagolysosomes, giant sized and often bizarre in shape, did not usually contain recognizable material. B cells were more nearly like those of normal synovia, except that they had more rough endoplasmic reticulum, often with dilated cisternae containing homogeneous material. Two synovia contained rough endoplasmic reticulum-bound material with electron-dense central and pale outer areas. Ribosomal stacking was frequent, and ribosomal rosettes were increased. Some B cells contained a few lysosomes. C cells appeared most like B cells, except that well-developed Golgi systems were observed in addition to the endoplasmic reticulum. The overlap between B and C cells was marked because both had filopodia and micropinocytotic vesicles as well as the other features listed below. Fig. 5. Montage of the synovial lining layer in JRA showing tightly packed pleomorphic cells. Necrotic lining cell (DLC) is separating from the surface (S). Lining cells are predominately phagocytic in type, with many primary and secondary lysosomes. Free myeloid membranes (arrows) and accumulations of fibrin (F) are shown. RBC, erythrocyte. x 3000.
margin. The lining layer (Fig. 5) was made up of A (phagocytic), B (synthetic), and C (intermediate) cells. I7 They were increased in numbers, and many were larger than their normal counterparts. The A cells were most numerous, followed by B and then C cells. A cells appeared to be most responsible for the lining cell hyperplasia. Fibrocytes were not observed.lL~‘s At the
Miscellaneous Features of All Cells of the Synovia Nuclei and nucleoli of the lining cells appeared normal. No nuclear inclusions were seen. Non-membrane-bound lipid was frequently observed in A, B, and C cells. Glycogen was noted in four synovia in the A cells and occasionally in the B cells. Myelinlike figures were most often seen in association with mitochondria, especially in cells of the lining layer; rarely they were seen in Golgi lamellae and associated with some phagolysosomes. Sometimes free or extracellular myeloid membranes were present. Mitochondria were more pleomorphic than in
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normal tissue. Round or swollen mitochondria were noted as well as the myeloid figures seen associated with them. One tissue showed mitochondria of low ADP or type IV configuration.‘” The more unusual mitochondria were usually seen in cells of the synovial surfaces. Crystals were not seen in any tissues. Microtubules were occasionally observed, primarily in relationship to centrioles. Microfilaments were seen in lining cells in subintimal and stromal fibroblasts, and in endothelial and smooth muscle cells of blood vessels. Centrioles were seen in all cell types in all synovia. No mitoses were observed ultrastructurally (although they were seen by light microscopy). Occasional free red cells were seen in two synovia. No siderosomes or ferritin particles were identified in synovial lining cell fibrolasts or macrophages. Mast cells, normally seen in healthy synovial membranes, were plentiful. They appeared similar to normal mast cells except that they were not observed to be releasing their fine fibrillar and homogeneous materials as often as normal, and they contained more lipidlike material. In one instance, granules with scroll-like configurations were seen. In another synovium, subintimal B-like cells were seen in close proximity to mast cells, with B-cell processes extending around the mast cells.
Inflammatory Cell Response Plasma cells were most frequently found around vessels (but never within their lumina), in close relationship to subintimal fibroblasts, macrophages, and sometimes A cells. All had typical prominent rough endoplasmic lamellae, and often dilated cisternae; they had the morphologic appearance of active protein synthesis. In most synovia some plasma cells contained Russell bodies. Free Russell bodies were not observed.20 These bodies were dark staining and homogenous and bound by rough endoplasmic reticulum. Paranuclear starlike well-developed Golgi lamellae and vesicles were usually present, especially in mature plasma cells. Mitochondria were plentiful and sometimes large; lipid drops and lysosomes were occasionally seen especially in young cells. Plasma cells appeared to have active surfaces with short filopodia. Binucleate plasma cells were frequently seen and showed all the cytoplasmic
WYNNE-ROBERTS
ET AL
