Clin. exp. Immunol. (1990) 80, 32-37

Soluble histocompatibility antigens in synovial fluids of patients with rheumatoid arthritis F. K. STEVENSON, W. A. DOUGLASS, M. B. SPELLERBERG, M. T. WALTERS* & M. I. D. CAWLEY* Lymphoma Research Unit, Tenovus Laboratory, and *Department of Rheumatology, Southampton General Hospital,

Southampton, England (Acceptedfor publication 3 November 1989)

SUMMARY Soluble histocompatibility antigens of the class II region have been detected in synovial fluids obtained from patients with rheumatoid arthritis. A capture immunoassay involving two monoclonal antibodies was used; interference by rheumatoid factor, which is a feature of such assays, was overcome by mild pretreatment of fluids with 2-mercaptoethanol. No HLA class II antigen could be detected in matched sera from patients, even when levels were high in synovial fluids. Released HLAclass II material was of high molecular weight (> 1000 kD) and was linked to HLA-class I antigen. However, no significant amounts of other common cell surface antigens were detected in the complex, suggesting a preferential release of MHC antigens from cells of the inflamed synovium. Attempts to induce production of similar material from a cell line which expresses HLA class II strongly at the cell surface, by stressing the cells in various ways did not succeed, indicating that release is an active process.

Keywords histocompatibility antigens rheumatoid arthritis synovial fluid

INTRODUCTION It is becoming clear that several cellular antigens normally considered as integral membrane proteins of the cell membrane can be released from the cell into the surrounding fluid. Among these are the CD8 and CD23 antigens and the interleukin-2 (IL-2) receptor (Rubin et al., 1985). In the case of the MHC antigens, production of a soluble form has been reported frequently in cell culture supernatants and also in the serum (Allison et al., 1977; Sachs, Kiszkiss & Kim, 1980). For the class I antigen there appear to be two distinct molecular species, one a cell surface integral protein, and the other a secreted form which lacks the hydrophobic tail (Krangel, Pious & Strominger, 1984). Recent studies have reported that these class I antigens can be detected in the circulation of liver-graft recipients as well as in normal donors (Davies, Pollard & Calne, 1989). In recipients, the material is derived at least in part from the graft and may provide a useful indicator of graft function. Less is known of the class II antigen, although the importance of circulating material potentially capable of blocking immune function has been pointed out (Pretell & Cone, 1985). However, the presence of soluble HLA class II in serum has been reported for patients with certain leukaemias (Herlyn et al., 1984; Stevenson et al., 1986).

In the inflammatory diseases such as rheumatoid arthritis, activation of various cell populations occurs in the synovial tissue and an enhanced amount ofclass II material has been seen in tissue sections (Duke et al., 1982). The source of this may include interdigitating cells which are known to increase in number in this disease, macrophages and possibly B and activated T lymphocytes. It was found in this laboratory that the class II antigen was present in synovial fluid in a soluble form (Stevenson et al., 1986), but further investigation was hampered by interference in capture immunoassays by rheumatoid factor (RF). The common RFs are pentameric IgM molecules which have affinity for IgG of both human and animal sources. Having bound to the capture antibody on a microtitre plate, the pentamer can form a direct bridge with the enzyme-conjugated detecting antibody thereby giving a false-positive result. Such interference presents a danger in all such assays and it is not confined to patients with overt autoimmune disease (Courtenay-Luck et al., 1987). We describe a mild method to overcome the problem which does not affect the class II antigen; this has allowed analysis of fluids for the presence of the antigen and further investigation of its molecular form.

Correspondence: Dr F. K. Stevenson, University of Southampton Lymphoma Research Unit, Tenovus Research Laboratory, Tremona Road, Southampton S09 4XY, UK.

