Clin. exp. Imniunol. (1977) 28, 474-483.
Membrane characteristics of adherent cells dissociated from rheumatoid synovial tissue T. G. ABRAHAMSEN, P. M. JOHNSON* & J. B. NATVIG Institute oj Immunology and Rheumatology, Rikshospitalet University Hospital, Oslo, Norway
(Received 7 January 1977) SUMMARY
Synovial tissues from rheumatoid-arthritis patients were dissociated by enzymes and the resulting cells incubated overnight in tissue-culture flasks. The adherent cell population was resuspended with EDTA-trypsin, and morphological examination showed 68-80% non-lymphoid cells, most of which had the appearance of synovial lining cells. The proportion of these cells increased during subsequent culture. Between 40 and 60% of the cells exhibited marked phagocytosis, and < 14% of the non-lymphoid cells could form EA rosettes. Further culture diminished this Fc-receptorbearing cell population. Indirect immunofluorescence studies with rabbit anti-human collagen sera revealed membrane staining for 30-60% of the cells; this proportion usually increased to >9000 after 6-14 days in culture. Omitting any changes of culture medium resulted in a marked decrease in the proportion of cells staining with anti-collagen sera, whereas the viability and phagocytic ability of the cells did not significantly alter. Subsequent cell passage was followed by an increase in the proportion of cells demonstrating membrane-associated collagen, and this effect was more pronounced when a high concentration (500%) of serum supplement was used. No clear definition could be made as to whether the membrane-associated collagen represents synthesis or phagocytosis of collagen by the cells. Faint membrane staining was also observed with non-immune rabbit serum for 4-20% of the cells after the initial overnight incubation, and this usually dropped to 99
+++
+++ +++ ++ ++ + ++ ++ + + ++ ++ ++ +++ + +
n.t.
n.t.
5 12 8 20 2 4 48 9 14 8 8 8 9 2
93
+++
n.t.
n.t.
>99
+++
n.t.
n.t.
>99 >99 >99
+++ ++ +++
n.t.
n.t.
>99 >99
+++ +++
n.t.
n.t.
>99 >99 > 99
+++ +++ +++
* Abbreviations as in Table 1. t Per cent of cells demonstrating positive membrane immunofluorescent staining. $ Scale of maximal membrane staining intensity, ranging from - to + + ±. § n.t. = Not tested.
n.t.
24
Intensity +
+ + +
+ + +
+ + + + + +
+ n.t. +
479
Rheumatoid tissue adherent cells
extracellular material that stained strongly (3+), whereas any lymphocytes in the preparations were negative with the anti-human collagen sera. Absorption of the anti-skin collagen serum with placental collagen virtually abolished the immunofluorescent staining. Adherent cells obtained from either adult or juvenile rheumatoid arthritis patients revealed similar results (Table 2). Incubation of the cells with antiserum at 4VC or at 370C was equally effective. The influence of EDTA-trypsin treatment for suspending cells from the plastic surface of the tissue-culture flasks was compared with physical methods such as scraping with a glass rod or the movement of a magnet within the flasks. Closely similar results were found when corrected for non-viable cells in suspensions obtained by the latter procedures. Adherent cells from enzyme-dissociated rheumatoid synovial tissue secrete collagenase into the culture medium (Dayer et aL, 1976). This led us to study such cells from one patient (I.N., juvenile rheumatoid arthritis) when medium changes and subculturing of adherent cells were omitted after the first passage. (Fig. 2). The viability of the cells, as judged by the trypan-blue exclusion test, was above 9500 during this 100
BeAA
\ A
0~~~~
50-
I~ ~ ~ ~ ~ ~ ~ ~ ~ ~/-= " 10
20 Time (days)
32
FIG. 2. Various parameters of non-lymphoid adherent cells from the synovial tissue of one patient (I.N., juvenile rheumatoid arthritis) in on-going culture. No medium change was performed between subcultures at day 6 and at day 27, which are indicated by arrows. All data are presented as percentages of resuspended cells. (A) Viable cells as determined by trypan-blue exclusion test (A) Cells staining with a rabbit anti-human skincollagen serum in indirect immunofluorescence; on day 27, one aliquot of cells was transfered to medium containing 50% foetal calf serum (.1), and compared with cells grown in medium containing 10% foetal serum (92). (A) Cells staining with serum from a non-immune rabbit in indirect immunofluorescence. (L) Cells phagocytosing latex beads (0-81 pm in diameter). (A) Non-lymphoid cells forming rosettes with IgGsensitized human erythrocytes (EA-RFC); Lymphocytes were excluded on the basis of morphology.