elements recorded above. No Mott cells were observed.” Lymphocytes were the second most commonly seen inflammatory cells. They were found around and in blood vessel walls, in the synovial lining and subintimal areas, and sometimes closely associated with A or B cells. The lymphocytes appeared to migrate through the endothelial cells of the vessels, especially the venules and veins. The lymphocytes in the vessel walls had well-developed filopodia. These lymphocytes often contained many large mitochondria and rough endoplasmic reticulum. Variations in size and shape of nuclei were seen, and nucleoli were often prominent. Larger lymphocytes with prominent nucleoli and variegated nuclei and active cytoplasmic elements were seen. Sometimes these cells were not easy to distinguish from monocytes, but the lymphocytes’ nuclear appearance helped to differentiate the two cell types. Thus lymphocytes more often appeared atypical (Fig. 6) or showed early blastic transformation. Centrioles were present, but mitoses were not observed. Cells with features of both lymphocytes and plasma cells were seen. Such cells had irregular nuclei and well-developed rough endoplasmic reticulum lamellae. Macrophages made up a large segment of the infiltrating cell population and were especially numerous in synovium from patient No. 3. They most closely resembled A cells, with many lysosomes and filopodia. Like A cells, macrophages were very pleomorphic. They contained many primary lysosomes, some of which appeared to be budding off the Golgi lamellae. The lysosomes were small (60-100 nm), dark staining, and membrane bound. Such lysosomes were also seen in the periphery of the cytoplasm of the macrophages. Some macrophages also contained small tubular structures of 22 36 nm outer diameter in their peripheral cytoplasm (Fig. 7A). Mitochondria, both rough and smooth endoplasmic reticula, and highly developed Golgi apparatuses were frequently seen. Some macrophages had features of the monocyte seen in the lumina of blood vessels. The mode of egress from vessel lumen to the tissue was by passage between (as opposed to through) endothelial cells of the vessel wall. Macrophages frequently showed evidence of
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Fig. 6. Parts of five cells. Macrophage (Mac 1) contains a large pleomorphic phagolysosome (PhL) and a small secondary lysosome (L). Lymphocyte (LY) shows early transformation with an irregular nucleus (N) and large mitochondria (M). Long cell process (CP), probably from a macrophage, extends in intimate contact along the lower margin of the lymphocyte. Large macrophage (Mac 2) contains a dark pleomorphic phagolysosome. Small dark granules (arrows) can be seen within the margin of the phagocytosad material. Parts of two other macrophages (Mac 3. Mac 4) are shown. C. collagen fibers; V. vacuoles. x 13,660.
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ET AL
Fig. 7. Two macrophages, one of which is in close contact with a lymphocyte. At points of cell contact desmosomelike junctions occur (arrows). Lysosomes IL), vacuoles P/I, mitochondria (MI. nuclei (N) are shown, x 14.000. Inset: Small lymphocyte and a small portion of a macrophage lie in relation to each other. Section of macrophage contains short tubular structures tarrows) of 22-36-nm outer diameter, with an occasional larger structure of diameter up to 73 nm. Ribosomes of the lymphocyte are about 15 nm. for comparison. x 13,680.
phagocytosis and contained variegated phagolysosomes of medium and giant size (Fig. 6). The material within the phagosomes ranged from unrecognizable, sometimes homogenous material to obvious fibrin, platelets, and plasma cells. The recently ingested plasma cells appeared somewhat necrotic, with clumped nuclear chromatin; these cells were seen in various stages of degradation. The plasma cell breakdown appeared as fragmentation and disorganization of the cells’ contents, with myeloid figure formation and shrinkage of the cell. Of interest, lysosomes were not specifically located in the vicinity of the degrading cells; however, sometimes dark-staining granules were seen around the edge of an ingested cell, most likely representing fusion of primary lysosomes with the phagosome. In other the close juxtaposition of instances, macrophage and plasma cell or lymphocyte was remarkable (Fig. 7). Sometimes a desmosomelike junction was present between the contiguous cells. Both macrophages and A cells
appeared to be by far the most active phagocytic cells in the inflammed synovia. Neutrophils were located both around vessels and scattered in the synovial lining and subintimal areas. Some had ingested fibrin or other material but otherwise appeared normal. They rarely were degranulated. Neutrophils were not numerous; they were occasionally observed to be ingested primarily by A cells of the lining layer. Platelets were seen in all synovia in the lumina of vessels, sometimes plugging gaps in their walls, or just outside them. “Inclusions” Tubularlike structures were seen in three synovia in the cytoplasm of B cells of the lining layer (patients No. I, 3, 14) (Table 1). The mean diameter of these short wavy tubules was 23 nm (Fig. 8). Blood Vessels Each synovium abnormal vessels.
contained both normal Some, notably venules
and and
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I:ig. 8.
SYNOVIUM:
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JRA VS. NORMAL
Part of a 8 cell containing fine wavy structures (arrows) of 23-nm diameter.
