Interleukin-10 Response Abnormalities in Systemic Lupus Erythematosus

Scand. J. Immunol. 46, 406–412, 1997

Interleukin-10 Response Abnormalities in Systemic Lupus Erythematosus A. E. MONGAN*, S. RAMDAHIN† & R. J. WARRINGTON*† Departments of *Immunology and †Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

(Received 25 March 1997; Accepted in revised form 25 June 1997)

Mongan AE, Ramdahin S, Warrington RJ. Interleukin-10 Response Abnormalities in Systemic Lupus Erythematosus. Scand J Immunol 1997;46:406–412 It has been previously reported that the production of interleukin-6 (IL-6) is often enhanced in systemic lupus erythematosus (SLE). The authors examined the secretion of IL-6, tumour necrosis factor-a (TNF-a), granulocyte–macrophage colony-stimulating factor, IL-1a and IL-4 by B cells and monocytes from lupus patients and compared this to the production in normal controls and in rheumatoid arthritis patients. IL-6 production was increased an average of 3.4-fold compared to that in normal subjects and 8.4-fold compared to rheumatoid arthritis patients. In SLE, a strongly positive correlation was found between the levels of IL-6 and TNF-a (R ¼ 0.8987, P ¼ 0.002). Since production of both IL-6 and TNF-a is regulated by IL-10, the enhancement of the production of these cytokines could reflect a defect in either IL-10 production or responsiveness. However, spontaneous production of IL-10 was enhanced in cultures of B cells and monocytes from lupus patients, compared to normal controls, the levels being increased 3.1- to 6-fold for monocytes and B cells, respectively. The finding of increased secretion of these cytokines implies an abnormality in IL-10-mediated suppression in SLE. To assess this possibility, the authors examined recombinant human IL-10-mediated suppression of IL-6 production by monocytes and B cells from lupus patients, compared to normal controls, and found that whereas IL-10 caused a concentration-dependent suppression of IL-6 production in normal B cells and monocytes, this suppression was deficient in B cells and monocytes from lupus patients. In SLE, it therefore appears that there may be an intrinsic defect in IL-10induced suppression of cytokine synthesis. This could explain the increased levels of IL-10 and IL-6 found in this condition, and may also be responsible for the characteristic polyclonal B-cell activation that is seen. Richard Warrington, Rheumatic Disease Unit Research Laboratory, Departments of Medicine & Immunology, University of Manitoba, RR014, 800 Sherbrook Street, Winnipeg, Manitoba, Canada R3A 1M4

INTRODUCTION Numerous studies have demonstrated increased levels of circulating interleukin-6 (IL-6) in systemic lupus erythematosus (SLE), or have found either enhanced production of this cytokine in vitro by mononuclear cells from lupus patients, or hyperactive B-cell responses to this cytokine [1–12]. Major sources of IL-6 are monocytes/macrophages and B cells. The production of IL-6 and associated cytokines, such as tumour necrosis factor-a (TNF-a), granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-1 etc., is regulated by the inhibitory cytokine IL-10 [13]. The abnormal increase of IL-6 in SLE could be the result of defects in IL-10-mediated regulation, such as defective IL-10 production or the presence of IL-10 inhibitors in SLE; or defective IL-10 responsiveness, resulting in lack of normal 406

inhibition of cytokine production. The first possibility is unlikely to occur, since increased IL-10 production has been reported in SLE by other investigators [14, 15]. The enhanced production of the above cytokines in SLE could have significant implications for B-cell hyperactivity and the induction autoimmune responses. Increased levels of IL-10 and IL-6, as well as the associated cytokines TNF-a and GM-CSF, could enhance B-cell proliferation and differentiation [16–19]. Exogenous IL-6 has been shown to promote murine lupus, while specific monoclonal antibodies (MoAb) to IL-6 and IL-10 delay the development of this disease in NZB/W F1 mice [20, 21]. An intrinsic overproduction of these cytokines might also explain the pre-existing B-cell hyperactive state reported in family members of SLE patients, as well as in lupus patients themselves [22–26]. Defects in responsiveness to IL-10 may enhance the generation q 1997 Blackwell Science Ltd

