Clin. exp. Immunol.

(1988) 71, 289-294

IgE rheumatoid factors: quantification in synovial fluid and ability to induce synovial mast cell histamine release
B. GRUBER, D. BALLAN, & P. D. GOREVIC Division of Allergy, Rheumatology and Clinical Immunology, Health Sciences Centre, State University of New York, Stony Brook, New York, USA

(Acceptedfor publication 6 August 1987)

SUMMARY IgE rheumatoid factor activity was found to be significantly elevated (P < 0-01) using an ELISA assay when paired synovial fluid and sera from 13 patients with active RA were compared to ten control samples. Synovial fluid IgE RF activity was higher than predicted by diffusion alone in 7/11 (64%) of the RA synovial fluids studied when coefficents of diffusion were determined. The specificity of IgE RF activity as measured by the ELISA was confirmed using immunoaffinity chromatography. Mast cells, obtained by enzymatic dispersion of rheumatoid synovial tissue, were sensitized with sera containing either IgE antibodies directed against ragweed or IgE with RF activity. Histamine was released upon challenge with anti-IgE antibodies (33-2% + 11), ragweed antigen E (34-6% + 11), or aggregated gamma globulin (47-6% + 17 9). No histamine release was observed if antigen challenge occurred in the absence of appropriate sensitization, with C3 anaphylatoxin, or after immunoadsorption of IgE from sera containing IgE RF activity.
Keywords IgE rheumatoid factor synovial mast cells synovial fluid IgE RF rheumatoid arthritis

INTRODUCTION
Mast cell hyperplasia is a characteristic feature of rheumatoid synovium (Crisp, 1964; Okada, 1973; Athreya, Moser & Schumacher, 1979; Sajveiya, 1983; Bromley, Fischer & Wooley, 1984; Crisp et al., 1984; Godfrey et al., 1984; Freemont & Denton, 1985; Gruber et al., 1986; Malone, Trani & Schwartz, 1986; Malone et al., 1987). Mast cells may subserve a pathogenic role in this chronic proliferative lesion via the modulation of a variety of proinflammatory processes (Wasserman, 1984). A role in connective tissue remodelling is suggested by the ability of mast cell products to stimulate and activate latent collagenase in vitro (Hausen, Lobb & Taylor, 1976; Dabbous et al., 1986; Dabbous, Tipton & Haney, 1986; Gruber, Marchese & Schwartz, 1987), and by their appearance at sites of tissue fibrosis (Wuchmann, 1955; Severson, 1969; Watanabe et al., 1974; Kawanami et al., 1979; Lykke, Schonell & Stewart, 1979; Hawkins et al., 1985). Mast cells accumulate in a number of other pathological states in which an influx of mononuclear cells into connective tissue is seen, such as allograft rejection, bullous pemphigoid, chronic urticaria, chronic peridontitis and Crohn's ileitis (McAulery & Sommers, 1961; Colvin & Dvorak, 1974;
Correspondence: Barry Gruber MD, Division of Allergy, Rheumatology and Clinical Immunology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794-8161,
USA.

Dvorak et al., 1978; Natbony et al., 1983; Shichikawa, TsujiNishioko, 1985; Dabbous et al., 1987). Several recent studies have documented the presence of IgE antiglobulins in the sera of a significant number of patients with active rheumatoid arthritis and rheumatoid vasculitis (Zuraw et al., 1981; Permin & Egeskjold, 1982; Merity et al., 1984; Chatpar et al., 1986). In this study, we test the hypothesis that these IgE rheumatoid factors may sensitize synovial mast cells in vitro and presumably in vivo. This provides a mechanism whereby immune complexes may then cause antigen-specific mast cell activation and secretion. The methodology we have employed involves enzymatic dispersion of rheumatoid synovial tissue to liberate the mast cells (Gruber et al., 1986), passive sensitization in vitro with specific IgE antibodies, and assessment of histamine release following antigenic challenge. Using an enzyme immunoassay previously described (Chatpar et al., 1986), we tested for the presence of IgE rheumatoid factor in synovial fluid, and assessed the ability of highly reactive sera and enriched fractions to sensitize synovial mast cells in short-term culture, and of aggregated IgG to cause immune-specific activation of synovial mast cells.
moto &

MATERIALS AND METHODS Histamine dihydrochloride, human serum albumin (HSA), diamine oxidase (porcine kidney), KC1, CaC12, MgCI, NaOH, chloroform, collagenase type II (Clostridia histolyticum) deoxyribonuclease, hyaluronidase, trypsin (bovine pancreas),

289

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B. Gruber, D. Ballan & P. D. Gorevic