Rough endoplasmic
reticulum her 1. nu-
clefus(N), mitochondria (MI indicated. x 21,780.
‘ig. 9. IA) Part of an andothalial cell of a venule showing rod bodies (RI, which are Sean at different tangents of sasction. tochondria (MI. nucleus (N). lumen of vassal (Lu), basal lumina (BL), myeloid figure (My) shown. x 41,040. (8) Regular array nembranes within the rod (RI arranged longitudinally. Rough endoplasmic reticulum her) indicated. x 74,250. (C) App #arant ralopmant of new rod bodies (arrows) by lateral budding from mature rods. x 31,920.
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296
capillaries, seemed to have the most changes. Endothelial cell hypertrophy and hyperplasia were sometimes present. The endothelial cells themselves appeared more active than normal. Large nuclei, well-developed rough endoplasmic reticulum, free ribosomes, and Golgi stacks were present in increased amounts. Mitochondria were plentiful, lysosomes were increased, and occasionally lipid drops were present. Rod bodiesz2 (Fig. 9), seen mainly in venules, were often numerous, especially on the luminal side of the vessel. Seen in longitudinal section they contained regular membranes oriented in the long axis of the rods (Fig. 9B). In cross section the rods contained approximately 8-12 welldefined membrane bound vesicles of 12 nm outer diameter. Rods occasionally showed lateral budding, which was best seen in longitudinal section (Fig. 9C). Endothelial cell microvillus projections into the vessel lumina were plentiful. Micropinocytotic activity was marked on both luminal and basal aspects of the vessels. Smooth muscle cells were hypertrophied and appeared actively synthetic. Increase in vessel wall thickness was principally due to increase in the ad-
Fig.
10.
membrane.
Capillary x 5600.
with
thickened
adventitial
sheath.
Two
ET AL
ventitial sheath, particularly of basal laminae and collagen (Fig. 10). These features were most often seen in veins and venules and capillaries of all synovia, even in those of most recent onset of juvenile rheumatoid synovitis (Table 1). Intercellular Matrix In addition to easily recognized collagen, fibrin often was seen between cells, in vacuoles of A cells, and ingested by neutrophils in the synovial lining and subintimal layers. The fibrin was seldom seen with banding of regular periodicity but generally was of the fine fibrillar kind, with slight beading. There was a considerable increase in intercellular amorphous and fine fibrillar materials. These materials were distinct from fibrin. Effete cells as well as debriding surface cells were occasionally found in all synovia. DISCUSSION
We have croscopic patients (4 of JRA
lymphocytes
studied the light- and electron-miappearances of synovia from 15 males, 11 females), with four forms (8 with monarticular, 1 with
are just
outside
the vessel
wall.
My,
free
myeloid
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SYNOVIUM.
JRA VS. NORMAL
oligoarticular, 4 with polyarticular arthritis, and 2 with Still disease), of age range 2.5516 yr. The findings were compared with synovia from seven patients (two female, five male), of ages 6-20 yr, with no synovial disease. I ’ The age and sex patterns of the two groups were not precisely matched. Females showed a preponderance over males by 2.5:1 in our JRA group; this experience is similar to that of others.‘-” Initially five of our patients (Table 1) did not fulfill the criteria for JRA, but they subsequently did so. Diagnois of JRA required the persistence of arthritis for 3 moor more and the elimination of other causes of arthritis. I3 Comparison of Synovial Membrane Morphology From JRA Within the Group of I2 Patients and With That of Adult RA The light-microscopic histology of the synovia from the different types of expression of JRA within our group were compared, after coding the tissues as unknown, and we were unable to discern any differences. Neither the clinical severity nor the duration of disease significantly affected the histology. The same procedure was adopted for comparison with a number of slides from patients with adult RA or with osteoarthritis; it was not possible to distinguish consistently thejuvenile from the adult form of RA. The greatest variations found in the synovial and inflammatory responses were within the tissues themselves, between one area and another, rather than from among different patients, Focal synovitis was a marked feature. This experience was similar to that recorded by Bywaters and Ansell.” Comparison With Reports of JRA Synovial Ultrastructure Borrachero et al.,s in their study of synovia from two children with Still disease, noted hyperplasia of the synovial lining layer and a marked plasma cell and lymphocytic response. Bierther and Schafer9 studied four children with JRA and recorded endothelial cell damage of capillaries and postcapillary vessels, a neutrophil response, and increase in mast cells. They did not comment on plasma cell, lymphocyte, or macrophage infiltration. The duration of disease in their four patients was not stated, but the predominance of neutrophils in the inflammatory response contrasted with our