IL-10 in SLE 407 of neoantigens on nucleic acids and nuclear proteins during apoptosis, because IL-10 also exerts an inhibitory effect upon the generation of reactive oxygen metabolites, which can modify such antigens [27–29]. Nuclear antigens expressed in a modified form on apoptotic cells may be immunogenic for T helper cells lacking tolerance to modified self, which could lead to the generation of high-affinity autoantibodies characteristic of SLE [30]. Thus IL-10 production and response abnormalities may have a central role to play in the pathogenesis of SLE. We therefore decided to test the hypothesis that enhanced cytokine production as indicated by the secretion of IL-6 might result from defective IL-10 responsiveness in SLE, by examining the pattern of cytokine production and the inhibitory effects of recombinant human IL-10 on spontaneous and lipopolysaccharide (LPS)-stimulated IL-6 production by B cells and monocytes in this disease, in comparison to control subjects.

MATERIALS AND METHODS Donors. Venous blood samples were obtained from patients with SLE, rheumatoid arthritis, or from normal donors. The diagnosis of disease in these patients was made based upon the revised American Rheumatism Association (ARA) criteria. The comparison between SLE patients and disease control rheumatoid arthritis patients was carried out on samples from patients judged to have active disease clinically, based upon the scoring index of Gawryl et al. [31] for lupus patients and for rheumatoid arthritis patients, the presence of six or more swollen joints tender to pressure, an hour or more of morning stiffness or a Westergren erythrocyte sedimentation rate > 30 mm/h. In the assessment of IL-10-induced suppression of IL-6 production, the lupus patients studied were heterogeneous in terms of disease activity. Their mean age was 43.6 6 15.1 years, disease duration 6.9 6 4 years, and only 20% of patients were judged to have active disease. Three patients were taking prednisone 20 mg per os daily or less, with two of these also on azathroprine, while one was receiving hydroxychloroquine. Reagents. Recombinant human IL-6 was obtained from Genzyme Corp. (Cambridge, MA, USA) and recombinant human IL-10 was kindly donated by Dr S. Narula (Schering-Plough Research Institute, Kenilworth, NJ, USA). MoAb to CD2, CD19, CD14 as well as isotype controls, were purchased from Cedarlane Laboratories Ltd (Hornby, Ontario, Canada). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6, IL-1, TNF-a, GM-CSF and IL-4 were obtained from R & D Systems (Minneapolis, MN, USA). The ELISA assay kit for IL-10 was obtained from Perspective Diagnostics (Cambridge MA, USA). Biotinlabelled goat anti-human IgG and IgM were purchased from ClonTech Laboratories (Palo Alto, CA, USA). Mouse anti-human IL-10 antibody was supplied by Serotec Ltd (Oxford, UK) and an isotype control, 3S3, was provided by Dr J. Wilkins (University of Manitoba, Canada). Peripheral blood mononuclear cell (PBMC) preparation. PBMC were prepared by Histopaque 1077 (Sigma Chemical Co., St. Louis, MO, USA) density gradient centrifugation and T cells were removed by sheep red blood cell-rosetting. Monocytes were purified from Histopaque 1077-isolated PBMC by adherence on 100 × 15 mm Petri dishes overnight at 378C in RPMI-1640 with 20% fetal calf serum (FCS; GibcoBRL, Grand Island, NY, USA). The washed unbound cells were then passed through a nylon wool column (Fenwall Laboratories, Deerfield, IL, USA), to separate T cells from B cells and the B cells were then eluted [32]. Purity of the cell preparation was determined by flow