culture period. There was an initial increase to 88% in the proportion of cells that gave positive immunofluorescence staining with the anti-human skin-collagen serum, but a marked decrease in this proportion was observed following more than 20 days after the cell passage. The percentage of cells capable of phagocytosing latex beads remained between 500% and 700. At this stage a passage of the cells was performed. One aliquot of the cells was grown in medium containing 500 foetal calf serum and was compared with a second aliquot grown in medium containing the usual 10% foetal calf serum. After three days incubation, an increase in numbers of cells staining with anti-human skin-collagen serum was observed for both groups, but was more marked for those cells grown in medium containing 500% foetal calf serum (Fig. 2). This effect was also observed for adherent cells obtained from synovial tissues of adult rheumatoid arthritis patients. Treatment of the resuspended adherent cells with purified bacterial collagenase, obtained from two different suppliers had no effect on the cell-membrane staining with anti-human collagen sera when compared with non-enzyme-treated cells. However, the staining of pronase-treated cells with these antisera was markedly diminished or abolished, but after overnight incubation at 370C in glass tubes the
480
T. G. Abrahamsen, P. M. Johnson & J. B. Natvig
cells regained positivity and showed no difference from non-enzyme-treated cells. It was also demonstrated, on cells from different tissues and stages of culture, that this effect of pronase on membrane anti-collagen staining was clearly proportional to the pronase concentration employed as well as to the degree of membrane staining on the cells prior to the enzyme treatment. Non-immune rabbit serum served as control in each experiment (Table 2). The average proportion of cells after the initial overnight incubation that showed any membrane staining with normal rabbit serum in indirect immunofluorescence was 12%y. However, this proportion rapidly decreased during on-going culture. There was no difference between non-immune sera obtained from the two separate rabbits. Rabbit antisera raised against human albumin, the F(ab')2-fragment of human IgG or human c2-macroglobulin also gave similar results, whereas the F(ab')2-fragment of rabbit IgG antibody to human x2-macroglobulin (employed at 2 mg/ml) was completely negative even at an early stage of culture. In contrast, a very high percentage of resuspended adherent cells at all stages of culture demonstrated membrane staining with an anti-human f32-microglobulin serum (Table 2). Capping experiments with the anti-collagen or anti-f2-microglobulin sera were performed by incubation of cells suspended in medium containing 20% foetal calf serum, at 370C for between 30 min and 2 hr before fluorescein-conjugated goat anti-rabbit immunoglobulin serum was applied. However, no clear membrane redistribution was seen. Indeed, intracellular fluorescence could be observed in some experiments, suggesting that phagocytosis of the stained material might occur during the incubation
period. Fc-receptor-bearing cells A small proportion of the resuspended, non-lymphoid adherent cells formed rosettes with antibodysensitized human erythrocytes (Table 1). The average value of these EA-RFC decreased during culture, but they were present in most cell suspensions tested. Overnight incubation of EDTA-trypsin suspended adherent cells at 370C in glass tubes also resulted in a decrease in the EA-RFC proportion. This may be due to an enhancing effect of trypsin treatment on the rosette formation as has been shown previously (Froland, Wisl0ff& Michaelsen, 1974). The non-lymphoid cells did not form rosettes with unsensitized sheep erythrocytes when incubated together for either 3 hr at 370C or overnight at 4VC. This contrasts with results reported for mouse L cells which exhibit a trypsin-insensitive marker for erythrocytes from sheep and other species (Papamichail et al., 1976). Adherent cells from non-rheumatoid patients Synovial tissues were obtained from four individuals undergoing reconstructive surgery following traumatic injury. However, the yield of cells obtained using the usual enzyme-dissociation procedure, was insufficient for investigation. For one (B.H.) of two patients studied with osteoarthritis, and showing a synovitis reaction that may occur in these patients (Sokoloff, 1972), an adequate number of adherent cells was obtained. Adherent cells from synovial tissue of a patient (D.A.) with ankylosing spondylitis were also investigated. The differential counts (Table 1) and immunofluorescent staining patterns (Table 2) for both of these non-rheumatoid cell preparations were similar to that observed with adherent cells from rheumatoid arthritis patients, as were the effects of no medium change (B.H.) and a high concentration (50%°) of foetal calf serum in the culture medium (D.A.). All cells from both preparations stained strongly with anti-fl2-microglobulin serum (Table 2).