297
findings of neutrophils being a lesser part of the response. Clawson et al.‘* reported an inflammatory and synovial lining cell response similar to ours. Vascular changes with endothelial swelling, necrosis, and scarring were seen. We have not observed necrosis or scarring in our cases. Schumacher and Kitridou’ studied synovitis of recent onset and included three patients with JRA. They noted that plasma cells and lymphoid cells were rare in early JRA or RA, but no particular differences between the two groups were recorded. In our series we had only one patient whose onset of disease was less than 1 mo prior to synovial biopsy (No. 3, Table 1). In this instance, we observed a chronic inflammatory response with marked macrophage activity, many plasma cells, and lymphocytes. In our other four patients with JRA of up to 2 mo duration plasma cell response was likewise established. Assessment of the Synovial and Inflammatory Responses in the Synovitis of 15 Patients With JRA and Comparison With 7 Control Synovia The inflammatory abnormalities seen in juvenile rheumatoid synovitis may be divided into two parts, those involving the cells indigenous to the synovium and those relating to the immigrant cell response. The morphologic response in JRA synovium is compared diagrammatically with normal synovial structure (Fig. 11). The striking differences between the inflammed and normal synovia were seen in all areas of the joint lining. The well-defined surface of the normal synovial lining layer was lost, being replaced by fibrin, sloughing cells, and neutrophils. The effete phagocytic cells, including neutrophils, most likely release lysosomal enzymes into the joint cavity that in turn could contribute to cartilage damage.23 The hypertrophy and mild hyperplasia of the lining cells suggested increased activities in response in part to unknown stimuli and in part to their marked phagocytic activity. Furthermore, their altered appearance with increased lipid and glycogen indicated changes in metabolism compared with normal. Increased anaerobic glycolysis and lactate production have been reportedz4 and enhanced oxygen uptake recorded in inflammed synovia.2” However, these observations are not unique to synovia;
298
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Joint Space
Joint Space
Venule
Fig. 11. (A) Diagrammatic concept of the structure of normal juvenile synovium. Fibrocvte IFc), phagocytic cell (A), synthetic cell (B), intermediate cell (CL amorphous material (AM). granulated mast cell, fibroblast (FL collagen (CL capillary (Cap). and a venule are shown. Interrupted lines from the vessel lumina to the synovial surface represent the sources and general direction of movement of fluid to which lining cells add hyaluronate, yielding highly viscous synovial fluid (SF). (B) Diagrammatic concept of the synovial and inflammatory responses in JRA. Synovial response consists of increase in numbers of phagocytic surface cells (A) and disappearance of fibrocytes (Fc). Synthetic cell (B). intermediate cell (CL debriding lining cell fdlc), lysosomes (Ly), fibroblast (F), collagen (C), and mast cells are shown. There is an increase in amorphous materials (AM). The immigrant inflammatory response is represented by fibrin (Fib), polymorphonuclear neutrophils (PMNL macrophages (ML lymphocytes (L). plasma cells (P), platelets (PI), and transformed lymphocytes (TL). Blood vessels show endothelial hyperplasia and thickened adventitial sheathes. Synovial fluid (SF) is formed as in normal synovium, except in increased amounts; to it are added some of the products of inflammation and increased amounts of hvaluronete
increased glycolysis has been noted in other inflammatory responses.‘6 The preponderance of A cells with many lysosomes in JRA contrasted with the B-cell predominance of the healthy tissues. This conversion of the synovial lining cells from mostly smaller synthetic-appearing ones to large active and predominantly phagocytic cells was remarkable. The possible origins of the increased numbers of A cells in the absence of increased mitoses may be speculated on. The fibrocytes”,‘* observed in normal synovia were no longer found; perhaps in the inflammatory state these cells may be the ones that differentiate into A cells. Alternatively, A cells may arise from C cells. Thirdly, some are possibly immigrant macrophages that in the lining layer become indistinguishable from indigenous A cells.