cytometry using fluorescein-isothiocyanate (FITC)-labelled mouse monoclonal anti-CD2, phycoerythrin-labelled anti-CD19, FITC-labelled anti-CD14, and biotin-labelled anti-human IgG and IgM, followed by avidin–FITC, using appropriate isotype controls. Purity of these preparations was shown to be > 90% for T cells and > 95% for B cells and for monocytes. The contaminating cells in the B-cell and monocyte fractions were T cells, which did not contribute to the total IL-6 production in vitro. IL-6 production by B cells and monocytes. T cell-depleted B cells and monocytes were incubated for 4 days initially in F24 wells at a cell density of 1 × 106 cells/ml, with RPMI-1640 and 10% heat-inactivated FCS, at the end of which time, supernatants were collected by centrifuging the cells and tested for IL-6, TNF-a, GM-CSF, IL-1 and IL-4 production. In subsequent experiments, purified preparations of B cells, monocytes and T cells were incubated at increasing cell densities from 625 to 104 cells per well in 96-well plates with RPMI-1640/10% FCS for 4 days, following which supernatants were removed for cytokine assay. When the effects of recombinant IL-10 on cytokine synthesis were assessed, IL-10 was added at varying concentrations from 5 to 15 ng/ml and the suppression of IL-6 synthesis, both spontaneous and in response to LPS (10 mg/ml; Sigma) was determined using the IL-6 responsive cell line B9. For statistical analysis of IL-10-induced suppression of IL-6 production, cultures that produced equivalent levels of IL-6 in the absence of exogenous IL-10 were compared. IL-6 production in the presence of increasing IL-10 concentrations at these cell densities was then assessed using monocytes and B cells from lupus patients and from normal subjects. Cytokine assays. Cytokines TNF-a, GM-CSF, IL-1 and IL-4 were measured in supernatants by ELISA (R & D Systems), according to the manufacturer’s instructions, at cell densities of 1 × 106 cells/ml. The sensitivities of these assays were as follows: TNF-a, 4.4 pg/ml; GMCSF, 1.5 pg/ml; IL-1, 0.3 pg/ml; IL-4, 3 pg/ml. IL-10 in the supernatants was measured by ELISA (Perspective) according to manufacturer’s instructions, using cell cultures containing 2.5 × 103 cells/well in 96well plates. The sensitivity of this assay was 5 pg/ml. IL-6 secretion was measured by the IL-6-dependent growth of the murine B-cell hybridoma cell line B9 (kindly provided by Dr L. van Aarden, Dutch Red Cross Laboratory, Amsterdam, the Netherlands) which was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma), a colorimetric assay for cell proliferation [33]. Quantification of IL-6 production was determined in relationship to recombinant human IL-6 (Genzyme). The specificity of this response was demonstrated using a rabbit anti-human IL-6 antibody (Genzyme). The sensitivity of the assay was 1.5 pg/ml. The B9 cell line did not respond to IL-10 alone at concentrations up to 15 ng/ml. However, in the presence of IL-6, B9 proliferation was enhanced by concentrations of IL-10 > 5 ng/ml. Levels of residual IL-10 in B-cell and monocyte culture supernatants tested after 4 days were always less than 1 ng/ml, in the cultures which received IL-10 at the start of the incubation period. Statistical analysis. This was carried out by multifactorial analysis of variance and linear regression analysis using the SAS System (University of Manitoba Statistical Service). Mean and standard deviations from the mean are shown.

RESULTS Spontaneous cytokine production in SLE, rheumatoid arthritis and normal donors Using T cell-depleted mixed B-cell and monocyte cell preparations from the three different donor groups, the spontaneous

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408 A. E. Mongan et al.

Fig. 2. Quantification of IL-10 secretion by B cells and monocytes from normal donors and lupus patients, constitutively and in response to LPS stimulation, showing significant enhancement of IL-10 production by lupus B cells and monocytes compared to normal B cells and monocytes (n ¼ 6).