DISCUSSION Immunofluorescence studies have indicated that a considerable proportion of adherent cells obtained from enzyme-dissociated rheumatoid synovial tissues express membrane-associated collagen. These cells have adherence properties, and become increasingly predominant during successive passages in culture. They resemble synovial-lining cells, both by histological examination and in their ability to phagocytose (Kinsella, Baum & Ziff, 1970; Traycoff, Pascual & Schumacher, 1976). The multi-nucleated cells
Rheumatoid tissue adherent cells
481
observed at all stages of the cultures are thought to be derived from synovial-lining cells (Zvaifler, 1973). Commonly, these cells also exhibited membrane-associated collagen and showed phagocytosis. Different rabbit antisera that had been raised against either human skin or placental collagen gave similar proportions of positively stained cells in indirect immunofluorescence. The membrane-staining pattern was speckled in appearance, as previously reported for staining with anti-collagen serum on mouse L cells (Faulk et al., 1975) and human fibroblasts (Lichenstein et al., 1976). Virtually no membrane staining of the cells was observed after absorption of the anti-human skin-collagen serum with human placental collagen. This may give additional evidence for the collagen specificity of the antisera employed. However, it can never be definitely excluded that they may possess some antibody specificities other than that for collagen. Pretreatment of resuspended adherent cells with purified bacterial collagenase had no demonstrable effect on the immunofluorescent staining with anti-human collagen sera, whereas pronase-treated cells showed a decreased or negative reaction. A possible explanation, previously invoked for similar observations with mouse L cells (Faulk et al., 1975), is that the anti-collagen sera are directed mainly against antigenic determinants on the nonhelical region of the collagen molecule that are susceptible to proteolytic enzymes. Bacterial collagenases produce several triple-helical fragments that can still retain antigenic determinants (Timpl, 1976). These observations could reflect the manner in which collagen is associated to the cell membrane, such that either the action of bacterial collagenase is blocked or that the resulting antigenic fragments contain binding sites to the cell membrane. The proportion of resuspended adherent cells that showed membrane staining with sera from nonimmune rabbits was always considerably lower than the percentage of cells staining with the anti-human collagen sera. Furthermore, in contrast to the increased proportion of anti-collagen positive cells, the number of cells exhibiting membrane staining with non-immune rabbit sera decreased during on-going culture. Similar results were also obtained with rabbit antisera to human albumin, cX2-macroglobulin or the F(ab')2 fragment of IgG, whereas the F(ab')2-fragment of rabbit IgG antibody to human a2macroglobulin was completely negative even in early cultures. This would indicate that these rabbit sera and antisera are reacting with cells which express membrane receptors for the Fc portion of IgG. The data obtained with anti-F(ab')2 serum may exclude the possible interference of collagencontaining immune complexes in interpreting the functional nature of these cells expressing membraneassociated collagen. Prolonged culture of the adherent cells without medium change resulted after 22 days in a marked decrease in the proportion of cells giving positive membrane fluorescence with anti-collagen sera. Since the viability of the cells remained greater than 95%0, it is possible that membrane-associated collagen had been digested by collagenolytic (Dayer et al., 1976) and/or proteolytic (Harris & Krane, 1972) enzymes released into the medium by cells present in the cultures. The inhibitory effect of serum, as described for synovial collagenase (Harris & Krane, 1974), may be saturated during prolonged culture. Indeed, a marked increase in the proportion of cells exhibiting membrane-associated collagen was commonly observed following subculture and growth in new medium containing 10% foetal calf serum. A higher serum concentration (50%0) was even more effective. The crude suspension of cells obtained after enzyme treatment of minced rheumatoid synovial tissue will contain various cell types that exhibit adherence properties. These would be expected to include synovial lining cells, macrophages, fibroblasts and endothelial cells. The membrane expression of 132-microglobulin has been observed for most human cell types (Nilsson, Evrin & Welsh, 1974). Using indirect immunofluorescence, more than 99%0 of the adherent cells at all stages of culture commonly exhibited strong, speckled membrane staining for /2-microglobulin. In contrast, antiserum to human a2-macroglobulin gave a staining pattern similar to sera from non-immune rabbits. A potent inhibitor of synovial collagenase is x2-macroglobulin (Harris & Krane, 1974), and its synthesis has recently been reported in cultured human-skin fibroblasts (Mosher & Wing, 1976), although its possible expression on the fibroblasts membrane was not studied. Nevertheless, human-skin fibroblasts synthesize collagen (Layman ct al., 1971), and the expression of collagen has also been observed on the fibroblast cell membrane (Lichenstein et al., 1976). Thus, our data could be interpreted as indirect evidence for a cell population related to fibroblasts in the culture of adherent cells from rheumatoid synovial tissue.
