Evidence for the presence of receptors for c3 and IgG Fc on human synovial cells.

1
ARTHRITIS
&
RHEUMATISM
OFFICIAL J O U R N A L OF T H E AMERICAN RHEUMATISM ASSOCIATION
SECTION OF T H E A R T H R I T I S F O U N D A T I O N
EVIDENCE FOR THE PRESENCE OF RECEPTORS
FOR C3 AND IgG Fc ON HUMAN SYNOVIAL CELLS
ARGYRIOS N. THEOFILOPOULOS, DENNIS A. CARSON, MEHDI TAVASSOLI, SUSAN F. SLOVIN,
WENDELL C. SPEERS, FRED B. JENSEN,and JOHN H. VAUGHAN
The presence of receptors for IgC Fc and fragments of C3 on primary cultures and cryostat sections
of normal and rheumatoid synovial tissues was assessed.
Significant proportions of large rounded cells with asteroid projections found in such cultures had receptors for
both IgC Fc and fragments of C3. Moreover, Cram negative bacteria that had fixed complement, but not EAC,
bound in a linear fashion on the superficial layers of synovial cryostat sections. On the basis of morphologic
and histochemical criteria, the cultured cells bearing
these receptors were tentatively determined to represent a subset of synovial lining cells. The possible role of
such receptors on synovial lining cells in the pathogenesis of rheumatoid arthritis is discussed.
From the Departments of Immunopathology, Clinical Immunology and Cellular and Developmental Immunology, Scripps
Clinic and Research Foundation, La Jolla, California.
Supported in part by NIH grants AM-21175, AM-25443,
AM-255 10, and AI-07007, and DOE contract DE-AS03-79EV00899.
Argyrios N. Theofilopoulos, MD: Associate Member, Department of Immunopathology (recipient of Research Career Development Award CA-00303); Dennis A. Carson, MD: Assistant Member 11, Department of Clinical Research Mehdi Tavassoli, MD:
Assistant Member 11, Department of Clinical Research; Susan F. Slovin, PhD: Research Fellow, Department of Clinical Research; Wendell C. Speers, MD: Research Fellow, Department of Immunopathology; Fred C. Jensen, DVM: Associate Member, Department of
Cellular and Developmental Immunology; John H. Vaughan, MD:
Member, Department of Clinical Research.
Address reprints to Argyrios N. Theofilopoulos, MD, Department of Immunopathology, Scripps Clinic Research Foundation,
10666 North Torrey Pines Road, La Jolla, California 92037.
Submitted for publication July 10, 1979; accepted in revised
form August 27, 1979.
Arthritis and Rheumatism, Vol. 23, No. 1 (January 1980)
The light and electron microscopic appearance
of human normal, osteoarthritic, and rheumatoid synovia has been described by various authors (14). In
general, these authors have described three types of synovial lining cells which have been classified as types A,
B, and C. The type A cell was described as a large
phagocytic cell, the type B as a large nonphagocytic cell,
and the type C as a small phagocytic cell which may
represent a monocyte-derived macrophage rather than a
true synovial lining cell.
Although there are several studies on the surface
markers of the human synovial infiltrating cells as well
as of the mononuclear cells in synovial fluids of rheumatoid patients (5-9), there is little information about
the types of surface receptors on human synovial lining
cells. In view of the role of cell surface receptors for IgG
Fc and C3 on cell-cell interactions and cell activation or
inactivation (lo), we decided to examine the presence of
such surface receptors on cells derived from primary
cultures of human synovial membranes. Our results indicate that the majority of large cells with finger-like
projections in such cultures do carry receptors for fragments of C3 and for IgG Fc and that based on histochemica1 and other criteria, these cells appear to be synovial
lining cells. The possible role of such receptors in the
pathogenesis of rheumatoid arthritis is presented.