Production of IL-6 by lupus and normal B cells and monocytes

Fig. 1. Spontaneous (open symbols) and LPS-stimulated (closed symbols) IL-6 production with increasing cell numbers, expressed as percentage of IL-6 standard containing 0.88 ng IL-6/ml, by (A) normal B cells, (B) lupus B cells, (C) normal monocytes, and (D) lupus monocytes (n ¼ 10 for normals, n ¼ 12 for lupus patients). IL-6 production by monocytes was significantly greater than that by B cells for both subject groups (P ¼ 0.002) and production by lupus B cells and lupus monocytes was significantly greater than production by normal B cells and monocytes (P < 0.001).

secretion of cytokines IL-6, TNF-a, GM-CSF, IL-1 and IL-4 was assessed. It was found that IL-6 production was increased in cell cultures from lupus patients (mean IL-6 production 4.7 6 1.94 ng/ml, n ¼ 16) compared to rheumatoid arthritis patients (0.56 6 0.15 ng IL-6/ml, n ¼ 13) or normal control donors (1.4 6 0.52 ng IL-6/ml, n ¼ 12) and the difference between lupus patients and the other two groups was statistically significant (P < 0.025). In the supernatants from eight lupus patients, TNF-a, GM-CSF, IL-1 and IL-4 were also measured. TNF-a was always found with IL-6 in the lupus B-cell and monocyte supernatants, in concentrations ranging from 41 to 215 pg/ml (mean ¼ 75.6 6 59 pg/ml) and the levels of these two cytokines significantly correlated with each other (R ¼ 0.8987, P ¼ 0.002). Of the other cytokines, GM-CSF and IL-1 were only detected when TNF-a was present in high concentrations and IL-4 was not detected.

IL-6 was produced spontaneously or on LPS stimulation only by B cells or monocytes and production was greatest by monocytes in both subject groups. A linear relationship existed between the cell density and production of IL-6. In lupus patients, both spontaneous and LPS-stimulated IL-6 production was significantly increased in B-cell and monocyte cultures (P ¼ 0.001), compared to normal controls (Fig. 1). IL-10 production by lupus and normal B cells and monocytes The levels of spontaneous and LPS-induced IL-10 production by monocytes and B cells from lupus patients were also significantly enhanced compared to normal controls. Again, production was greatest in monocyte cultures and with LPS-stimulation. Spontaneous IL-10 production by lupus monocytes and B cells was 66.25 6 16.34 pg/ml and 30 6 13.7 pg/ml, respectively, compared to 21.25 6 8.92 pg/ml and 5 6 5.6 pg/ml for normal donors. In LPS-stimulated cultures, the differences were also significant, being 101.25 6 18.83 pg/ml for lupus monocytes and 81.25 6 16.72 pg/ml for lupus B cells, compared to 62.5 6 5.59 pg/ml and 33.75 6 7.39 pg/ml for cells from normal donors. These differences were statistically significant, as shown in Fig. 2. IL-10-induced suppression of IL-6 production in lupus and normal B-cell and monocyte cultures In the presence of exogenous recombinant human IL-10 at concentrations from 5 to 15 ng/ml, IL-6 production by normal B cells or monocytes at cell densities from 625 to 104 cells per well was suppressed and this suppression was dose-dependent. Because the production of IL-6 by lupus B cells or monocytes

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IL-10 in SLE 409 Table 1. IL-10-induced suppression of constitutive and LPS-stimulated IL-6 production by B cells and monocytes from normal subjects or from lupus patients

IL-6 production (ng/ml) Cell type

þ/–

IL-10 0 ng/ml

IL-10 5 ng/ml (%)

IL-10 10 ng/ml (%)

IL-10 15 ng/ml (%)

