Department of Rheumatology, Guy's, King's and St Thomas' School of Medicine, King's College, Guy's Campus, London SE1 9RT, UK and
1 Aristotle University of Thessaloniki, Fourth Department of Medicine, Hippocration General Hospital, Thessoloniki 540 06, Greece
Correspondence to:
V. M. Corrigall, Department of Rheumatology, 5th Floor Thomas Guy House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
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Abstract |
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Methods. The concomitant production of interleukin (IL)-2, IL-10 and lymphocyte proliferation were studied by enzyme-linked immunosorbent assay and [3H]thymidine uptake, respectively, in 12 normal controls and eight RA patients.
Results. An inverse correlation was found between IL-10 production and proliferation to PPD. The proliferative response was shown to be critically affected by the IL-2:IL-10 ratio so that absolute levels of secreted IL-2 or IL-10 correlated non-significantly with lymphocyte proliferation.
Conclusion. The deficient T-cell proliferation in RA peripheral blood mononuclear cells is related to the relative proportions of IL-2:IL-10 rather than the absolute amounts secreted.
KEY WORDS: T lymphocyte, IL-2, IL-10, Recall antigens
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Introduction |
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The course of many infectious diseases is determined by the ability of the host to mount an efficient Th1 response with eventual eradication of the infective agent [7]. IL-10, which may be produced by both Th1 and Th2 cells in addition to other cells such as macrophages and B cells, has a pivotal role in determining the outcome of disease. In recovery from Mycobacterium leprae infection, IL-2 production was seen in patients who mounted a strong T-cell response via the CD4+ T cells augmented by monocyte production of IL-12, whilst progressive disease was associated with CD8+ T cells, IL-4 and greatly increased IL-10 production by macrophages [8]. Similarly, HIV type 1 gp 120 altered PBMC responses in that, while resting mononuclear cells (MC) produced increased amounts of IL-10, interferon gamma (IFN-) and TNF-
, CD3-stimulated proliferation was deficient as a consequence of reduced IL-2 production. Addition of recombinant IL-2 only partially restored T-cell activity [9]. These observations suggest that the ratio of IL-2:IL-10 may be crucial in determining the degree of T-cell proliferation.
There are numerous studies of serum levels of IL-10 in patients with RA. The findings are contradictory, with investigators finding elevated, normal, reduced or undetectable levels of IL-10 [1013]. However, where IL-10 was present, most researchers agree that it did not correlate with disease activity or drug therapy [10, 13]. An explanation for this may be polymorphic genetic control of the production of IL-10. Three polymorphisms of the IL-10 promoter have been described and these have been suggested to be correlated with in vitro production by concanavalin A-stimulated PBMC [14].
In the present study, we investigated the relationship of IL-2 and IL-10 production and lymphocyte proliferation of RA and control PBMC to phorbol myristate acetate (PMA) plus ionomycin (IONO), as well as to tuberculin PPD.
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Materials and methods |
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PBMC cultures
Heparinized blood, from the patients or normal control subjects, was separated by density centrifugation using lymphoprep (Nycomed, Birmingham, UK). PBMC were washed and cultured in 96- or 24-well flat-bottomed plates at 2x105 cells/well or 106 cells/well, respectively, in tissue culture medium (TCM) (RPMI 1640 supplemented with heat-inactivated 10% fetal calf serum) (Life Technologies, Paisley, UK) either alone or with PPD (20 µg/ml) (Central Veterinary Laboratories, Weybridge, UK), PMA (10 ng/ml) (Sigma, Poole, UK) and IONO (25 ng/ml) (Sigma) at optimal concentrations established in preliminary experiments (data not shown). The cultures in 96-well plates (Costar, High Wycombe, UK) were pulsed with tritiated thymidine (0.2 µCi/well) (Amersham, Little Chalfont, UK) 24 h prior to harvesting with distilled water, using a Skatron harvester, and counted in a liquid scintillation counter (Taurus, ICN, USA) to detect proliferation. Supernatants were aspirated from the cultures in 24-well plates, aliquoted and stored at -70°C until required for cytokine estimation. All cultures were harvested at 24, 72, 120 and 168 h for proliferation and cytokine estimation.
Recombinant human (rhu) IL-10 (0.1100 ng/ml) (Pharmingen, Cambridge Biosciences, Cambridge, UK) and neutralizing anti-IL-10 (clone JES3-1283) at 2 µg/ml (Pharmingen) were added to the cultures as required.
Cytokine estimation
Supernatants were harvested at 24 h for IL-2 and 72 h for IL-10, which were maximal for cytokine detection as shown by time course experiments. The cytokine concentration was estimated by enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies, recombinant human cytokines and conditions as directed by the suppliers (IL-2; Duoset, Genzyme, West Malling, UK) (IL-10; Pharmingen).
Statistics
Proliferation results were expressed as the mean±S.D. of triplicates. Cytokine concentrations were expressed as the mean of duplicate samples. Results were analysed using a paired Student's t-test.