MATERIALS AND METHODS
Synovial tissues. Synovial tissues from 3 patients with
rheumatoid arthritis, 2 patients with osteoarthritis, and 2 normal persons with traumatic arthritis provided the source for
THEOFILOPOULOS ET AL
2
Figure 1. Monolayers of cultured synovial cells stained with Wright-Giemsa. Elongated and rounded cells with fine projections are
noted (A, x 250; B, x 400).
synovial cells. Minced synovial tissues were suspended in 50
ml of minimum essential medium (Eagle’s MEM) containing
1,500 units of clostridial collagenase (Sigma) and 0.25% trypsin and were incubated under constant stirring at 37OC for 1
hour. Cells isolated from the digested synovium were washed
3 times with the medium supplemented with 10% fetal bovine
serum to inhibit digestion, then washed twice, resuspended to
1 X lo6 cells/ml and distributed into 60 mm Petri dishes. In
certain dishes sterile cover slips were added at the start of the
culture. Cell cultures were examined daily under the phase inverted microscope. Within 6-7 days the cells became confluent and at that time they were harvested and used in the experiments reported in the following section.
Frozen tissue sections of 6-8p thickness from normal
and rheumatoid synovia were obtained by use of a microtome,
mounted on slides, and stored at -70°C.
Cell surface receptor identification. Cultured cells
were examined for various receptors either as monolayers on
coverslips or as suspension after nonattached cells were
washed out and carefully scraped off. Coverslips were washed
3 times with medium, mounted on slides, and overlaid with either a suspension of sheep red blood cells (SRBC) sensitized
with IgM rabbit hemolysin and mouse complement (EAC) or
with red blood cells sensitized with IgG rabbit anti-ox red
blood cell antibody (EA). B lO.D,/old mouse serum was used
as a source of complement to form EAC. These intermediates
are immune adherence negative and therefore they detect primarily C3d receptors (1 1,12). Methods of preparation of those
reagents and of the IgG ox cells have been described elsewhere (1 1-13). After incubation in a humidified box, the coverslips were washed 3 times with large volumes of medium to
remove nonattached red cells and mounted on slides under
glycerogel. The cells were examined under a phase contrast
microscope and the percentage of rosette forming cells was recorded. Controls included cells incubated with nonsensitized
SRBC or ox red blood cells. Cultured synovial cells in suspension were examined for rosetting with neuraminidasetreated SRBC and for the presence of surface bound Ig by
use of a polyvalent fluorescein isothiocyanate (FITC) conjugated rabbit antiserum to human immunoglobulins (Cappel
Laboratories) as described (14). Synovial cells in suspensions
were also first reacted with FITC anti-human Ig antibody at
4°C for 30 minutes and after 3 washes they were incubated
with EAC or EA to determine if rosette forming cells had Ig
on their surface. On occasion the presence of C3 receptors on
cultured synovial cells in suspension or monolayers was determined using FITC conjugated Salmonella typhii which had reacted with fresh human serum as a complement source (15).
The human serum used in these experiments as a complement
source had been prescreened for Salmonella febrile agglutinins, group D, by bentonite flocculation. Only a serum with
undetectable agglutinins was used. Soluble human C3b and
F(ab’)* fragments of an FITC-rabbit anti-human C3 antibody
were prepared as described and binding of soluble C3b to cultured synovial cells in suspension was examined by immunofluorescence (14).
RECEPTORS FOR C3 AND IgG Fc
3
Figure 2. IgG Fc (A) and C3 receptors (B, C) on cultured synovial cells (A,
Frozen synovial tissue section studies. Frozen synovial tissue sections, 6 to 8p thick, on glass slides, were overlaid
with fluorescein (F1TC)-conjugated Salmonella typhii that had
fixed human complement (5 X lo9 particles/ml in tissue culture media with 5% bovine serum albumin to reduce nonspecific binding). After incubation at room temperature for 20
minutes, the slides were dipped vigorously in phosphate-buffered saline to wash free nonspecifically bound bacteria, and
the tissue was then examined as a buffered glycerol wet mount
under phase and fluorescence light microscopy. Control preparations were similarly incubated with nonopsonized bacteria.