B cells Normal

–

0.5548 6 0.29

Lupus

–

1.0186 6 0.22

Normal

þ

1.1253 6 0.64

Lupus

þ

1.927 6 0.7752

0.3773 6 0.21 (68) P ¼ 0.049 0.8888 6 0.25 (87.3) P ¼ 0.47 0.8752 6 0.53 (77.8) P ¼ 0.23 1.7188 6 0.74 (89) P ¼ 0.53

0.2877 6 0.16 (52) P ¼ 0.004 0.8268 6 0.23 (82) P ¼ 0.26 0.7406 6 0.43 (65.8) P ¼ 0.066 1.6439 6 0.73 (85.3) P ¼ 0.4

0.2415 6 0.16 (43.5) P ¼ 0.0008 0.7763 6 0.2 (76) P ¼ 0.154 0.6112 6 0.36 (54.3) P ¼ 0.016 1.5841 6 0.72 (82) P ¼ 0.3

Monocytes Normal

–

1.2179 6 0.79

Lupus

–

1.3957 6 0.3774

Normal

þ

1.3763 6 0.4463

Lupus

þ

1.7768 6 0.5834

0.8466 6 0.44 (69.5) P ¼ 0.26 1.1994 6 0.42 (86) P ¼ 0.27 1.1672 6 0.31 (84.8) P ¼ 0.2 1.6672 6 0.65 (94) P ¼ 0.71

0.8546 6 0.6 (66.7) P ¼ 0.154 1.173 6 0.38 (84) P ¼ 0.21 1.0014 6 0.36 (72.8) P ¼ 0.022 1.5446 6 0.59 (87) P ¼ 0.43

0.6873 6 0.46 (56.4) P ¼ 0.04 1.0466 6 0.38 (75) P ¼ 0 053 0.8247 6 0.35 (60) P ¼ 0.001 1.4784 6 0.62 (83) P ¼ 0.31

was enhanced, compared to normal donors, we compared where possible, IL-10-induced suppression of IL-6 production at cell densities producing approximately comparable levels of IL-6 in the absence of IL-10. For all lupus cultures, this meant comparing suppression at lower cell densities than for normal controls, i.e. the levels of exogenous recombinant IL-10 per cell added were greater for lupus cells than normal cells. Despite this, there was a significant reduction in IL-10-induced suppression of IL-6

production by B cells and monocytes from lupus patients, whether spontaneous or LPS-stimulated, compared to normal donors (Table 1). The difference in IL-10-induced suppression of IL-6 production in SLE patients compared to normal controls was most evident for unstimulated B cells and for LPS-stimulated monocyte cultures. Indeed, although the levels of IL-6 production decreased slightly with increasing exogenous IL-10 concentrations in lupus B-cell and monocyte cultures, these

Table 2. Percentage suppression of IL-6 production by IL-10 (5 ng/ml) in B-cell and monocyte cultures from normal donors (n ¼ 6) and lupus patients (n ¼ 6) as determined in the B9 cell assay after neutralization of residual IL-10