Patients Twenty-four patients attending the Rheumatology Unit at Southampton General Hospital were entered into the study. All

MATERIALS AND METHODS

32

Soluble HLA antigens in rheumatoid arthritis had classical or definite rheumatoid arthritis according to ARA criteria (Ropes et al., 1958). Their mean age was 60 years (range 24 5-79) and the mean duration of disease was 10 5 years (range 0 33-16 0). The majority were seropositive for IgM RF. All patients were taking non-steroidal anti-inflammatory drugs (NSAIDs) and most were also undergoing therapy with one of the following disease-modifying anti-rheumatic drugs: gold, sodium thiomalate, D-penicillamine, azathioprine or hydroxychloroquine. Each patient had synovitis of at least one knee joint considered sufficiently severe to require aspiration of the effusion and intra-articular injection of a corticosteroid. No patient had received such an injection in the 3 months prior to aspiration for the assay. Synovial fluids were sent directly to the laboratory in a sterile container together with a serum sample taken at the same hospital visit. Fluids were centrifuged at 1500 g and the supernatant stored at 40C with sodium azide (10 mM) as preservative. In some cases the pH of the fluid was adjusted to 5 8 and was treated with testicular hyaluronidase (Sigma Chemical Co., Poole, UK) at 20 yg/ml for 30 min at 370C to reduce viscosity.

Cultured human B cell line (FLE) The FLE cell line has been derived from human B cells-by Epstein-Barr virus (EBV) transformation and is used as a homozygous typing cell (HLA-DR4, DQw3, DwI3) in the laboratory of Dr H. Festenstein, who kindly supplied it to our laboratory. The cells express HLA-class II strongly at the cell surface and were used to assess release of this material in shortterm culture following exposure to various kinds of stress. These included inhibition of protein synthesis by cycloheximide (5 mM) irradiation (2000 rad for 40 min) and water lysis. Cells were cultured at 107/ml for 6 h with gentle swirling, and centrifuged, and supernatants were analysed by immunoassay for HLA-class II. Monoclonal antibodies Ascitic fluid containing a rat monoclonal antibody against human HLA-DR (class II) antigens (MAS054) obtained from Sera-Lab (Crawley Down, UK) was precipitated with 50% saturated ammonium sulphate solution, spun and redissolved at 1 mg/ml in 0-02 M Tris-HCl/0 I M NaCl/0 02% NaN3, pH 8-0. The antibody recognizes a monomorphic determinant of both the DR and DP subloci. The second antibody with class II specificity (WR1 8) was raised in this laboratory and recognizes the beta-chains of DR, DQ and DP antigens (Stevenson et al., 1986). Monoclonal antibodies against the MHC class I (HB95), CD3 (OKT3, CRL8001), CD2 (OKTl 1, CRL8027) and CDl (OKT6, CRL8020) were all obtained from the American Type Culture Collection (Rockville, MD). Monoclonal antibodies against CD37 (WR17) and CD45 (WR6) were raised in this laboratory and assigned at the 3rd International Conference on Human Leucocyte Differentiation Antigens. ELISA Class II antigens. A double-determinant assay already described was used to measure levels of class II antigen (Stevenson et al., 1986). Briefly, the rat anti-class II antibody was used at 2 yg/ml to coat the wells of a microtitre plate. After washing, antigen-containing fluids were added and incubated

33

for 90 min at 37°C. Plates were washed again and exposed similarly to the second (mouse) anti-class II antibody (WR1 8) at 2 Mg/ml. Bound mouse IgG was detected by horseradish peroxidase-conjugated (HRP) rat anti-mouse IgG (Jackson ImmunoResearch Labs., Avondale, PA), at 1/500 dilution. Controls were carried out either by using no mouse antibody, or by using a subclass-matched (IgG2a) mouse antibody of irrelevant specificity, an anti-idiotype antibody against a guinea pig lymphoma. Colour was developed with the substrate ophenylene diamine. The assay had previously been standardized by using a lysate of cells from a patient with a B cell leukaemia and it was possible then to use one of the positive synovial fluids as a secondary standard.

Investigation of other molecules associated with class II Molecules which may be linked to class II antigen were investigated by a modification of the assay above. The class IIcontaining antigen was first bound to the antibody on the microtitre plate. Associated antigens such as class I were then detected by exposure to an appropriate mouse monoclonal antibody in the same way as for the double determinant assay described. Concentrations of antibodies were saturating for cell antigens as judged by immunofluorescence and were usually 2-10 pg/ml of IgG. Detection and removal of RF activity RF activity in this system was defined as the colour yield obtained in the assay when only coating rat anti-class II antibody, patient fluid and detecting HRP-rat anti-mouse antibody were used. Since the two rat antibodies do not interact, no colour should be developed unless there is a bridging molecule such as RF in the patient fluid. The only other possible interfering molecule would be pre-existing human anti-rat antibody in the patient, and although this was detected in some fluids, it was rare ( < 1% of patients) and those with significant levels were eliminated from the study. In contrast, RF was found frequently but was removed from synovial fluids or sera by incubation with 2-mercaptoethanol (20 mM final concentration) in 0.5 M Tris-EDTA, pH 8 0, for 30 min at 37°C. Released -SH groups were blocked by incubation with iodoacetamide (22 mM) for 10 min at 37°C.

