Rheumatol Int (1991) 10:235-239

RheuUOIoOUY

Clinical and Experimental Investigations

© Springer-Verlag 1991

Induction of IgM and IgM-rheumatoid factor synthesis in vitro by indomethacin J. Hassan 1,, A. Whelan 1, B. Bresnihan g, and C. Feighery 1 1 Department of Immunology, St. James's Hospital, Dublin 8, Republic of Ireland 2 Department of Rheumatology, St. Vincent's Hospital, Dublin 4, Republic of Ireland Received May 31, 1990/Accepted September 12, 1990

Summary. Indomethacin, which is thought to exert its therapeutic effect by inhibiting the synthesis of PGE2, is a commonly used first-line agent in the treatment of rheumatoid arthritis (RA). However, the effect of this drug on the humoral immune response in RA remains unclear. In this study, modulation of the in vitro synthesis o f IgM and IgM-rheumatoid factor (RF) by indomethacin and prostaglandin E2 was examined in 11 patients with active RA and 10 normal controls. Indomethacin at a final concentration of 1 gg/ml significantly enhanced IgM production ( P < 0.01) and R F production ( P < 0 . 0 2 ) in Staphylococcus aureus Cowan I (SAC) stimulated RA cultures when compared to controis in whom no net enhancement effect was observed. In the patients, this increase in IgM production was more pronounced than the corresponding increase in R F synthesis (P = 0.078), suggesting that IgM and IgM-RF-secreting RA plasma cells have different susceptibilities to P G E 2 mediated suppression. Nonetheless, addition of P G E 2 (10-8 M final concentration) to the cultures inhibited IgM and R F production to a similar degree in the patient and control cultures. These findings demonstrate that P G E z causes suppression of IgM and IgM auto-antibody production in vitro and that inhibition of endogenous P G E 2 synthesis in RA patients treated with indomethacin results in a marked increase in the production o f these antibodies.

thesis of PGE2 [2]. The prostanoid, PGE2, has been reported to have a variety of immunomodulatory effects: these include suppression of lectin-induced mitogenesis [3]; inhibition of B cell proliferation [4, 5]; down-regulation of Ia expression [6, 7]; and inhibition of interleukin-2 (IL-2) and gamma-IFN production [8, 9]. Furthermore, increased levels of P G E 2 have been detected in the joints of patients with RA, and PGE2 has been shown to stimulate osteoclastic activity and resorption of bone when added in vitro to bone culture systems [10]. Hypergammaglobulinaemia and the presence of circulating rheumatoid factor (RF) are characteristic features of RA. However, the mechanisms regulating their production remain unclear. Although the results of some studies have indicated that P G E 2 causes increased immunoglobulin [11, 12] and R F production [11] by unseparated peripheral blood mononuclear cells (PBMCs) stimulated by pokeweed mitogen (PWM), other more recent reports have shown significant inhibitory effects of PGEa on immunoglobulin and R F secrection in vitro [5, 13]. In this study, the effect of indomethacin on IgM and IgMRF production in vitro was investigated in an attempt to understand the role of this drug and P G E 2 in patients with active RA.

Key words: Indomethacin - Prostaglandin E 2 - IgM IgM-rheumatoid factor

Patients and controls. Eleven RA patients with active disease classi-

Materials and methods

Indomethacin has been widely used as an anti-inflammatory agent in the treatment of rheumatoid arthritis (RA) [1]. This and similar drugs are thought to act by blocking the cyclo-oxygenase pathway, thereby inhibiting the syn-

fied according to the ARA criteria [14] were recruited for the study. Disease activity was assessed using standard clinical and laboratory indices: duration of morning stiffness, pain (100 mm visual analogue scale), Richie articular score [15], functional score (according to Steinbroker and colleagues [16]), grip strength, joint circumference, erythrocyte sedimentation rate and serum C-reactive protein levels. All patients were seropositive on routine RF latex investigation. None of the patients had received remittive therapy for 3 months prior to entry to the study. Normal controls used were healthy laboratory staff members and were age and sex matched for the patients.