482
T. G. Abrahamsen, P. M. Johnson & J. B. Natvig
However, human fibroblasts, definitely derived from tissues such as skin, are not generally acknowledged with the ability to phagocytose, in contrast to a proportion of the synovial-lining cells (Traycoff et al., 1976). Between 40 and 7000 of the adherent cells from synovial tissue were clearly capable of phagocytosing latex beads, and there was only a small decrease in this percentage during prolonged culture. Phagocytosis is a property of macrophages, and such cells express membrane-bound Fc receptors (Davey & Asherson, 1967). Only a small percentage of the resuspended adherent, non-lymphoid cells formed EA rosettes at any time during culture. Furthermore, the proportion ofcells exhibiting peroxidasestaining was also small in early cultures, and this marker disappeared during in vitro growth. Peroxidasecontaining granules are found in neutrophilic granulocytes and monocytes, whereas macrophages rapidly lose this feature (Van Furth, Hirsh & Fedorko, 1970). Normal synovial tissues were collected from individuals undergoing reconstructive surgery; the yield of adherent cells was, however, very small. This is consistent with the lack of synovial hypertrophy and mononuclear cell infiltration in these tissues (Zvaifler, 1973). Sufficient numbers of adherent cells for limited immunofluorescence studies were obtained from synovial tissues of two patients with chronic non-rheumatoid synovitis. Anti-collagenpositive cells could be demonstrated in proportions comparable to the adherent cells from rheumatoid tissue. This indicates that there are only minor differences between rheumatoid synovial tissues with respect to these cells, a finding which is in agreement with studies on collagenase production in explant cultures of synovial tissues from patients with various inflammatory joint diseases (Harris, Cohen & Krane, 1969). Although no definitive conclusion can be drawn concerning the identity of the adherent cells staining with anti-collagen sera, it is tempting to speculate that some, or all, are synovial-lining cells. The data on the expression of membrane-associated collagen during prolonged in vitro growth under various culture conditions indicates that a proportion of the synovial-lining cells synthesize collagen, a feature commonly associated with fibroblasts (Layman et al., 1971; Lichenstein et al., 1976). However, some synoviallining cells may also be capable of ingesting collagen released into the medium from by-stander synthetic cells. Such ingestion of collagen has previously been described for macrophages (Parakkal, 1969). Thus, demonstrable membrane-associated collagen could represent the attachment phase of its ingestion. Clearly, further investigations are required to determine whether the membrane-associated collagen is related to collagen synthesis or also phagocytosis in order to clarify the functional role of those adherent cells from synovial tissues. We are indebted to Professor W. P. Faulk, Dr H. K. Beard and Dr A. J. Bailey for the preparation and gifts of collagen and anti-collagen sera, and to B.Sc. Leifur Thorsteinsson for performing the peroxidase-staining experiments. Furthermore, we thank the doctors at the Department of Surgery, Oslo Sanitetsforenings Rheumatism Hospital, the Department of Plastic Surgery, Rikshospitalet University Hospital and Kronprinsesse Marthas Institute for their generous supply of synovial tissues. The skilful technical assistance of Ms Kari Ulrichsen is gratefully acknowledged. This study was supported by grants from the Norwegian Women's Public Health Organization and the Royal Society, London.
REFERENCES by isolated adherent rheumatoid synovial cells. Proc. nat. ABRAHAMSEN, T.G., FR0LAND, S.S., NATVIG, J.B. & PAHLE, Acad. Sci. (Wash.), 73, 945. J. (1975) Elution and characterization of lymphocytes from rheumatoid inflammatory tissue. Scand. 7. Immunol. DUKSIN, D.A., MAOZ, A. & FuCHs, S. (1975) Differential cytotoxic activity of anticollagen serum on rat osteoblasts 4, 823. and fibroblasts in tissue culture. Cell, 5, 83. CASTOR, C.W. (1971) Abnormalities of connective tissue cells cultured from patients with rheumatoid arthritis. II. EISEN, A.Z., JEFFREY, J.J. & GROSS, J. (1968) Human skin collagenase. Isolation and mechanism of attack on the Defective regulation of hyaluronate and collagen formacollagen molecule. Biochim. Biophys. Acta (Amst.), 151, tion... Lab. Clin. Med. 77, 65. 637. CHUNG, E. & MILLER, E.J. (1974) Collagen polymorphisms: characterization of molecules with chain composition EVANSON, J.M., JEFFREY, J.J. & KRANE, S.M. (1967) Human collagenase: identification and characterization of an [al(l 1 1)]3 in human tissues. Science, 183, 1200. enzyme from rheumatoid synovium in culture. Science, DAVEY, M.J. & ASHERSON, G.L. (1967) Cytophilic antibody. 158, 499. I. Nature ofthe macrophage receptor. Immunology, 12, 13. DAYER, J.-M., KRANE, S.M., RUSSELL, G.G. & ROBINSON, FAULK, W.P., CONOCHIE, L.B., TEMPLE, A. & PAPAMICHAIL, M. (1975) Immunobiology of membrane bound collagen D.R. (1976) Production of collagenase and prostaglandins