Cytochemistry and histochemistry. Cells grown on
coverslips were used for cytochemical studies. They were fixed
before or after rosetting with EAC using formalacetone (for
acid phosphatase studies, methanolacetone was used). For
histochemistry, unfixed cryostat sections of the synovial tissues were used. They were stained with Wright-Giemsa, Sudan Black B, peroxidase, periodic acid-Schiff (PAS), alkaline
phosphatase, and pyronine. Acid phosphatase staining was
carried out according to a modified method of Barka and Anderson by use of naphthol AS-BI phosphate and hexagotized
pararosanilin (16). Tartrate inhibition of acid phosphatase
was carried out by adding 0.02M liter (+) tartrate to the staining solution. Esterase reactions were carried out according to
X
630; B,
X
400; C, x 630).
the method of Li et a1 (17) with a naphthyl butyrate (for nonspecific esterase, NSE) and naphthol AS D-chloracetate (chloracetate esterase, CAE) as substrates and freshly made hexaxotized new fuschin or pararoseaniline, respectively, as couplers. Staining for acid mucopolysaccharides was done using
1% alcian blue 86 times in 3% acetic acid (18).
Electron microscopic studies. Synovial cells on plastic
substratum were rosetted with EAC as described previously.
Modified Karnovsky’s fixative was used initially, followed by
2% O,O, in 0.125M cacodylate buffer after fixation, staining
with saturaged thiocarbohydrazide, and 2% 0,04 fixation,
again. Staining with 4% uranyl acetate was performed after
partial dehydration to 30% alcohol. After complete dehydration the monolayer was embedded in Epon. Perpendicular
sections were mounted on coated grids and then stained with
2% uranyl acetate and 1% lead citrate.
RESULTS
Morphology of cultures. In the primary cultures
and at the sixth to seventh day, the synovial cells were
of three types: rounded large cells with some asteroid
projections, spindle-like cells, and a variable percent of
Figure 3. Cultured synovial cells with fluorescinated attached Gram negative bacteria that had fixed complement (A,
400. B. x 630).
X
THEOFILOPOULOS ET AL
4
Figure 4. Electron micrographs of cultured synovial cells with attached EAC.
fibroblast-like cells (Figure lA, B). The rounded cells
with projections comprised about 40-50% of the total
population as estimated by phase microscopy. Occasionally, these rounded cells clustered into groups but most
often they were single or in pairs. There were no apparent morphologic differences between the synovial cell
cultures derived from normal, osteoarthritic, or rheumatoid individuals. However, rheumatoid synovial cultures
appear to have a higher proliferative capacity and exhibit higher cellularity. Subcultured synovial cells at the
fifteenth subpassage appear elongated with many projections, an appearance similar to that described by
Smith (19).
Cell surface receptors. Monolayers of rheumatoid, osteoarthritic, and normal synovial cells exposed to
EAC or EA showed almost no attachment (only 2-5%
rosetting cells) to spindle-like cells or fibroblasts. In
contrast, over 85% of the rounded cells with asteroid
projections formed rosettes with both types of the sensitized cells (Figure 2A, B, C) as well as with Gram negative bacteria that had fixed complement (Figure 3A, B).
Interiorized EA or EAC were se'en very frequently. The
percentage of synovial celis in suspension obtained from
similar monolayer cultures forming rosettes with EAC
and EA was similar (50-60%) to the percentage of
rounded cells seen in monolayers. There was no significant difference in the percentage of cells expressing C3
and IgG Fc receptors among synovial cells derived from
normal controls, osteoarthritic or rheumatoid patients. No cells were found to form rosettes with control
nonsensitized SRBC or ox red blood cells. Approximately 2-6% of detached synovial cells obtained from 6
to 7 day monolayers were found to bear surface Ig.
However, these Ig positive cells did not form rosettes
with EAC or EA, a finding which suggested that they
were B cells in an advanced maturational stage. When
synovial cells in suspension were incubated with soluble
isolated human C3b or inulin-activated human serum,
35-45% of cells in all cultures studied were found to be
surface positive when stained with FITC anti-human
C3 antibody. Control cells incubated with medium, instead of C3b, were negative when reacted with the
above antiserum.