* To whom offprint requests should be sent at: Children's Research Centre, Crumlin, Dublin 12, Republic of Ireland

heparinised venous blood from patients and controls. PBMCs were

Introduction

Separation of peripheral blood mononuclear cells. Weobtained4Oml

236 isolated by Ficoll-Hypaque density gradient centrifugation using Boyum's method [17]. Interface cells were washed twice and resuspended in RPMI 1640 (Gico, Scotland) supplemented with 10% fetal calf serum (Randox Labs, Belfast, Northern Ireland), 2 gg/ml gentamycin (Gibco), 2 gg/ml fungizone (Gibco) and 1% glutamine (Gibco). All cultures were grown in this medium.

Table 1. IgM and IgM-RF synthesis in vitro: response of spontaneous (SPON), PWM and SAC-stimulated cultures in five control subjects and five RA patients

Controls

RA patients

SPON

PWM

SAC

SPON

PWM

SAC

1.28" 1.13

1.16 1.06

4.60 1.40

1.58 1.47

1.48 1.45

3.74 1.60

Cell culture. We plated 200 gl of PBMCs at 1 x 106/ml into roundbottomed 96-well microtiter plates (Nunc, Denmark). Preliminary cell culture experiments were performed to optimise the doses of SAC (Staphylococcus aureus Cowan I, Behring Diagnostics, La Jolla, Calif.), PWM (pokeweed mitogen, Gibco), PGE 2 and indomethacin to be examined. SAC and PWM were investigated at three different doses and the optimum stimulatory concentration was found to be 0.02% final concentration for SAC and 1 : 700 final dilution for PWM. PGE 2 (Sigma, Dorset, UK) was examined at five doses ranging from 10-6 to 10-to M and indomethacin (Sigma) at five doses ranging from 0.1 to 10 lag/ml. Cultures were incubated at 37 °C in a humidified 5% CO z/95 % air incubator. The supernatants were harvested after 7 days by centrifuging out cells at 400 g for 10 min. Supernatants were aspirated and stored at -20 °C until assayed for total IgM and IgM-RF.

ELISA assays. ELISA assays were performed as previously described [18]. Briefly, microtiter plates (Nunc) were coated with 100 gl of goat anti-human IgM (Cappel Laboratories, Cochranville, USA) at a concentration of 1 gg/ml in a coating buffer (carbonatebicarbonate, pH 9.6) and incubated overnight at 4 °C. After washing with phosphate-buffered saline containing 0.05% v/v Tween-20, pH 7.4 (PBS-Tween), 100 gl of neat supernatant was added. Standard dilutions of IgM (Behring) ranging from 0.01 to 5 gg/ml were included on every plate. All samples were assayed in triplicate. After incubation at 37 °C for 30 min, the plates were washed with PBSTween. We then added 100 gl of peroxidase-conjugated goat antihuman IgM (Cappel) at a 1:1000 dilution in PBS-Tween. After further incubation at 37 °C for 30 min, the plates were washed again with PBS-Tween, following which 100 gl of freshly prepared substrate solution (ortho-phenylenediamine in phosphate-citrate buffer, pH 5.0, containing 0.012% H 2 0 2 ) was added. The enzyme reaction was allowed to proceed for 5 min. The reaction was stopped by the addition of 100 ~1 of 2.5 M H2SO 4. The plates were read on a spectrophotometer (Dynatech minireader) at a wavelength of 492 nm and the results were expressed as ELISA Index (mean of the sample/mean of the background). IgM-RF assays were performed similarly except human IgG (1 mg/ml) was used for coating and the peroxidase conjugated goat anti-human IgM F(ab)e was used at a 1:10000 dilution. The reaction time was 20 min and results were expressed as ELISA Index.

Stat&tical analysis. Different groups of experiments were compared using the Wilcoxon-Rank test since the data showed non-parametric distribution. Where appropriate, paired analysis was performed.

Results

I g M and I g M rheumatoid factor synthesis in vitro P r e l i m i n a r y e x p e r i m e n t s were p e r f o r m e d to o b t a i n the o p t i m a l c o n d i t i o n s for e x a m i n i n g the m o d u l a t o r y effects of PGE 2 and indomethacin. Cultures of PBMCs isolated f r o m five n o r m a l a n d five active R A p a t i e n t s were incub a t e d w i t h o u t stimulus ( s p o n t a n e o u s ) o r with S A C o r P W M as stimuli. Table 1 shows t h a t S A C - i n d u c e d cultures gave the o p timal IgM response whereas PWM-stimulated or spontan e o u s cultures p r o d u c e d m i n i m a l levels o f I g M . A s reg a r d s I g M - R F synthesis, similar t r e n d s to t h o s e seen for