Rheumatoid tissue adherent cells in mouse fibroblasts in tissue culture. Nature (Lond.), 256, 123. FAULK, W.P. & JOHNSON, P.M. (1977) Immunological studies ofhuman placentae. Identification and distribution of proteins in mature chorionic villi. Clin. exp. Immunol. 27, 365. FR0LAND, S.S. (1972) Binding of sheep erythrocytes to human lymphocytes. A probable marker for T lymphocytes. Scand. 7. Immunol. 1, 269. FROLAND, S.S. & NATVIG, J.B. (1970) Effect of polyspecific rabbit anti-immunoglobulin antisera on human lymphocytes in vitro. Int. Arch. Allergy, 39, 121. FROLAND, S.S., WISLOFF, F. & MICHAELSEN, T.E. (1974) Human lymphocytes with receptors for IgG. A population of cells distinct from T- and B-lymphocytes. Int. Arch. Allergy, 47, 124. F0RRE, 0., NATVIG, J.B., FR0LAND, S.S. & JOHNSON, P.M. (1976) Distribution of heavy-chain variable-region (VH) subgroups on human lymphocytes. Scand. 3. Immunol. 5, 1221. HARRIS, E.D., JR., COHEN, G.L. & KRANE, S.M. (1969) Synovial collagenase: its presence in culture from joint disease of diverse etiology. Arthr. and Rheum. 12, 92. HARRIS, E.D., JR., KRANE, S.M. (1972) An endopeptidase from rheumatoid synovial tissue culture. Biochim. Biocphys. Acta (Amst.), 258, 566. HARRIS, E.D., JR & KRANE, S.M. (1974) Collagenases. New Engl. 3. Med. 291, 557. JOHNSON, P.M. & FAULK, W.P. (1976) Immunological studies of human placentae. Lectin binding to villous stroma and to trophoblastic basement membrane. Clin. exp. Immunol. 24, 435. KAPLOW, L.S. (1965) Simplified myeloperoxidase stain using benzidine dihydrochloride. Blood, 26, 215. KINSELLA, T.D., BAUM, J. & ZIFF, M. (1970) Studies of isolated synovial lining cells of rheumatoid and nonrheumatoid synovial membranes. Arthr. and Rheum. 13, 734. LAYMAN, D.L., McGOODWIN, E.B. & MARTIN, G.R. (1971) The nature of the collagen synthesized by cultured human fibroblasts. Proc. nat. Acad. Sci. (Wash.) 68, 454. LICHTENSTEIN, J.R., BAUER, E.A., HOYT, R. & WEDNER,
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H.J. (1976) Immunologic characterization of the membrane-bound collagen in normal human fibroblasts: Identification of a distinct membrane collagen... exp. Med. 144, 145. LUSTIG, L. (1970) Cytotoxic action of an antiserum to soluble collagen on tissue culture of fibroblasts. Proc. Soc. exp. Biol. (N.Y.), 133, 207. MOSHER, D.F. & WING, D.A. (1976) Synthesis and secretion of cx2-macroglobulin by cultured human fibroblasts. J. exp. Med. 143, 462. MUNTHE, E. & NATVIG, J.B. (1971) Characterization of IgG complexes in eluates from rheumatoid tissue. Clin. exp. Immunol. 8, 249. NILSSON, K., EVRIN, P.-E. & WELSH, K.I. (1974) Production of f2-microglobulin by normal and malignant human cell lines and peripheral lymphocytes. Transplant. Revt. 31, 53. PAPAMICHAIL, M., GUTIERREZ, C., TEMPLE, A. & FAULK, W.P. (1976) Spontaneous rosette formation by mouse L cells. Immunology, 30, 129. PARAKKAL, P.F. (1969) Role of macrophages in collagen resorbtion during hair growth cycle. 7. Ultrastruct. Res. 29, 210. SOKOLOFF, L. (1972) The pathology and pathogenesis of osteoarthritis. Arthritis and Allied Conditions (ed. by J.L. Hollander and D.J. McCarty, Jr), p. 1009. Lea & Febiger, Philadelphia. STOSSEL, T.P. (1974) Phagocytosis. New Engl. J. Med. 290, 833. TIMPL, R. (1976) Immunological studies on collagen. Biochemistry of Collagen (ed. by G.N. Ramachandran and A.M. Reddy), p. 319. Plenum Press, New York. TRAYCOFF, R.B., PASCUAL, E. & SCHUMACHER, H.R., JR. (1976) Mononuclear cells in human synovial fluid. Identification of lymphoblasts in rheumatoid arthritis. Arthr. and Rheum. 19, 793. VAN FURTH, R., HIRSCH, J.G. & FEDORKO, M.E. (1970) Morphology and peroxidase cytochemistry of mouse promonocytes, monocytes and macrophages. ]. exp. Med. 132, 794. ZVAIFLER, N.J. (1973) Immunopathology of joint inflammation in arthritis. Advanc. Immunol. 16, 265.
Membrane characteristics of adherent cells dissociated from rheumatoid synovial tissue T. G. ABRAHAMSEN, P. M. JOHNSON* & J. B. NATVIG Institute oj Immunology and Rheumatology, Rikshospitalet University Hospital, Oslo, Norway
(Received 7 January 1977) SUMMARY
Synovial tissues from rheumatoid-arthritis patients were dissociated by enzymes and the resulting cells incubated overnight in tissue-culture flasks. The adherent cell population was resuspended with EDTA-trypsin, and morphological examination showed 68-80% non-lymphoid cells, most of which had the appearance of synovial lining cells. The proportion of these cells increased during subsequent culture. Between 40 and 60% of the cells exhibited marked phagocytosis, and < 14% of the non-lymphoid cells could form EA rosettes. Further culture diminished this Fc-receptorbearing cell population. Indirect immunofluorescence studies with rabbit anti-human collagen sera revealed membrane staining for 30-60% of the cells; this proportion usually increased to >9000 after 6-14 days in culture. Omitting any changes of culture medium resulted in a marked decrease in the proportion of cells staining with anti-collagen sera, whereas the viability and phagocytic ability of the cells did not significantly alter. Subsequent cell passage was followed by an increase in the proportion of cells demonstrating membrane-associated collagen, and this effect was more pronounced when a high concentration (500%) of serum supplement was used. No clear definition could be made as to whether the membrane-associated collagen represents synthesis or phagocytosis of collagen by the cells. Faint membrane staining was also observed with non-immune rabbit serum for 4-20% of the cells after the initial overnight incubation, and this usually dropped to 99
+++
+++ +++ ++ ++ + ++ ++ + + ++ ++ ++ +++ + +
n.t.
n.t.