The tremendous increase in lysosomes and phagocytic activity seen in both A cells and immigrant macrophages was probably partly induced by the presence of increased numbers of effete cells, fibrin, and other intercellular materials such as immune complexes. This process would be in keeping with the mechanism of removal of unwanted biologic materials described by Axline and Cohn.“’ B cells were larger than normal and showed much more endoplasmic reticulum with dilated cisternae. Possibly these cells were secreting more hyaluronate and other mucopolysaccharides into the adjacent matrix and the synovial effusion accompanying the synovitis.” Two rheumatoid tissues contained much electrondense amorphous material similar to that recorded in tissue-cultured fibroblasts derived
JUVENILE
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from a patient with JRA who had a high hyaluronate synthesis rate and similar to material noted by others. 2g The significance of the unusual appearance of the rough endoplasmic reticulum contents is unknown but may be due partly to the effects of tissue processing. Of particular interest were the blood vessel changes, especially endothelial proliferation, evidence of increased cytoplasmic activities and adventitial sheath hypertrophy. These represented changes due to stimulation of the blood vessels that apparently take part in the overall hyperactivity of the synovium. Focal necrosis of part of a vessel wall or of whole vessels, with or without thrombosis, was not seen in our sampling. Despite the perivascular location of the invasive inflammatory response and of evidence of migration of inflammatory cells through the vessel walls, morphologic evidence of damage to the vessels, that is, a vasculitis, was not present. Vasculitis has been considered in adult RA by Goldie and co-workers,““~3’ who noted abnormalities by light microscopy. No firm conclusions were drawn by these authors about the role of a vasculopathy in RA. However, Marin et al.“” found vasculitis as a primary lesion; certainly in more malignant forms of RA, vasculitis may be a very prominent feature. The immigrant inflammatory cell response consisted of neutrophils, macrophages, and lymphocytes. The role of the neutrophil appeared to be that of scavenger in and on the surface of the synovial lining layer, whereas the macrophage phagocytized cells and debris both in the lining and subintimal areas. The frequent intimate relationship of macrophage and plasma cell or lymphocyte raised the question of passage of informational or other material between the intimate cells. The role of the macrophage in immunity has been widely explored and it has been shown that macrophages can ingest some organisms and can excrete antigens derived from the ingested organisms.33 The monocyte-macrophage response in JRA is a prominent feature of the inflammatory response. Perhaps this response has an important role to play in processing and modifying an antigen. The continual production of antigenic stimuli is suggested by the chronicity of the active inflammatory response. We may speculate on whether or not the
299
phagocytic behavior of lining A cells (matching that of macrophages) is accompanied by an ability on the part of A cells to “process” some infectious agents and produce antigen in a way similar to that documented for macrophages.33 Such an adaptation could be an important part of the synovitis of JRA. Our impression of marked increase of lysosomes in the inflammed synovial tissue was based only on morphologic observation because we have not specifically measured lysosomal increase using an enzyme marker. Our observations are similar to those who have studied the synovitis of adult RA. 34*35Primary lysosomes were seldom seen in the vicinity of phagosomes; the small dense granules lining membranebound material suggested that fusion of primary lysosomes with phagosomes occurred with the formation of secondary phagolysosomes. Autophagic vacuoles, residual bodies, and extracellular free membranes were all seen, indicating that the sequence of events of digestion of materials documented by deDuve and Whattiaux had taken place.“” Several authors have specifically studied the plasma cell component of the inflammatory response.2”~97~38 Curtiss noted in a review”!’ that plasma cell response does not occur early in the synovitis of RA but is established after 4-5 mo of active disease. We saw a well-established response of plasma cells and lymphocytes in our early cases of JRA (Table I). This is in keeping with the early findings of immunologically induced arthritis in rabbits.40 Furthermore, it has been shown that IgM and then IgG antibodies occur in response to a specific antigenic stimulus within 14 days from primary immunization by the antigen. 41 Therefore any primary antigenic stimulus (or stimuli) in RA or JA should attract antibody-forming cells, and antibodies might be expected to be produced within 3 wk of the onset of disease. Some have commented on the presence of Russell bodies, which have been shown to contain gamma globulins, within plasma cells.*“~~” We, too, have observed Russell bodies, but never large nor found outside cells.“’ In the tissues of our study, as in those of adult RA, all plasma cells appeared morphologically to be very active in protein synthesis, as indicated by well-developed ergastoplasm and Golgi systems. While we observed frequent close rela-