Synovial cells Synovial tissue samples were obtained from patients at the time of open orthopaedic surgical procedures with informed consent under a protocol approved by our Institutional Human Subjects Committee. The tissue was immediately placed in calcium-free Tyrode's buffer and minced into small fragments. Enzyme digestion using collagenase type II (10 mg/ml), trypsin (10 mg/ml) hyaluronidase (10 mg/ml) and deoxyribonuclease (10 mg/ml) proceeded for 45 min at 22C, with gentle stirring. The dispersed cells were then passed over a 200 ym pore nylon mesh (Nytex, Tarrytown, NY), washed and centrifuged at 200g for 10 min. The cells were placed in RPMI media (Gibco Labs, Grand Island, NY) supplemented with 10 mm glutamine, 10% fetal calf serum and 1% antibiotics (penicillin, fungizone and streptomycin; Gibco Labs, Grand Island, NY) and cultured overnight in 5% C02-humidified air. Cytospun smears (Shandon Corp., Sewickley, PA) of the freshly digested cells were prepared and stained with either toluidine blue or Giemsa. These preparations contained approximately 10% mast cells by specific stains, with < 1% contaminating basophils.
Histamine release studies The following day the cells were harvested, washed twice, and passively sensitized by incubation with reactive serum at 1:10 dilution in Tyrodes buffer at 37C for 2 h. The cells were washed and divided into samples of 2 x 105 cells/ml in HEPES buffer. Specific antigens were added to each sample and incubated for 30 min at 37C. The cells were then centrifuged (250 g for 5 min) and the supernatant assayed for histamine and LDH release (Hitachi 75 Autoanalyzer). Total histamine content was determined by boiling the cell suspension for 20 min. Histamine assay Histamine was measured by the double radioenzymatic assay as described by Shaff & Beaven (1979). Histamine-N-methyl transferase (HNMT) was prepared from freshly obtained rat kidneys. All samples were done in duplicate and specificity of the assay was confirmed following digestion with histaminase (diamine oxidase, Sigma). Total histamine was determined by boiling the cells for 10 min. The net % histamine release was determined by (histamine released in sample-spontaneous release) (total histamine) x 100%. The sensitivity of this assay allowed detection of > 0 5 ng/ml of histamine.
RESULTS

HEPES, and alkaline phosphatase type VII were all purchased from Sigma Chemical Co. (St Louis, MO). Sepharose 4-B (Pharmacia Div., Pharmacia Fine Chemicals, Piscataway, NJ) was used to prepare immunosorbent columns by CNBr covalent coupling (Ayen, Porath & Ernback, 1967). Calcium-free Tyrodes buffer was prepared by adding NaCl 8 g, KCI 0-2 g NaH2PO4 0 05 g, glucose 1 0 g, gelatin 1 0 g, deoxyribonuclease 15 mg, at pH 7-4 in I litre of distilled water and filter sterilized. HEPES buffer contained NaCl 8 g, KCl 0 37 g, CaC12 003 g, MgCl2 0-02 g, HEPES 2-4 g, and human serum albumin 0 3 g in I litre of distilled water at pH 7-4. Secretagogues The specificity of the polyclonal rabbit anti IgE and murine monoclonal FCE-specific antibodies used for the ELISA assay has been previously described (Chatpar et al., 1986). Monoclonal anti-IgE (kindly supplied by Dr Andrew Saxon, UCLA School of Medicine) was conjugated with alkaline phosphatase for the ELISA assay used to prepare immunoabsorbents for affinity purification studies, and to remove residual IgE from aggregated IgG used for the ELISA assay. Serum samples with high IgE RF activity were used at 1:10 dilution in phosphate buffered saline (PBS) for synovial mast cell release studies. Serum was obtained from a ragweed antigen E sensitive individual as confirmed by skin tests and used for passive sensitization experiments. Aggregated gamma globulin was prepared by heating Cohn fraction II in PBS at 63C for 30 min; ragweed antigen E was obtained from NIH, Bethesda, MD. The antigens for mediator release studies were diluted in HEPES buffer before use. C3a and C3a des arg were kindly supplied by Dr Anthony Hugh (Scripps Clinic, LaJolla, CA).
Assays for antiglobulin activity and specific proteins The enzyme-linked immunoabsorbent assay used for detection of IgE rheumatoid factor in serum or synovial fluid from patients with seropositive RA has been described (Chatpar et al., 1986). Synovial fluid samples were treated with hyaluronidase (I mg/ml for 30 min at 370C) to reduce viscosity before performing the ELISA. This treatment had no effect on the OD405. Samples were also tested for IgM rheumatoid activity by latex fixation and rate nephelometry (Beckman Instruments, Palo Alto, CA) and total IgE by the PRIST assay (Pharmacia, Piscataway, NJ). Serum and synovial fluid albumin (Sfalb) and c2 macroglobulin levels (Sf 2m) were determined by an automated chemstation (Coulter-DACOS Analyzer, Hialeah, FL) and radial immunodiffusion (CalBiochem Diagnostics, San Diego, CA) respectively. The levels of IgE in the synovial fluids were predicted on the basis of the relative levels of alpha 2-macroglobulin and albumin in the respective samples, using the formula of Hunder & Gleich (1974); Sp x 1/2 (Sfa/Sa + SF