5 12 8 20 2 4 48 9 14 8 8 8 9 2
93
+++
n.t.
n.t.
>99
+++
n.t.
n.t.
>99 >99 >99
+++ ++ +++
n.t.
n.t.
>99 >99
+++ +++
n.t.
n.t.
>99 >99 > 99
+++ +++ +++
* Abbreviations as in Table 1. t Per cent of cells demonstrating positive membrane immunofluorescent staining. $ Scale of maximal membrane staining intensity, ranging from - to + + ±. § n.t. = Not tested.
n.t.
24
Intensity +
+ + +
+ + +
+ + + + + +
+ n.t. +
479
Rheumatoid tissue adherent cells
extracellular material that stained strongly (3+), whereas any lymphocytes in the preparations were negative with the anti-human collagen sera. Absorption of the anti-skin collagen serum with placental collagen virtually abolished the immunofluorescent staining. Adherent cells obtained from either adult or juvenile rheumatoid arthritis patients revealed similar results (Table 2). Incubation of the cells with antiserum at 4VC or at 370C was equally effective. The influence of EDTA-trypsin treatment for suspending cells from the plastic surface of the tissue-culture flasks was compared with physical methods such as scraping with a glass rod or the movement of a magnet within the flasks. Closely similar results were found when corrected for non-viable cells in suspensions obtained by the latter procedures. Adherent cells from enzyme-dissociated rheumatoid synovial tissue secrete collagenase into the culture medium (Dayer et aL, 1976). This led us to study such cells from one patient (I.N., juvenile rheumatoid arthritis) when medium changes and subculturing of adherent cells were omitted after the first passage. (Fig. 2). The viability of the cells, as judged by the trypan-blue exclusion test, was above 9500 during this 100
BeAA
\ A
0~~~~
50-
I~ ~ ~ ~ ~ ~ ~ ~ ~ ~/-= " 10
20 Time (days)
32
FIG. 2. Various parameters of non-lymphoid adherent cells from the synovial tissue of one patient (I.N., juvenile rheumatoid arthritis) in on-going culture. No medium change was performed between subcultures at day 6 and at day 27, which are indicated by arrows. All data are presented as percentages of resuspended cells. (A) Viable cells as determined by trypan-blue exclusion test (A) Cells staining with a rabbit anti-human skincollagen serum in indirect immunofluorescence; on day 27, one aliquot of cells was transfered to medium containing 50% foetal calf serum (.1), and compared with cells grown in medium containing 10% foetal serum (92). (A) Cells staining with serum from a non-immune rabbit in indirect immunofluorescence. (L) Cells phagocytosing latex beads (0-81 pm in diameter). (A) Non-lymphoid cells forming rosettes with IgGsensitized human erythrocytes (EA-RFC); Lymphocytes were excluded on the basis of morphology.
culture period. There was an initial increase to 88% in the proportion of cells that gave positive immunofluorescence staining with the anti-human skin-collagen serum, but a marked decrease in this proportion was observed following more than 20 days after the cell passage. The percentage of cells capable of phagocytosing latex beads remained between 500% and 700. At this stage a passage of the cells was performed. One aliquot of the cells was grown in medium containing 500 foetal calf serum and was compared with a second aliquot grown in medium containing the usual 10% foetal calf serum. After three days incubation, an increase in numbers of cells staining with anti-human skin-collagen serum was observed for both groups, but was more marked for those cells grown in medium containing 500% foetal calf serum (Fig. 2). This effect was also observed for adherent cells obtained from synovial tissues of adult rheumatoid arthritis patients. Treatment of the resuspended adherent cells with purified bacterial collagenase, obtained from two different suppliers had no effect on the cell-membrane staining with anti-human collagen sera when compared with non-enzyme-treated cells. However, the staining of pronase-treated cells with these antisera was markedly diminished or abolished, but after overnight incubation at 370C in glass tubes the
480
T. G. Abrahamsen, P. M. Johnson & J. B. Natvig
cells regained positivity and showed no difference from non-enzyme-treated cells. It was also demonstrated, on cells from different tissues and stages of culture, that this effect of pronase on membrane anti-collagen staining was clearly proportional to the pronase concentration employed as well as to the degree of membrane staining on the cells prior to the enzyme treatment. Non-immune rabbit serum served as control in each experiment (Table 2). The average proportion of cells after the initial overnight incubation that showed any membrane staining with normal rabbit serum in indirect immunofluorescence was 12%y. However, this proportion rapidly decreased during on-going culture. There was no difference between non-immune sera obtained from the two separate rabbits. Rabbit antisera raised against human albumin, the F(ab')2-fragment of human IgG or human c2-macroglobulin also gave similar results, whereas the F(ab')2-fragment of rabbit IgG antibody to human x2-macroglobulin (employed at 2 mg/ml) was completely negative even at an early stage of culture. In contrast, a very high percentage of resuspended adherent cells at all stages of culture demonstrated membrane staining with an anti-human f32-microglobulin serum (Table 2). Capping experiments with the anti-collagen or anti-f2-microglobulin sera were performed by incubation of cells suspended in medium containing 20% foetal calf serum, at 370C for between 30 min and 2 hr before fluorescein-conjugated goat anti-rabbit immunoglobulin serum was applied. However, no clear membrane redistribution was seen. Indeed, intracellular fluorescence could be observed in some experiments, suggesting that phagocytosis of the stained material might occur during the incubation