Cell type

Donors

Spontaneous or LPS stimulated

B cells

Normal Lupus

Spontaneous Spontaneous

58.8 6 5.0 24.9 6 10.5

P < 0.001 P < 0.001

Normal Lupus

LPS-stimulated LPS-stimulated

55.7 6 8.7 29.0 6 9.3

P < 0.001 P < 0.001

Normal Lupus

Spontaneous Spontaneous

61.7 6 6.8 31.3 6 9.9

P < 0.001 P < 0.001

Normal Lupus

LPS-stimulated LPS-stimulated

54.1 6 10.1 26.4 6 9.7

P < 0.001 P < 0.001

Monocytes

q 1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 46, 406–412

% suppression 6 SD

P value

410 A. E. Mongan et al. changes never reached statistical significance for any of the lupus cell populations, whereas a progressive and eventually significant increase in IL-10-induced suppression of IL-6 production was seen in normal B cells and monocyte cultures, whether unstimulated or LPS-stimulated. This difference between normal subjects and lupus patients was not the result of the increased levels of residual IL-10 in the supernatants from lupus patients, since the levels present were below those required to enhance the B9 response to IL-6, i.e. > 5 ng/ml, and these differences persisted, and were in fact enhanced, when this residual IL-10 was neutralized by a specific MoAb to IL-10 (Table 2). The IL-10specific antibody was used at a concentration sufficient to neutralize 12.5 ng of IL-10/ml in the B9 assay and was compared to an isotype control. DISCUSSION SLE is a disease that is characterized by the production of autoantibodies of multiple specificities, although typically, such antibodies are directed at groups of nucleus-related antigens such as DNA, RNA and nuclear proteins [34–39]. Although there is evidence for polyclonality in this response, molecular analysis of autoantibodies suggests an antigen and T cell-driven process, because these antibodies show evidence of somatic mutation in their complementarity-determining regions [15, 40–42]. SLE, both in murine models and in humans, is also associated with a general B-cell hyperactivity, and the presence of this abnormality in family and twin studies of individuals without actual disease suggests that this may be an intrinsic abnormality [15, 22–26]. Additional factors therefore are implicated in the progression to autoimmune disease which presumably occurs on this background of polyclonal B-cell hyperactivity. A number of studies have demonstrated increased IL-6 production in SLE, which could play a role in the enhancement of B-cell differentiation in this condition [1–12]. IL-6 is one of several cytokines including TNF-a and GM-CSF, whose secretion is inhibited by IL-10 and so the possibility exists that enhanced IL-6 secretion in SLE is the result of either reduced IL-10 production or deficient IL-10 responsiveness [13]. But since other investigators have shown that IL-10 production is frequently enhanced in SLE, it would be more likely that the abnormality is in IL-10 responsiveness [14, 15]. IL-6 levels in SLE correlate with disease activity and the administration of IL-6 promotes disease in NZB/WF1 mice, while anti-IL-10 can retard the development of disease in this model [1–12, 16, 17]. The latter findings would not support the presence of IL-10 response defects in SLE, unless this is selective, affecting cytokine production but not IL-10 effects upon B-cell expansion and maturation [13, 21]. Llorente et al. [43] have shown that in vitro antibody production is indeed dependent upon the autosecretion of IL-10 by mononuclear cells from lupus patients. We assessed spontaneous cytokine production by B cells and monocytes in patients with lupus or rheumatoid arthritis and in normal donors and showed that IL-6 production was significantly

enhanced in lupus, compared to the other two donor groups, confirming previous findings by other investigators [1–9]. In addition, we found a direct correlation between secreted IL-6 levels in lupus B-cell/monocyte supernatants and the levels of TNF-a, while other cytokines such as IL-1 and GM-CSF were only detected in the presence of high levels of TNF-a. This linkage suggested that there might be a defect in IL-10 responsiveness, since this cytokine is known to suppress the production of the above group of cytokines. A deficient response to extrinsic IL-10 could reflect either defective IL-10 responsiveness because of some intracellular or receptor abnormality or be due to the occupation of IL-10 receptors by intrinsically produced IL-10. It was found that IL-10 production, both spontaneous and LPS-induced, was increased in lupus B-cell and monocyte preparations, compared to cells from normal donors, but the levels of production were approximately 1000-fold less than the concentrations of recombinant IL-10 required to cause significant suppression of IL-6 secretion in vitro. Nevertheless, the chronic hyperproduction of IL-10 resulting in a reduced expression or persistant occupation of IL-10 receptors in SLE remains to be excluded. The enhancement of IL-10 production, shown by ourselves and others previously, could itself be a reflection of deficient IL-10 responsiveness, since IL-10 exerts self-regulatory effects [13–15]. We next determined the levels of IL-10-induced suppression of IL-6 production by monocytes and B cells in lupus, both spontaneous and LPS-stimulated, and compared this to the suppression induced in cells from normal donors. It was found that a significant defect in IL-10-induced suppression of IL-6 production was present in lupus and this defect occurred in both B-cell and monocyte preparations, being seen when both spontaneous and LPS-induced IL-6 production by monocytes and B cells were assessed. This abnormality was not the result of increased levels of residual IL-10 in supernatants assayed for IL-6, because it persisted when the IL-10 was specifically neutralized with anti-human IL-10 antibody. This defect in IL-10 responsiveness could result in the presence of increased levels of other cytokines, such as TNF-a, IL-1 and GM-CSF. Overproduction of the cytokines IL-6, TNF-a and IL-10 could lead to a polyclonal hyperactivity of B cells [1, 10, 11, 16, 19]. IL-10 may exert an effect upon NFkB production intracellularly, and this action is possibly mediated by an inhibition of the production of reactive oxygen metabolites (ROM) within the cell [30, 44–47]. Defects in the IL-10-induced inhibition of ROMs might affect apoptosis, with the generation of cryptic epitopes or novel fragments of nuclear antigens capable of activating autoreactive T cells [27–29]. Apoptotic keratinocytes have been found to express nucleosomal DNA, Ro, La and small nuclear ribonucleoproteins in membrane blebs and such complexes appear to be necessary for the induction of autoantibodies against nuclear antigens [30, 48–50]. It remains to be determined if the defect in IL-10 responsiveness in lupus is general or restricted to these two cell populations and if such a deficiency would indeed induce polyclonal B-cell activation and an enhanced risk of autoimmunity.