a-a2m/Sa-a2m).
Patients Paired serum samples and synovial fluid aspirates collected from patients for diagnostic purposes were centrifuged at 300 g for 20 min to remove debris. The supernatants were stored at - 20C. All patients in this study fulfilled criteria for definite rheumatoid arthritis (RA) (Committee of the American Rheumatism

Association (1958).

Quantification of IgE rheumatoid factor activity in paired sera and synovialfluids from patients with RA Thirteen paired specimens from patients with active RA were compared with matched synovial fluid and sera from 10 patients with mechanical orthopaedic disorders or degenerative disease. Whereas all control specimens had negligible IgE RF activity (OD405 < 0 1 at 60 min), significantly elevated values (P < 0 01) were found in both synovial fluid (mean OD 405 = 0 31 + 017) and serum (mean OD 405 = 0 37 + 022) of patients with RA compared to controls. These results are compatible with a larger study previously reported (Chatpar et al., 1986). The IgE RF activity (OD450) appeared comparable in the matched sera and synovial fluid samples (P> 0 1). In order to further evaluate this observation, 11 synovial fluid samples were analysed for diffusable proteins. The predicted values were subtracted from

IgE rheumatoidfactor: quantification in synovialfluid

Table 1. Paired sample analysis

291

Patient

ODsf (actual)
0260 0-143 0-193 0-756 0-58 0353 0299 0-319 0 374 0286 0465

ODs 0350 0-195 0-228 1-080 0-234 0352 0304 0-425 0-410 0297 0376

ODsf (predicted)
0 192 0-181 0 216 0-356 0-119 0 120 0456 0-242 0-246 0095 0312

Difference
+0068 -0038 -0 023 +0 400 -0-061 +0233 -0-157 +0 077 +0-128 +0 191 +0 153

I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

A.F. R.L. V.C. A.V. G.B. D.L. B.L. R.A. R.G. E.S. R.P.

OD450 determined by ELISA for IgE RF activity. Actual readings at 60 min given for synovial fluid (sf) samples and matched sera (s). This is compared to the OD450 of synovial fluid predicted by measurements of diffusable proteins (see Materials and Methods). The last column represents the difference of the measured OD450 for synovial fluid and the predicted GD450, suggesting local production if (+).
the actual measured activities in synovial fluid and the difference calculated (Table 1). Seven of the eleven samples measured were higher than predicted by these calculations, thus suggesting local production of IgE-RF. The enzyme immunoassay used was linear over the values examined when myeloma IgE was used as substrate.

Im

'I

-P 0

.a *f

Specificity of IgE RF activity Several markedly positive sera were selected to further define the specificity of our assay. Samples of 1 ml were passed over a monoclonal anti-IgE immunoadsorbent Sepharose 4-B column (10 ml bed volume) and eluted with 3 M potassium thiocyanate. Both the column wash and the eluate were concentrated by Amicon ultrafiltration to the original volume and then tested for IgE RF activity. Results of a representative experiment are shown in Fig. 1 and were duplicated with two other samples. IgE RF activity was removed by passage over an anti IgE immunoadsorbent or by pre-incubation overnight at 40C with aggregated y-globulin (I mg/ml). Similar results were obtained with a positive synovial fluid sample. Both sera and synovial fluid IgE RF activity were unaffected by passage over an immunoabsorbent specific for , heavy chain determinants. Furthermore, total IgE assayed by PRIST was 260 ng/ml in the applied material and became undetectable (< 10 ng/ml) in the effluent of the anti-IgE column. IgM latex activity was not significantly diminished by the IgE adsorption (Fig. 1). In separate experiments the serum containing high IgE RF activity was subjected to heating at 63C for 1 h to inactivate IgE. The IgE RF activity was no longer detectable ( < 0 1 at 60 min) by ELISA (data not shown).
Synovial mast cell preparations High titre and control sera were then selected for passive sensitization of synovial mast cells (Lichtenstein & Osler, 1964). First, dispersed cells from seven rheumatoid synovia were incubated with sera which contained IgE antibodies specific for ragweed antigen E. Samples containing 106 cells were challenged with monospecific polyclonal anti-IgE (1 pg/ml), ragweed