300
tionship of plasma cells to either A cells or macrophages and ingestion of plasma cells by phagocytes, only one other author has recorded plasma cell phagocytosis by histiocytes,J2 but others have noted the close juxtaposition of the two cell types.20,43 We may speculate on why plasma cells were ingested. ‘It may simply be to remove old or effete cells, because the life span of some plasma cells is relatively short,44 about 1 wk, or perhaps selected plasma cells are being removed from the inflammatory scene by phagocytosis because their products are injurious. Binucleate (telophase) plasma cells were noted frequently in the synovitides, which suggested that replication of plasma cells occurred. Binucleate and multinucleate plasma cells have been reported in other inflammatory situations.44 Our findings in JRA agree with those of others4” that plasma cells dominate the inflammatory responseof RA. Lymphocytes, like plasma cells, were observed in close juxtaposition with phagocytic cells; however, ingested lymphocytes were not detected. Three explanations can be considered for this latter observation. First, most lymphocytes survive longer than plasma cells; second, perhaps many were transformed into plasma cells; third, some lymphocytes may reenter the circu1ation.l” We noted that lymphocytes once outside the circulation often appeared atypical and that some were similar to those described as transformed.“X We did not observe mitoses among the lymphocyte series, although in vitro they have been reported to occur 667 days after phytohemagglutinin stimulation.“’ That the small lymphocyte was most likely the source of plasma cells was supported by our never visualizing intraluminal plasma cells and by our seeing all intermediate stages of transformation between lymphocyte and plasma cell. This behavior of many lymphocytes therefore placed them in the B (bone marrow) dependent category of the immune defense were not distinguished system. 48 T lymphocytes from untransformed B lymphocytes. The question of an infectious agent or agents as the possible cause(s) of RA has been raised by many; and we have raised it with respect to the etiology of JRA. Neumark and Farkas have consistently reported nuclear particles that they considered to be viral.4g In 1973 Neumark et
WYNNE-ROBERTS
ET AL
al.“” noted cytoplasmic viral particles in 42 rheumatoid patients and not in 17 nonrheumatoid controls. We have carefully studied their work and reviewed our synovia for similar particles but have not seen them. Neumark et al. stated specifically that they were able to distinguish the particles of size 120-220 nm from small lysosomes. We have seen membrane-bound dark-staining particles predominantly in phagocytes. They appeared to be derived from Golgi systems, and therefore we thought them most likely primary lysosomes. Morphologically these lysosomes appeared quite similar to elementary bodies observed by Schumacher and Kitridou7 in the cytoplasm of a perivascular cell from a patient with transient synovitis. Clearly, it is extremely difficult to distinguish single viruses or certain mycoplasmal organisms from normal cell organelles in some instances, in contrast to viral particles that form lattices. Rod bodies have previously been considered abnormalZZ and possibly an infective agent but most likely a normal component of at least some small blood vessels.“’ Rod bodies were more plentiful than normal, and it was interesting to note that they appeared to replicate by budding. Their functional significance is not known. Tubularlike structures were seen by Helder et al.,“2 and we saw similar structures in three synovia. Certainly these structures were reminiscent of some viral configurations. They were unlike the lacey tubular structures seen in the endothelial cells of blood vessels of patients with systemic lupus erthematosus or polymyositis.“’ Interestingly, the morphologic appearance of the tubular structures seen by us had staining characteristics suggesting they comprised primarily membrane material. The tubules of about 30-nm outer diameter frequently seen in the peripheral cytoplasm of macrophages were longer than the wavy 23-nm structures seen in synovial lining cells and may have been an abnormal component of the macrophage because we have found no documentation of these tubuIes in other situations. The tubules just discussed appeared different from the microtubules of 1526-nm diameter seen as a normal cytoplasmic component of all cells. The presence of iron in rheumatoid synovia
JUVENILE
SYNOVIUM:
JRA VS. NORMAL
301
has been extensively reported by Muirden in his studies of chronic anemia of RA. Two of our patients were anemic, and four of the group had active arthritis for more than 4 yr. Prussian blue staining for iron was present by light microscopy, but neither siderosomes nor ferritin granules were seen ultrastructurally in any cells, This negative result represents a problem
of sampling, since sequestration occurred in those with JRA.
of iron clearly
ACKNOWLEDGMENT
Dr. J. Cassidy of the Rackham
Arthritis Research Unit, of Michigan, Ann Arbor, kindly reviewed the manuscript. Dr. W. Green and Dr. H. Swensen of the Orthopedic Department of Presbyterian University Hospital, University of Pittsburgh, kindly obtained some of the synovia for our study. University
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