Electron microscopic studies. By transmission
electron microscopy the rosetted synoviocytes resembled type A cells of human synovium and the macrophage-like cells of human fetal synovium and rabbit
synovium (20-22). The most characteristic feature was
the numerous delicate filopodia that extended to contact
and finally engulf the erythrocytes (Figure 4A, B).
Points of actual contact between the erythrocyte and
synoviocyte membranes were relatively small and infrequent until the red cell had become completely internalized. A second feature was the abundance of lucent
vacuoles, occasionally more than a micron in diameter,
immediately beneath the plasma membrane, especially
in areas with numerous filopodia. A third common feature was the phagocytic vacuoles; these were occasionally very large and often contained obvious partially degraded cellular debris. The nuclei of the rosetted cells
were often deeply indented, but other ultrastructural
features were not particularly characteristic. Many rosetted and fibroblast-like nonrosetted cells contained
numerous lipid inclusions approximately 1p in diameter. Most of the rosetted cells had a generally spherical
configuration; however, two very elongated cells with
RECEPTORS FOR C3 AND IgG Fc
5
Table 1. IgG Fc and C3 receptors and histochemical characteristics of synovial cells
Sections
(superficial
layers)
Rounded cells
(cultured
monolayers)
(40-50%)
EA
EAC
C-fixing
Gram neg
bacteria
Acid
phosphatase
Neg
Neg
+++
+++
Neg
+
+
Neg
Neg
Neg
Neg
+++
+++
+++
+++
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Alkaline
Nonspecific
phosphatase
esterase
numerous attached and engulfed red cells were seen. In
other respects these cells resembled the spherical cells.
This suggests that some of the elongated “fibroblastlike” cells in the cultures were in fact derived from the
rounded cells.
Cytochemistry and histochemistry. These studies were carried out to further characterize the IgG Fc
and C3 receptor-bearing cells. The results are summarized in Table 1. The EAC and EA rosetting normal
and rheumatoid cultured cells were equally strongly
stained for acid phosphatase (Figure 5). The upper layers of synovial tissue cryostat sections were also stained
strongly for acid phosphatase (Figure 6A,B,C) but the
intensity of reaction was noticeably higher in synovial
sections from rheumatoid arthritis patients as compared
to normal controls. The acid phosphatase activity was
almost totally inhibited by L-tartrate. Although the covering layer of the synovial tissue showed some positive
reaction for nonspecific esterase, the cultured cells were
completely negative. Similarly, some pyroninophilia
was noted in tissue cells, whereas this was absent in cultured cells. However, the upper layers of the tissue sections as well as the cultured cells were negative when
stained with peroxidase, chloracetate-reacting esterase,
PAS, alkaline phosphatase, Sudan black B, and alcian
blue.
When cryostate synovial sections from normal
and rheumatoid patients were incubated with FITCconjugated Salmonella typhii that had fixed human
complement and were then examined under the fluorescent microscope, attachment of the bacteria along the
upper layer of the section in a discrete linear pattern
was observed (Figure 7A,B). No such attachment was
seen in sections incubated with Salmonella typhii without fixed complement or with EAC.
DISCUSSION
In this report we describe the presence of IgG Fc
and C3 receptors on the surface of a portion of cells cul-
Pyroninophilia
Sudan
black
B
Peroxidase
PAS
Alcian
blue
tured from normal and rheumatoid synovia. The receptor-bearing cultured cells had characteristics similar to
the type A lining cells as indicated by cytochemical and
electron microscopic studies.
Extensive studies have been conducted by others
on the surface markers characteristic of cells infiltrating
rheumatoid synovia and of mononuclear cells in synovial fluids. These studies have indicated the presence of
monocyte-derived macrophages, lymphoblasts, and lining cells in synovial fluids and the predominance of T
Figure 5. A cultured synovial cell, EAC-rosetted and stained for acid
phosphatase. The nucleus (N)is identified. Perinuclear area is heavily
stained with acid phosphatase containing granules (Cyt) indicating
the presence of abundant lysosomes. Two EAC rosettes are identified
intracellularly (arrows) and the cytoplasm of the synovial cell in these
areas is particularly rich in lysosomal acid phosphatase ( X 1500).