8c
. z w

JL1

(I mg/mi) Fig. 1. The specificity of the ELISA for IgE RF activity (0) is indicated by immunoadsorption of a markedly positive serum sample over an anti-IgE absorbant. The latex fixation (LF) activity (-) was not significantly changed, as compared to the marked decrease in IgE RF activity. The activity was recovered in the eluate. The IgE RF and LF activity of the same serum was inhibited by preincubation overnight with soluble aggregated globulin, as shown.

following IgE immunoodsorpion

RA serum

RA serum with oggregatd

y

globulin

individual subsequently responded to challenge with antigen E. Cells incubated with control serum followed by ragweed challenge released less than 5% total histamine (Fig. 2). Both C3a and C3a Des Arg failed to cause significant histamine release, even at 10-5 M concentration (n = 8), even though the C3a induced an immediate wheal and flare reaction in volun-

antigen E (1 ,ug/ml), or with C3a (I0-5-10 -M). As indicated in Fig. 2, synovial mast cells from seven RA patients released a mean of 33-2% ( 11 -0) and 34-6% ( 11 -0) of total histamine following challenge with anti-IgE and ragweed antigen E. The requirement for specific IgE was confirmed by sensitizing half of the synovial cells with control serum and the remainder with serum containing IgE antibodies against ragweed antigen E. Only the cells sensitized with serum from a ragweed-sensitive

292
Sensitization (60 min)
None

B. Gruber, D. Ballan & P. D. Gorevic

Cha l lenge (30 min)
0
9

% Histamine release
10
I

20
I

30
I

40
I

50
I

None

Control

serum

Anti-IgE

/Lg/ml
i

IgE anti-ragweed AgE

Ragweed AgE

plig/ml

Control

serum

Ragweed AgE

-5

Control serum

C3a

(10

M)

Fig. 2. Net % histamine release following sensitization and stimulation is indicated. These data represent six separate experiments in which dispersed rheumatoid synoviocytes were challenged with specific secretagogues. The bar is the mean + s.e.m. Spontaneous release was <5%.

Sensitization (60 min)

None

Cha l lenge (30 min) 0

10

% Histamine release 30 20
I

40
I

50
I

None

IgE RF

serum

None

IgE RF

serum

Agg.f globulin

Lg/ml

Control

serum

Agg.

r globulin lp g/ml

Agg./ globulin /g/ml IgE RF serum after a IgE absorbent Fig. 3. Net % histamine release following sensitization and stimulation as indicated. The bar represents the mean + s.e.m. histamine release from six synovium, except in the IgE RF sera depleted of IgE (n = 3). Spontaneous release was < 5% in all the experiments.
to the same concentration of aggregated y-globulin. A specific

teers upon intradermal challenge and caused histamine release

from normal human blood basophils (Hugli & Muller-Eberhard, 1978). Sera found by ELISA assay to have high IgE RF activity were then used to passively sensitize dispersed synovium from five different RA patients. In each experiment, cells were challenged with aggregated y-globulin (1 pg/ml). Controls included challenge with the same concentration of IgG either without prior sensitization or following sensitization with control sera. A mean of 47-6% + 17 9 of total histamine was released following sensitization with IgE RF serum (Fig. 3). Cells from nine different dispersed synovium sensitized with control serum released less than 2% of histamine when exposed

role for IgE antibodies in the sensitization obtained with RA sera was further indicated by the observation that the same serum immunoabsorbed over an anti IgE column (Fig. 1) failed to release histamine to any significant extent (Fig. 3). Furthermore, antigens were not cytotoxic to the cells since > 95% of the cells excluded trypan blue after activation, and no LDH could be detected in the culture supernatants.