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Results |
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After stimulation with tuberculin PPD, there was no significant difference for IL-10 production between normal and RA patients. However, there was significantly less IL-2 produced by RA as compared with control MC (960±600 vs 5031±4055 pg/ml, respectively; P=0.004). Hence, the IL-2:IL-10 ratio was significantly depressed in RA patients (controls 15.0±14.4 vs RA 3.2±2.6; P=0.04) (Table 1).
Correlation of PBMC proliferation and IL-10 production after stimulation with PPD.
The hypothesis behind this study is that levels of IL-10 secreted by PBMC will correlate inversely with their proliferative capacity. However, there was no correlation between the stimulation of proliferation of control PBMC by PPD and the production of IL-10 (Fig. 1A). By contrast, a significant negative correlation (r=-0.895, P<0.01) was seen with RA PBMC (Fig. 1B
) irrespective of whether production of IL-10 was examined at 24, 72 or 120 h and compared with the proliferation at 120 h (data for 24 and 120 h not shown).
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The addition of neutralizing anti-IL-10 antibody (1 µg/ml) to control PBMC cultured with PPD had no effect on proliferation (Fig. 3). This supports the notion that secreted IL-10 was not limiting for T-cell proliferation in the control subjects. By contrast, addition of the JES3-1283 antibody to RA PBMC culture significantly enhanced the PPD response at 120 h (P=0.008) (Fig. 3
), supporting the notion that a relative excess of IL-10 limited T-cell proliferation.
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Discussion |
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RA PBMC activated maximally with PMA+IONO showed no defect in their proliferative response, although there was a significant reduction in both IL-2 and IL-10 production. Despite this, the IL-2:IL-10 ratio remained similar to that of the controls (RA 2399±1156 vs control 2172±1648). When PBMC were stimulated with tuberculin PPD, which stimulates via the CD3/TCR complex, there was significantly deficient IL-2 production by RA PBMC, but the IL-10 secretion was comparable to control levels. Consequently, the IL-2:IL-10 ratio was significantly lower in RA (control 15±14.4 vs RA 3.2±2.6; P=0.04). This suggested that the deficit in RA T-cell proliferation may reside between the CD3/TCR complex on the cell surface and protein kinase C within the cell. Therefore, to study events distal to protein kinase C, maximal stimulation of PBMC by PMA+IONO was used, thus bypassing the CD3/TCR complex and causing direct cellular activation via protein kinase C and Ca2+ flux. Under these circumstances, the RA PBMC proliferative response was equivalent to normal. Therefore, despite a significant reduction in the absolute amounts of both IL-2 and IL-10 produced by RA patients, a favourable IL-2:IL-10 ratio was maintained, identical to that seen with control MC.
To investigate this phenomenon further, the IL-2:IL-10 ratio was manipulated in vitro. The addition of rhu IL-10 to control PBMC cultures stimulated by tuberculin PPD caused a dose-dependent inhibition of proliferation, indicating that there was a threshold of IL-2:IL-10 ratio below which the IL-2 concentration was insufficient to promote T-cell activation.
Conversely, the addition of neutralizing anti-IL-10 antibody to RA and control PBMC increased T-cell activation in all the RA PBMC cultures, but not the control PBMC response. Control PBMC remained unaffected by the addition of neutralizing anti-IL-10 because there was already a relative excess of IL-2. By contrast, the neutralization of IL-10 antibody in the RA cultures increased the IL-2:IL-10 ratio and increased lymphocyte proliferation without a concomitant increase in the absolute concentration of IL-2. These findings support the hypothesis that the regulation of T-cell proliferation to recall antigens is dependent on a complex balance of IL-2:IL-10 within the cultures.
Since the addition of rhu IL-2 [1] or the addition of excess neutralizing anti-IL-10 antibody could, only partially, restore the defective response of RA PBMC to tuberculin PPD, other factors should be considered. Previous workers have failed to find any correlation between clinical disease activity and serum and/or synovial fluid IL-10 concentration in RA [10, 13]. One reason for this may be regulating factors outwith the activation state of the cell influencing IL-10 production. A recent publication has implied that production of IL-10 may be under genetic control via a polymorphism at a functionally active site (-1087) on the IL-10 promoter gene [14]. There are three possible genotypes: GG, GA and AA; the former being the high-producing genotype and the two latter for the low producers of IL-10. However, our study of control and RA patients shows, on analysis, no evidence for gross genetic control over the production of IL-10 [15] and Keijers et al. [16] in fact suggest that the A allele might be associated with high IL-10 production. With the present conflicting results in this field, the defined IL-10 polymorphisms are unlikely to be the explanation for defective proliferation of RA T cells.
In conclusion, this study indicates the complexities of the responses to recall antigens. The proliferative response remains independent of the actual amount of IL-10 produced. Therefore, the determining factor is the threshold of the IL-2:IL-10 ratio below which deficient T-cell responses are observed.
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Acknowledgments |
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References |
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