THEOFILOPOULOS ET AL
6
Figure 6. Cryostat section of a normal (A) and a rheumatoid synovium (B and C) stained for acid phosphatase. Note the intense staining at the lining areas.
cells in the lymphocytic infiltrates of synovial tissues in
rheumatoid arthritis (5-9). Several studies have been focused on the light and electron microscopic appearance
of synovial cells ( 1 4 ) and in one study the authors examined the presence of IgG Fc and C3 receptors on cultures of rabbit synovial tissues (21). These authors
found that in rabbit synovial explants about 10% of the
cells were round and formed rosettes with IgG and C3
markers, whereas the remaining cells were stellate and
had no receptors for IgG and C3. The results obtained
with rabbit synovial cells are similar to our results with
human synovial cells, the only difference being that the
proportion of rounded cells bearing these receptors was
much higher in human synovial cultures than the reported percentages in rabbit synovial cultures.
Our electron microscopic studies have indicated
the resemblance of the cultured rosetting synoviocytes
with type A cells of human synovium (2) and the macrophage-like cells of human fetal synovium (20) and
rabbit synovium (21). Furthermore, the mesothelial nature of the rounded cells bearing the IgG Fc and C3 re-
ceptors was demonstrated using cytochemical procedures. Thus, these cells with Wright-Giemsa staining
had mesothelial appearance and were positively stained
for acid phosphatase, indicating the presence of abundant lysosomes and thereby establishing their macrophage-like nature. However, these cultured cells were
negative for peroxidase, alkaline phosphatase, esterases,
and PAS, arguing against their granulocytic, monocytic,
lymphocytic, or endothelial origin. Additionally, these
cells were not noticeably pyroninophilic nor sudanophdic thus making it unlikely that they were RNA enriched
cells, plasma cells, or fat cells. On the other hand, the
rosetting cultured cells appeared to be identical to some
of the lining cells since, like the former cells, the lining
cells in synovial cryostat sections were also found to be
strongly positive for acid phosphatase and Gram negative bacteria with fixed complement attached to the lining areas in a discrete linear pattern. The stronger intensity of staining for acid phosphatase observed with
rheumatoid synovial tissues over that with normal synovium can be attributed to the uptake of rheumatoid fac-
Figure 7. Cryostat section of a normal synovium with attachment on the lining areas of fluorescinated Gram negative bacteria
that had fixed complement as viewed by fluorescence (A) and phase (B) microscopy.
RECEPTORS FOR C3 AND IgG Fc
tors and immune complexes in vivo which might have
induced the enzyme. The almost total inhibition of the
acid phosphatase activity by L-tartrate further distinguishes these cells from such bone-associated macrophages as osteoclasts in which the acid phosphatase is
tartrate-resistant (23). Also, the positive staining with
nonspecific esterase suggests the presence of some
monocyte-macrophages (possibly type C cells) in the superficial layers of synovial tissues.
It should be noted that in contrast to the linear
attachment of Gram negative bacteria that had fixed
complement on the superficial layers of synovial cryostat sections, no attachment of EAC was observed. This
result suggests that either the binding of complementfixing bacteria was not mediated via true C3 receptors
or that the spatial arrangement and density of the C3
fragments on the bacteria were more conducive to their
interaction with cellular C3 receptors than that on EAC.