IgM IgM-RF

a Results are expressed as the mean ELISA Index. An ELISA Index of 1.0 represents background values

I g M were o b s e r v e d . A g a i n , S A C - d r i v e n systems s h o w e d m a x i m a l s t i m u l a t i o n a n d t h e r e f o r e a l l o w s for a b e t t e r analysis o f the effects o f P G E 2 a n d i n d o m e t h a c i n . C o n s e quently, all f u r t h e r results refer to S A C - i n d u c e d P B M C cultures only. O f interest, cells f r o m three o f the five c o n t r o l s s h o w e d d e t e c t a b l e I g M - R F levels in their supern a t a n t s w h e n S A C was u s e d as the stimulus.

Effect of indomethacin on I g M and I g M - R F synthesis in vitro. F i g u r e 1 a shows the effect o f i n d o m e t h a c i n o n I g M p r o d u c t i o n in S A C d r i v e n c u l t u r e systems in 11 active R A p a t i e n t s a n d 10 c o n t r o l subjects. Total I g M synthesis was significantly e l e v a t e d in r h e u m a t o i d cultures in the presence o f i n d o m e t h a c i n w h e n c o m p a r e d to the c o n t r o l subjects w h o s h o w e d either little e n h a n c e m e n t or i n h i b i t i o n ( m e a n % e n h a n c e m e n t _ + S E : R A : 7 9 . 3 + 3 1 . 4 % ; controls: - 1 2 . 1 + 5.6%; P < 0 . 0 1 ) . S i m i l a r t r e n d s were also o b s e r v e d w h e n I g M - R F p r o d u c t i o n was e x a m i n e d (Fig. 1 b). I g M - R F levels were significantly i n c r e a s e d in the presence o f i n d o m e t h a c i n in R A p a t i e n t s w h e n c o m p a r e d to n o r m a l c o n t r o l s ( m e a n % e n h a n c e m e n t _ _ S E : R A : 27.6+_14.7%; controlS: 11.9 _+5.8%; P < 0 . 0 2 ) . It s h o u l d be n o t e d t h a t in the R A p a t i e n t s , i n d o m e t h a c i n a p p e a r s to e n h a n c e I g M p r o d u c tion to a g r e a t e r e x t e n t t h a n the e n h a n c e m e n t o f I g M - R F , a l t h o u g h this d i d n o t r e a c h statistical significance ( P = 0.078).

Dose response effect of P G E 2 on SAC-induced I g M synthesis. A d o s e r a n g e o f 1 0 - l O M to 1 0 - 6 M final c o n c e n t r a t i o n o f P G E z was e x a m i n e d in five c o n t r o l s a n d five R A patients. F i g u r e 2 shows the r e d u c t i o n in the level o f I g M secreted ( r e p r e s e n t e d as the E L I S A Index), as the d o s e o f P G E 2 was i n c r e a s e d to 1 0 - 6 M in cultures. P G E 2 at 1 0 - lo M d i d n o t alter the a m o u n t o f I g M p r o d u c e d in cultures. H o w e v e r , as the c o n c e n t r a t i o n o f P G E z was i n c r e a s e d f r o m 10 - 9 M to 10 - 6 M , a stepwise r e d u c t i o n in d e t e c t a b l e I g M levels was o b s e r v e d . I n s u b s e q u e n t exp e r i m e n t s , P G E 2 was u s e d a t 1 0 - S M final c o n c e n t r a tion.

Influence o f P G E z on I g M and R F synthesis. F u r t h e r cultures were also p e r f o r m e d in the s a m e 10 c o n t r o l s a n d 11 R A p a t i e n t s to investigate the role o f p r o s t a g l a n d i n E 2 ( 1 0 - S M final c o n c e n t r a t i o n ) o n S A C - s t i m u l a t e d I g M a n d R F p r o d u c t i o n (Fig. 3). A m o d e s t P G E z - m e d i a t e d i n h i b i t o r y effect was n o t e d in b o t h c o n t r o l a n d p a t i e n t g r o u p s . I n these cultures, a s i m i l a r level o f i n h i b i t i o n was

237 a)lgM P~ 0.01 i

b)lgM RF P

Induction of IgM and IgM-rheumatoid factor synthesis in vitro by indomethacin.

Indomethacin, which is thought to exert its therapeutic effect by inhibiting the synthesis of PGE2, is a commonly used first-line agent in the treatme...
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