Gel filtration of synovialfluids This was carried out on synovial fluids precipitated in 50% ammonium sulphate solution in which the class II material was insoluble. After precipitation it was redissolved in Tris buffer and loaded (1 ml) onto a column of AcA 22 (Ultrogel) (1 -6 x 80 cm), equilibrated with 0-2 M Tris buffer, pH 8-0. Fractions (2-5 ml) were collected and assayed for class II antigen.

RESULTS Measurement of class II antigen The results of measuring class II antigen in untreated synovial fluids from 11 patients with rheumatoid arthritis are shown in Table 1. In the fluids which contained RF activity, the levels of class II antigen in untreated fluids had to be calculated from the difference in colour yield obtained in the ELISA in the presence and the absence of the second mouse anti-class II antibody as described in Materials and Methods. Exposure of the fluids to reducing agent generally removed this 'background' activity

F. K. Stevenson et al.

34

Table 1. Levels of HLA-class II antigen in matched synovial fluids and sera from patients with rheumatoid arthritis: effect of reduction

Serum (U/ml)

Synovial fluid (U/ml) Untreated Patient no.

Class II

RF

1 2 3 4 5 6 7 8 9 10 11 12 13 14

487 1650 170 91 33 67 52 204 48 284 17

567 0 128 14 16 1-7 11 0 83 28 30

Reduced*

Class II

458 1780 191

58 15 61 39 171 58 279 15 52 54 62

Table 2. Association of other cell surface molecules with class II antigen in synovial fluids of patients with rheumatoid arthritis

Reduced

RF

49 0 24 0 0 05 54 0 0 0 0

Class II

0 1-3

1.0 0 0 0 0 0 0 16

* Reduction was with 2-mercaptoethanol (20 mM), 30 min at 370C. Serum values are quoted only after reduction. RF, rheumatoid factor.

without affecting net levels of class II antigen. The actual levels of RF before and after reduction are also shown in Table I and indicate almost total removal by this procedure. In a minority of cases, such as Patient 7 in Table 1, a high percentage of apparent RF persisted after reduction, and other factors such as IgG RF or antibody against rodent IgG may be involved. Similar results showing almost complete loss of RF after reduction were obtained for serum samples. As seen in Table 1, the levels of class II antigen are very variable between patients. Similarly the RF levels are variable and there is no apparent correlation between the amounts of class II and RF detected.

Comparison of SF and serum Matched sera were obtained for 10 of the patients and were examined for class II antigen after removal of RF. As also shown in Table 1, no significant amounts of this material could be detected in the serum samples even when levels were high in synovial fluids. Association of class II antigen with other molecules By manipulating the ELISA it was possible to examine the association between class II antigen and other molecules detectable with suitable monoclonal antibodies. Class II antigen was first allowed to bind to the anti-class II antibody attached to the microtitre plate. Associated antigens were than detected by adding a second monoclonal antibody as described in Materials and Methods and profiles obtained are listed in Table 2. The antigen found regularly complexed to class II was MHC class I antigen, and the ratio ofclass II: class I varied between 1 -7: 1 and 6: 1 with a mean of 3 5. Other common cell surface antigens were also sought with the results given in Table 2. The T cell antigens

HLA antigens (U/ml)

Patient no.