period. Fc-receptor-bearing cells A small proportion of the resuspended, non-lymphoid adherent cells formed rosettes with antibodysensitized human erythrocytes (Table 1). The average value of these EA-RFC decreased during culture, but they were present in most cell suspensions tested. Overnight incubation of EDTA-trypsin suspended adherent cells at 370C in glass tubes also resulted in a decrease in the EA-RFC proportion. This may be due to an enhancing effect of trypsin treatment on the rosette formation as has been shown previously (Froland, Wisl0ff& Michaelsen, 1974). The non-lymphoid cells did not form rosettes with unsensitized sheep erythrocytes when incubated together for either 3 hr at 370C or overnight at 4VC. This contrasts with results reported for mouse L cells which exhibit a trypsin-insensitive marker for erythrocytes from sheep and other species (Papamichail et al., 1976). Adherent cells from non-rheumatoid patients Synovial tissues were obtained from four individuals undergoing reconstructive surgery following traumatic injury. However, the yield of cells obtained using the usual enzyme-dissociation procedure, was insufficient for investigation. For one (B.H.) of two patients studied with osteoarthritis, and showing a synovitis reaction that may occur in these patients (Sokoloff, 1972), an adequate number of adherent cells was obtained. Adherent cells from synovial tissue of a patient (D.A.) with ankylosing spondylitis were also investigated. The differential counts (Table 1) and immunofluorescent staining patterns (Table 2) for both of these non-rheumatoid cell preparations were similar to that observed with adherent cells from rheumatoid arthritis patients, as were the effects of no medium change (B.H.) and a high concentration (50%°) of foetal calf serum in the culture medium (D.A.). All cells from both preparations stained strongly with anti-fl2-microglobulin serum (Table 2).
DISCUSSION Immunofluorescence studies have indicated that a considerable proportion of adherent cells obtained from enzyme-dissociated rheumatoid synovial tissues express membrane-associated collagen. These cells have adherence properties, and become increasingly predominant during successive passages in culture. They resemble synovial-lining cells, both by histological examination and in their ability to phagocytose (Kinsella, Baum & Ziff, 1970; Traycoff, Pascual & Schumacher, 1976). The multi-nucleated cells
Rheumatoid tissue adherent cells
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observed at all stages of the cultures are thought to be derived from synovial-lining cells (Zvaifler, 1973). Commonly, these cells also exhibited membrane-associated collagen and showed phagocytosis. Different rabbit antisera that had been raised against either human skin or placental collagen gave similar proportions of positively stained cells in indirect immunofluorescence. The membrane-staining pattern was speckled in appearance, as previously reported for staining with anti-collagen serum on mouse L cells (Faulk et al., 1975) and human fibroblasts (Lichenstein et al., 1976). Virtually no membrane staining of the cells was observed after absorption of the anti-human skin-collagen serum with human placental collagen. This may give additional evidence for the collagen specificity of the antisera employed. However, it can never be definitely excluded that they may possess some antibody specificities other than that for collagen. Pretreatment of resuspended adherent cells with purified bacterial collagenase had no demonstrable effect on the immunofluorescent staining with anti-human collagen sera, whereas pronase-treated cells showed a decreased or negative reaction. A possible explanation, previously invoked for similar observations with mouse L cells (Faulk et al., 1975), is that the anti-collagen sera are directed mainly against antigenic determinants on the nonhelical region of the collagen molecule that are susceptible to proteolytic enzymes. Bacterial collagenases produce several triple-helical fragments that can still retain antigenic determinants (Timpl, 1976). These observations could reflect the manner in which collagen is associated to the cell membrane, such that either the action of bacterial collagenase is blocked or that the resulting antigenic fragments contain binding sites to the cell membrane. The proportion of resuspended adherent cells that showed membrane staining with sera from nonimmune rabbits was always considerably lower than the percentage of cells staining with the anti-human collagen sera. Furthermore, in contrast to the increased proportion of anti-collagen positive cells, the number of cells exhibiting membrane staining with non-immune rabbit sera decreased during on-going culture. Similar results were also obtained with rabbit antisera to human albumin, cX2-macroglobulin or the F(ab')2 fragment of IgG, whereas the F(ab')2-fragment of rabbit IgG antibody to human a2macroglobulin was completely negative even in early cultures. This would indicate that these rabbit sera and antisera are reacting with cells which express membrane receptors for the Fc portion of IgG. The data obtained with anti-F(ab')2 serum may exclude the possible interference of collagencontaining immune complexes in interpreting the functional nature of these cells expressing membraneassociated collagen. Prolonged culture of the adherent cells without medium change resulted after 22 days in a marked decrease in the proportion of cells giving positive membrane fluorescence with anti-collagen sera. Since the viability of