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IL-10 in SLE 411 The demonstration of this abnormality in cytokine suppression may lead to additional insights into the pathogenesis of SLE and suggest new modes of therapy. ACKNOWLEDGMENTS We acknowledge the support of the Arthritis Society (Manitoba Division) and the Department of Medicine, University of Manitoba. A. E. Mongan was supported by the SUDR project, Indonesia. We thank Heather Gray, who typed the manuscript and the Statistical Services, University of Manitoba, who analysed the data. We also express our appreciation to Dr Edward Rector, Department of Immunology, University of Manitoba Flow Cytometry Facility. The recombinant human IL-10 was provided by the Schering-Plough Research Institute (Kenilworth, NJ, USA). REFERENCES 1 Linker-Israeli M, Deans RJ, Wallace DJ et al. Elevated levels of endogenous IL-6 in systemic lupus erythematosus. J Immunol 1991;147:117–23. 2 Spronk PE, Borg EJ, Limburg PC et al. Plasma concentration of IL-6 in systemic lupus erythematosus; an indicator of disease activity? Clin Exp Immunol 1992;90:106–10. 3 Al-Janadi M, Al-Balla S, Al-Dalaan A et al. Cytokine profile in systemic lupus erythematosus, rheumatoid arthritis, and other rheumatic diseases. J Clin Immunol 1993;13:58–67. 4 Ogawa N, Itoh M, Goto Y. Abnormal production of B cell growth factor in patients with systemic lupus erythematosus. Clin Exp Immunol 1992;89:26–31. 5 Linker-Israeli M. Cytokine abnormalities in human lupus. Clin Immunol Immunopathol 1992;63:10–11. 6 Metsarinne KP, Nordstrom DC, Konttinen YT et al. Plasma interleukin-6 and renin substrate in reactive arthritis, rheumatoid arthritis, and systemic lupus erythematosus. Rheumatol Int 1992;12:93–6. 7 Swaak AJG, van Rooyen A, Aarden LA. Interleukin-6 (IL-6) and acute phase proteins in the disease course of patients with systemic lupus erythematosus. Rheumatol Int 1989;8:263–8. 8 Hirohata S, Miyamoto T. Elevated levels of interleukin-6 in cerebrospinal fluid from patients with systemic lupus erythematosus and central nervous system involvement. Arthritis Rheum 1990;33:644– 9. 9 Alcocer-Varela J, Aleman-Hoey D, Alarcon-Segovia D. Interleukin1 and interleukin-6 activities are increased in the cerebrospinal fluid of patients with CNS lupus erythematosus and correlate with local late T-cell activation markers. Lupus 1992;1:111–17. 10 Kitani A, Hara M, Hirose T et al. Autostimulatory effects of IL-6 on excessive B cell differentiation in patients with systemic lupus erythematosus: analysis of IL-6 production and IL-6R expression. Clin Exp Immunol 1992;88:75–83. 11 Kitani A, Hara M, Hirose T et al. Heterogeneity of B cell responsiveness to interleukin 4, interleukin 6, and low molecular weight B cell growth factor in discrete stages of B cell activation in patients with systemic lupus erythematosus. Clin Exp Immunol 1989;77:31– 6. 12 Pelton BK, Hylton W, Denman AM. Activation of IL-6 production by UV irradiation of blood mononuclear cells from patients with systemic lupus erythematosus. Clin Exp Immunol 1992;89:251–4.

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