Class II

1 2 3 6 7 8 13 14 15 16 17 18 19 20 21 22 23 24

487 1650 170 67 39 204 54 62 10 30 56 28 15 119 26 43 14 13

Class I

24 80 86 90 17

CD3

CD2

CD1

CD45

0 0 0 0

0 0 0 0

0 0 0

136

5.0 0

0

0

1.5

3.5 10 11 28 27 14 10

5.5

0 0

0 0

CD37

0 4-3 0 0 0 0 0 0

165

0

1H1 0 0

0

0 0 67 0 83 28

0 0 0 0 0 0

7-6

CD3 and CD2, and the B cell associated antigen CD37 were not found in significant amounts; CD1 antigen, found on interdigitating cells, was also very low and variable; CD45, the leucocyte common antigen, was present at levels > 2 U/ml in six out of 12 cases and in only two of these did it reach 20% of the total detectable class II material. Thus the only consistently associated antigen was class I. Molecular size In order to estimate molecular size of the class II-containing complex, synovial fluids were first precipitated with 50% saturated ammonium sulphate solution and then passed down an AcA 22 sizing gel column. All the class II material was precipitable with the ammonium sulphate solution and this provided a simple procedure for partial purification and concentration. A typical elution profile obtained from the column separation is shown in Fig. 1. The class II antigen was eluted at the void volume ahead of pentameric IgM, indicating a molecular weight of > 1000 kD. The peak from six fluids was similar in being slightly asymetric with a trailing shoulder, and as shown in Fig. 1, the class II and class I antigens eluted together. No effect of reduction with 2-mercaptoethanol on the elution profile of either antigen was noted. Release of class II antigen from a cell line In order to gain insight into the ability of dispersed cells of known phenotype to release class II antigen into the surrounding fluid, a cell line (FLE) was studied. It is an EBV-transformed human B cell and, as is typical of such lines, expresses large amounts of class II antigen at the cell surface when examined by immunofluorescence. Analysis of cells, lysed in 0 5% NP40, by the ELISA gave a figure of 173 U/107 cells for total cellular class II antigen. Cells were incubated in medium alone with swirling for 6 or 21 h and the amount of MHC antigen in the supernatant

Soluble HLA antigens in rheumatoid arthritis 120

35 0

100

20

-2 80 N

1D

40

B-! C,

(n

cn 0

._e(n

60 60

c E

(n

U 40 0

80

20

100 0

Tube number

Fig. 1. Separation of synovial fluid by gel filtration. Synovial fluid was precipitated with ammonium sulphate (50% saturation) and passed through a column of AcA22 (1-6 x 80 cm). Fractions (2 5 ml) were monitored for protein (% transmission) at 280 nm (-) and assayed for HLA class II (0) and class I (-) by ELISA. Table 3. Effects of stress on release of HLA antigens from a human B cell line Soluble (U/107 cells)

Incubation time* Stress

(h)

Class II

Class I

Viability (%)

None

6 21

08 17

0 0

80 70

Cycloleximide

6 21

0-2 17

0 02

80 0

Irradiation

6 21

04 2-4

0 09

50 30

Water lysis

0

0

0

0

* Cells were incubated at 37°C for the times indicated in medium alone, or in the presence of cycloleximide, or following irradiation. Cells exposed to water lysis were not incubated.

was measured by immunoassay. As shown in Table 3, little material was released spontaneously during this time even though the viability fell to 70% at the cell density used (107/ml). If the protein synthesis inhibitor cycloheximide was included in the culture medium, viability after 21 h was essentially zero, but there was still no significant release of MHC antigens. Similarly, prior irradiation of the cells which induced an early death did not bring about production of soluble antigen. Finally, complete lysis of cells by exposure to water did not generate soluble MHC material. Thus there was little spontaneous release of MHC antigens by this cell line and it was not possible to induce release by various kinds of cell trauma.

DISCUSSION There is considerable interest in the apparent ability of cells to release molecules, usually identified as integral membrane proteins, into the surrounding fluids. Since some of these molecules have described functions as cellular receptors, the potential biological importance of this 'soluble' material is clear. Thus one of the polypeptide chains of the IL-2 receptor is found in supernatant fluids from activated T lymphocytes and is able to bind IL-2, thereby reducing its availability to local cells (Robb & Kutny, 1987). The molecular nature of the released receptor, however, is not clear since a variety of molecular weights have been reported suggesting either polymerisation or binding to other molecules, and at least some of the released protein appears to arise by proteolysis (Robb & Kutny, 1987). Since several types of receptor may be produced during cell activation, measurement of 'soluble' receptors could provide some index of disease activity in inflammatory disorders such as rheumatoid arthritis. The availability of monoclonal antibodies against a variety of receptors has allowed the establishment of immunoassays where a 'capturing' antibody is coated to a plastic plate and the bound antigen, which could be a soluble receptor molecule, is then detected with a second enzyme-labelled antibody. The major problem with this technique is that IgM RF, found frequently in serum and fluids of patients with autoimmune diseases, will link together the capturing antibody and detecting antibody in the complete absence of putative antigen. The presence of these RFs in healthy subjects or in patients has been pointed out in relation to problems consequent on administration of xenogeneic monoclonal antibodies, particularly for tumour localization (Courtenay-Luck et al., 1987). However, the potential interference in the multiplicity of assays now available for various serum markers has received little attention,