the cells remained greater than 95%0, it is possible that membrane-associated collagen had been digested by collagenolytic (Dayer et al., 1976) and/or proteolytic (Harris & Krane, 1972) enzymes released into the medium by cells present in the cultures. The inhibitory effect of serum, as described for synovial collagenase (Harris & Krane, 1974), may be saturated during prolonged culture. Indeed, a marked increase in the proportion of cells exhibiting membrane-associated collagen was commonly observed following subculture and growth in new medium containing 10% foetal calf serum. A higher serum concentration (50%0) was even more effective. The crude suspension of cells obtained after enzyme treatment of minced rheumatoid synovial tissue will contain various cell types that exhibit adherence properties. These would be expected to include synovial lining cells, macrophages, fibroblasts and endothelial cells. The membrane expression of 132-microglobulin has been observed for most human cell types (Nilsson, Evrin & Welsh, 1974). Using indirect immunofluorescence, more than 99%0 of the adherent cells at all stages of culture commonly exhibited strong, speckled membrane staining for /2-microglobulin. In contrast, antiserum to human a2-macroglobulin gave a staining pattern similar to sera from non-immune rabbits. A potent inhibitor of synovial collagenase is x2-macroglobulin (Harris & Krane, 1974), and its synthesis has recently been reported in cultured human-skin fibroblasts (Mosher & Wing, 1976), although its possible expression on the fibroblasts membrane was not studied. Nevertheless, human-skin fibroblasts synthesize collagen (Layman ct al., 1971), and the expression of collagen has also been observed on the fibroblast cell membrane (Lichenstein et al., 1976). Thus, our data could be interpreted as indirect evidence for a cell population related to fibroblasts in the culture of adherent cells from rheumatoid synovial tissue.
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T. G. Abrahamsen, P. M. Johnson & J. B. Natvig
However, human fibroblasts, definitely derived from tissues such as skin, are not generally acknowledged with the ability to phagocytose, in contrast to a proportion of the synovial-lining cells (Traycoff et al., 1976). Between 40 and 7000 of the adherent cells from synovial tissue were clearly capable of phagocytosing latex beads, and there was only a small decrease in this percentage during prolonged culture. Phagocytosis is a property of macrophages, and such cells express membrane-bound Fc receptors (Davey & Asherson, 1967). Only a small percentage of the resuspended adherent, non-lymphoid cells formed EA rosettes at any time during culture. Furthermore, the proportion ofcells exhibiting peroxidasestaining was also small in early cultures, and this marker disappeared during in vitro growth. Peroxidasecontaining granules are found in neutrophilic granulocytes and monocytes, whereas macrophages rapidly lose this feature (Van Furth, Hirsh & Fedorko, 1970). Normal synovial tissues were collected from individuals undergoing reconstructive surgery; the yield of adherent cells was, however, very small. This is consistent with the lack of synovial hypertrophy and mononuclear cell infiltration in these tissues (Zvaifler, 1973). Sufficient numbers of adherent cells for limited immunofluorescence studies were obtained from synovial tissues of two patients with chronic non-rheumatoid synovitis. Anti-collagenpositive cells could be demonstrated in proportions comparable to the adherent cells from rheumatoid tissue. This indicates that there are only minor differences between rheumatoid synovial tissues with respect to these cells, a finding which is in agreement with studies on collagenase production in explant cultures of synovial tissues from patients with various inflammatory joint diseases (Harris, Cohen & Krane, 1969). Although no definitive conclusion can be drawn concerning the identity of the adherent cells staining with anti-collagen sera, it is tempting to speculate that some, or all, are synovial-lining cells. The data on the expression of membrane-associated collagen during prolonged in vitro growth under various culture conditions indicates that a proportion of the synovial-lining cells synthesize collagen, a feature commonly associated with fibroblasts (Layman et al., 1971; Lichenstein et al., 1976). However, some synoviallining cells may also be capable of ingesting collagen released into the medium from by-stander synthetic cells. Such ingestion of collagen has previously been described for macrophages (Parakkal, 1969). Thus, demonstrable membrane-associated collagen could represent the attachment phase of its ingestion. Clearly, further investigations are required to determine whether the membrane-associated collagen is related to collagen synthesis or also phagocytosis in order to clarify the functional role of those adherent cells from synovial tissues. We are indebted to Professor W. P. Faulk, Dr H. K. Beard and Dr A. J. Bailey for the preparation and gifts of collagen and anti-collagen sera, and to B.Sc. Leifur Thorsteinsson for performing the peroxidase-staining experiments. Furthermore, we thank the doctors at the Department of Surgery, Oslo Sanitetsforenings Rheumatism Hospital, the Department of Plastic Surgery, Rikshospitalet University Hospital and Kronprinsesse Marthas Institute for their generous supply of synovial tissues. The skilful technical assistance of Ms Kari Ulrichsen is gratefully acknowledged. This study was supported by grants from the Norwegian Women's Public Health Organization and the Royal Society, London.
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