DISCUSSION In previous studies, we noted the presence of significant numbers of mast cells in rheumatoid synovium, and demon-

IgE rheumatoidfactor: quantification in synovialfluid

strated responsiveness of these cells to specific secretagogues and a histamine-releasing cytokine. Immunofluorescence studies demonstrated binding of monoclonal IgE to the surface of these cells (Gruber et al., 1986). In the present report, we have presented data showing that these cells are capable of releasing histamine following sensitization with specific IgE antibodies, including RA sera found to have high titres of IgE antiglobulins by an ELISA assay. Rheumatoid factors of the IgE isotype have been shown by several groups to be present in the sera of a significant percentage of RA patients by both ELISA and radioimmunoassays (Zuraw et al., 1981; Permin & Egeskjold, 1982; Merity et al., 1984; Chatpar et al., 1986). The significance of these antibodies has remained uncertain. Although Permin et al. (1978; 1983) have reported immune-specific triggering of basophils isolated from peripheral blood of patients with RA, the percent release was modest and, in our hands, negligible (unpublished observations). By contrast, synovial mast cells sensitized with RA sera containing IgE RF activity by ELISA assay released a mean of 47-6% total histamine in response to IgG. Quantification of IgE RF relative to specific proteins in synovial fluid and serum suggested local production in 64% (7/11) of paired samples analysed. By contrast, none of 10 synovial fluid samples from patients with degenerative joint disease or postraumatic arthritis had detectable IgE RF activity. Further understanding of the contribution of local production to elevated titres in synovial fluid will require study of short term explants or cultured synovial mononuclear cells, as has been demonstrated for rheumatoid factors of other isotypes (Hannestad & Mellbye, 1967; Munthe & Natvig, 1972; Smiley et al., 1985). IgE RF secreted by synovial plasma cells may bind to Fc epsilon receptors on neighbouring mast cells. Aggregation of IgE receptors and subsequent membrane events which lead to degranulation might then be initiated following exposure to an antigen complex such as self-aggregating IgG (Munthe & Natvig, 1972). Complement activation, which has been well documented in the rheumatoid joint space (Perkin & Zvaifler, 1964; Morley & Ruddy, 1985; Mollaes & Paris, 1986) provides a potentially important alternative pathway to mast cell degranulation via the generation of fragments with anaphylatoxic activity (C3a, C5a, C4a). However, C3a and C3a Des Arg had no apparent effect on dispersed synovial mast cell preparations. We cannot exclude the possibility that higher levels of C3a ( > 10- I M) may be generated in vivo contiguous to synovial mast cells and thus induce degranulation, or that C3a receptors are degraded during enzymatic digestion of our synovial tissue preparations. Nevertheless, our results are consistent with reports of other investigators studying mast cells derived from pulmonary and intestinal tissue (Warner et al., 1987). In fact, at the present time, only human dermal and cardiac mast cells have been shown to respond to the complement-derived anaphylatoxins (Hugli & Muller-Eberhard, 1978) and this may be mediated indirectly (Yancey et al., 1987). Increased numbers of mast cells have been documented in both RA synovium and joint fluid (Crisp, 1964; Okada, 1973; Athreya, Moser & Schumacher, 1979; Sajveiya, 1983; Bromley, Fischer & Wooley, 1984; Crisp et al., 1984; Godfrey et al., 1984; Freemont & Denton, 1985; Gruber et al., 1986; Malone et al.,

293

1987). These cells may influence the local vasculature by enhancing permeability, thus allowing macromolecules and effector cells to enter into synovial tissue from the vascular space. Products released from mast cells may lead to an increase in fibroblast activity and may stimulate endothelial cell migration (Wasserman, 1984). In addition, mast cell lysates have been shown to induce synovial adherent cells to release collagenase and PGE, and a specific mast cell protease (tryptase) is capable of activating latent collagenase (Gruber et al., 1987). The precise role of these cells in the remodelling ofconnective tissue is under current investigation in our laboratory. If mast cells and their products contribute to the pathogenesis of rheumatoid synovitis, it is necessary to propose pathways for activation and secretion, and document the presence of activated mast cells in situ. We have presented data in this report to support our contention that synovial mast cells are capable of IgE-mediated degranulation in vitro, and have previously documented mast cell activation in situ by ultrastructural analysis (Gruber et al., 1986). Approximately one-third of rheumatoid patients have detectable circulating IgE RF in the serum by our assay (Chatpar et al., 1986) and here we show comparable levels in the synovial fluid. Aggregated or complexed IgG, at levels comparable to that used in our challenge studies, may be generated locally in the rheumatoid joint space, as has been shown for IgG and IgM rheumatoid factors (Munthe & Natvig, 1972). Alternative pathways to mast cell secretion include biologically active specific cytokines (Gruber, Haak-Frendscho & Kaplan, 1985) or autoantibodies directed against IgE (Gruber et al., in press). In addition, IgE RF might sensitize circulating basophils and contribute to extra-articular features of RA, as suggested by an increased incidence of IgE RF in rheumatoid vasculitis (Merity et al. 1984).

ACKNOWLEDGMENTS
This study was supported by grants available through the Veterans Administration and NIH AM 26588.

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