36

F. K. Stevenson et al.

apart from a few cautionary tales on specific assays (De Clerck et al., 1988). Thus, unless steps are taken to remove RF or to control the assay by leaving out antigen, results will be misleadingly positive and may well show correlation with disease as can levels of RF. We describe a simple procedure, which is a modification of that used previously to detect IgG RFs (Carson, 1985), which removes the problem. Another potential source of interference is the presence of antibody against rodent IgG in patients' fluids. Antibodies against mouse IgG have been reported to interfere with commercial kits designed to measure human serum chorionic gonadotrophin (Clark, Raggatt & Price, 1985). In our experience with patients with rheumatoid arthritis, this has been only a minor problem. One group of cell surface glycoproteins which is released by activated cells is that associated with the MHC. Class I and II antigens have been found in supernatants from activated cells of both mouse and human origin (Emerson, Murphy & Cone, 1980; Capobianchi et al., 1985), and similar material has been detected in serum (Allison et al., 1977). Actual membrane vesicles have been identified in the blood of patients with various types of leukaemia (Carr, Dvorak & Dvorak, 1985) and we and others have reported the presence ofclass II antigen in the serum of some patients with acute lymphoblastoid leukaemia (Herlyn et al., 1984; Stevenson et al., 1986). This might reflect the large numbers of blast cells in the circulation of such patients which may be shedding cell surface components, since in normal subjects clearance of injected synthetic vesicles is rapid (LopezBerestein et al., 1984). For patients with rheumatoid arthritis no class II could be detected in serum and it is possible that locally produced activation products either do not enter the blood, or that they are rapidly cleared. Thus the class II material does not present a useful monitor of disease in the serum but the variable levels in joint fluid might provide an indicator of the inflammatory process. Correlations of levels in SF with clinical status are being carried out both on defined patient groups and on individual patients during various therapeutic schedules. The large size of the class II-containing material suggests that it is shed from cells as has been observed for other cell types (Emerson et al., 1980). Previous reports have indicated that the process of release is an active one which ceases in dying cells (Sachs et al., 1980; Black, 1980), and also that certain cell surface components are released preferentially (Black, 1980). Although it was not possible to mirror the complex cell populations in the synovium, the failed attempts to bring about release of class II antigen from a B cell line by applying various forms of stress supports the view that cell death does not give rise to 'soluble' material. It also appears that released class II is not as random cell surface vesicles, carrying multiple cell surface antigens, but is selective for the class II antigen together with some class I. A physical association of class II with some class I antigen at the cell surface has been indicated by co-capping experiments and this may have functional significance (Neppert & MuellerEckhardt, 1984). The physiological importance of the shed material is not yet understood although its ability to induce allogeneic cytotoxic T cell responses has been described (Meeusen, 1987). The 'soluble' class II antigen could have a role in presenting antigen to T cells and it has been established using labelled antigen that release can occur from macrophages in a vesicular form (Harding, Leyva-Cobian & Unanue, 1988). Thus it can be conjectured that the presence of large amounts of available class

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SACHS, D.H., KiSzKiss, P. & KIM, K.J. (1980) Release of Ia antigens by a cultured B cell line. J. Immunol. 124, 2130. STEVENSON, F.K., GEORGE, A.J.T., WALTERS, M.T. & HAMBLIN, T.J. (1986) Analysis of soluble HLA class II antigenic material in patients with immunological diseases using monoclonal antibodies. J. immunol. Methods, 86, 187.

Soluble histocompatibility antigens in synovial fluids of patients with rheumatoid arthritis.

Soluble histocompatibility antigens of the class II region have been detected in synovial fluids obtained from patients with rheumatoid arthritis. A c...
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