Furthermore, since Gram negative bacteria activate the
alternative complement pathway whereas IgM antibody-sensitized sheep red blood cells activate the classic
complement pathway, the former indicator particles
may carry, apart from the C3 fragments, additional
complement components not present on EAC. These
components may stabilize the C3 fragments (24) or
play a role in the interaction of bacteria with synovial
lining cells. It is also of importance to note that Traycoff
et a1 (8) found that in contrast to a large percentage
(> 80%) of sudanophilic cells that were considered to
represent monocytes and monocyte-derived macrophages, a very small proportion (3%) of cells considered
to be true lining cells derived from collagenase digested
fresh human synovial membranes did form rosettes with
EAC. However, as stated previously in our study with
primary cultures of synovial cells, the EAC rosetting
cells were not sudanophilic, thus making it unlikely that
they were infiltrating macrophages similar to those described by Traycoff et al (8). Possibly, in our study we
observed high frequency of EAC-rosette forming cells
either because of an expansion of the complement-receptor bearing cell population during in vitro culturing
or because we used EAC and Gram negative bacteria
heavily coated with complement. It is known that EAC
rosetting is linearly related to the amount of complement fixed on the indicator particles (10). At any rate,
further work is needed to determine with certainty if indeed synovial lining cells have in situ IgG Fc and IC receptors.
The presence of IgG Fc and C3 receptors in synovial lining cells would certainly have much impor-
7
tance in mediating phagocytosis of both complement
fixing and noncomplement fixing complexes. It is
known that synovial tissues of rheumatoid patients are
abundant in Ig and complement deposits (25,26) and
that synovial fluids contain aggregates of Ig and complement, rheumatoid factor-gamma globulin complexes
capable of fixing C lq, nuclear antigen-antibody complexes, low complement levels, and high levels of breakdown products ( C ~ CC3d)
,
of the third complement
component (27-29). Phagocytosis of particulate immune complexes has been shown to be mediated by IgG
Fc receptors, and complement promotes attachment of
immune complexes to cells (30, 31). Moreover, recent
studies have shown phagocytosis of soluble immune
complexes via C3 receptors without the necessity of Fc
receptor participation (32). It cannot be concluded from
the present study whether the C3 receptors observed on
synovial cells are CR1 (C3b, immune adherence receptors), CR2 (C3d receptors), or CR3 (C3bi receptors)
since the binding of soluble C3b described can be mediated via the fixation of the C3d portion of the molecule
on the C3d receptors (10,33,34). Inhibition studies with
isolated fragments of C3 and various types of EAC are
needed to answer this question. At any rate, immune
complexes formed in the synovial fluids of rheumatoid
patients may bind to Fc and C3 receptors of lining cells,
thereby being phagocytosed. The result would be release of granules and lysosomal enzymes. Similar release of lysosomal enzymes may occur after interaction
of macrophage-like cells with soluble C3 breakdown
products (35). Such enzymes in turn would cause the
cleavage of fluid phase C3, resulting in the production
of C3b which could then bind to the lining cells and
stimulate release of enzymes, thus leading to chronic inflammation. This concept is similar to that advanced by
Schorlemmer et a1 (35) for explaining the role in inflammatory processes of the activation of macrophages by
soluble C3b.
Additionally, in view of the recent findings by
Alspaugh et a1 (36) of a high frequency in rheumatoid
arthritis of antibodies to Epstein-Barr virus (EBV) induced nuclear antigens and the findings of Jondal and
coworkers (37) indicating that the receptors for C3 are
closely associated or identical to receptors for EBV, synovial lining cells with C3 receptors may be a susceptible target for EBV infection. Experiments in our laboratory are aiming at further determining the true nature
of the Fc and C3 receptor bearing cells, in examining
the biologic effects of immune complex interaction with
cultured synovial cells, and in assessing the presence of
THEOFILOPOULOS ET AL
8
EBV antigens on synovial lining cells from rheumatoid
patients and the susceptibility of these cells to EBV infection.
ACKNOWLEDGMENTS
The excellent editorial and secretarial assistance of Ms
Phyllis Minick and Ms Lorene Masewicz is gratefully acknowledged.
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ARA National Scientific Meeting May 28-31, Hyatt Regency Hotel, Atlanta, Georgia
Information o n registration, hotel reservations, and the various sessions t o be held during the
week of the National Scientific Meetings of the American Rheumatism Association will be available in March and will be sent t o all ARA members. Non-members may write t o the ARA Assistant
Executive Secretary, Angel Fortenberry, for information.
Specific details on times, topics, and speakers for the ARA Workshops and Postgraduate Clinical
Seminars will be announced in future issues of Arthritis and Rheumatism.