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Rheumatology 1999; 38: 1203-1207
© 1999 British Society for Rheumatology

The relative proportions of secreted interleukin-2 and interleukin-10 determine the magnitude of rheumatoid arthritis T-cell proliferation to the recall antigen tuberculin purified protein derivative

V. M. Corrigall, A. Garyfallos1 and G. S. Panayi

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.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To investigate the mechanisms of the deficient proliferative responses by rheumatoid arthritis (RA) peripheral blood T cells to the recall antigen tuberculin purified protein derivative (PPD).

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


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) patients show depressed cutaneous delayed-type hypersensitivity (DTH) responses and decreased lymphocyte proliferation to recall antigens [1–3]—reminiscent of the situation in certain infectious diseases. The exact mechanism of the in vitro RA peripheral blood mononuclear cells' (PBMC) hyporesponsiveness to recall antigens, such as tuberculin purified protein derivative (PPD), is not fully understood. We and others have shown that the defective proliferation is only partially corrected by the addition of recombinant human interleukin (IL)-2 [2]. Reduced IL-2 production, therefore, does not fully explain their deficient reactivity. Additional mechanisms resulting in deficient proliferation may include chronic exposure to tumour necrosis factor alpha (TNF-{alpha}) [4], or other production of a type 2 cytokine, such as IL-10 [5], with a consequent inhibitory effect on T-cell proliferation [6] by altering the IL-2:IL-10 ratio.

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-{gamma}) and TNF-{alpha}, 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 [10–13]. 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.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients and normal control subjects
RA patients and normal control subjects were recruited from the out-patient clinics at Guy's and Lewisham hospitals. The age range for controls was 21–77 yr (mean 51.6 yr), male:female ratio 7:5, and that for RA patients 33–77 yr (mean 59.8 yr), male:female ratio 3:5. RA patients were on a range of non-steroidal anti-inflammatory and/or disease-modifying drugs; 2/8 were on methotrexate, 2/8 on no treatment and 4/8 on non-steroidal anti-inflammatory drugs.

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 (0.1–100 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.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
PBMC proliferative responses to PMA+IONO and PPD
Table 1Go shows the results of PBMC proliferation following stimulation by PMA+IONO for 72 h or PPD for 120 h. The maximum stimulation produced by PMA+IONO resulted in similar proliferation by control and RA PBMC (100695±21292 and 79903±17207, respectively; P=not significant). As previously reported [2, 12], the RA PBMC responses to PPD were significantly lower (P=0.019) than for controls (3200±1236 and 16564±13421 d.p.m., respectively). Although the RA patients were on different drug therapies or untreated, no correlation was observed with proliferative response (results not shown).


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  		TABLE 1.  The proliferation, and IL-10 and IL-2 production of peripheral blood mononuclear cells from normal (N) controls and rheumatoid arthritis (RA) patients. The proliferation was measured by uptake of tritiated thymidine and is expressed as d.p.m. IL-2 and IL-10 were measured by ELISA after 24 and 72 h, respectively, and expressed as the concentration shown
 
Proliferative responses and secretion of IL-2 and IL-10
Production of IL-2 and IL-10.
Following stimulation with PMA+IONO, the concentrations of IL-2 and IL-10 produced by RA PBMC (Table 1Go) were within the normal range, but significantly lower (P=0.004 for IL-2; P=0.006 for IL-10) than those of controls. However, the ratio of IL-2:IL-10 was comparable for the two groups (controls 2172±1648 vs 2399±1156; P=not significant).

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 1Go).

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. 1AGo). By contrast, a significant negative correlation (r=-0.895, P<0.01) was seen with RA PBMC (Fig. 1BGo) 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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  		FIG. 1.  Correlation between IL-10 production and PBMC proliferation. The correlation of production of IL-10 at 72 h of culture with proliferation to tuberculin PPD at 120 h. (A) Ten control subjects (r=0.02, P=NS); (B) six patients with rheumatoid arthritis (r=0.895, P<0.01). Note the difference in the y ordinate between (A) and (B).

 
Addition of rhu IL-10 to control PBMC cultures.
According to the hypothesis, the failure to show a correlation between the proliferative response to PPD and the amount of IL-10 secreted by control PBMC may be due to a relative excess production of IL-2, as shown by a high IL-2:IL-10 ratio (Table 1Go). In order to test this possibility, rhu IL-10 was added to five control PBMC stimulated with PPD so as to reduce the IL-2:IL-10 ratio. Figure 2AGo shows the dose-dependent reduction in proliferation following the addition of rhu IL-10 (0.1–100 ng/ml) to two representative control cultures. The reduction was significant at doses >1.0 ng/ml. In Fig. 2BGo, the percentage inhibition of lymphocyte proliferation induced by rhu IL-10 in all five control subjects is shown; it is of interest to note that the IC50 was remarkably similar for the five subjects studied, being 4.2±1.5 ng/ml.



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  		FIG. 2.  Inhibition of PBMC proliferation in normal controls following addition of rhu IL-10. (A) The proliferation of control PBMC stimulated by tuberculin PPD for 120 h in the absence or presence of increasing concentrations of rhu IL-10 (0.1–100 ng/ml) in two representative control subjects out of five tested. (B) The mean percentage inhibition of proliferation caused by the addition of increasing concentrations of rhu IL-10 (0.1–100 ng/ml) to control PBMC stimulated by tuberculin PPD (20 µg/ml) in the same five subjects.

 
Addition of neutralizing anti-IL-10 antibody to RA and control PBMC cultures.
The converse experiment was to investigate the effect of the addition of the neutralizing anti-IL-10 antibody, JES3-1283, to RA PBMC, as in this situation the poor proliferative response may be due to excessive IL-10 production, as suggested by the low IL-2:IL-10 ratio (Table 1Go).

The addition of neutralizing anti-IL-10 antibody (1 µg/ml) to control PBMC cultured with PPD had no effect on proliferation (Fig. 3Go). 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. 3Go), supporting the notion that a relative excess of IL-10 limited T-cell proliferation.



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  		FIG. 3.  The effect of the addition of anti-IL-10 on N and RA PBMC proliferation. The proliferation by PBMC from RA patients and N controls to PPD (15 µg/ml) in the presence and absence of anti-IL-10 (1 µg/ml). The proliferation by RA PBMC was significantly increased (P=0.008) by the addition of anti-IL-10, while the response by N control PBMC was unchanged (P=0.15 NS).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
A characteristic feature of T-cell function in RA is the reduced proliferative capacity for stimulation via the CD3/T-cell receptor (TCR) complex. The mechanism for this defect is unknown. In this study, we have investigated the hypothesis that differences in IL-2 and IL-10 production, and hence the ratio of IL-2:IL-10, are the determining factors in the T-cell defect.

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.


   Acknowledgments
 
We would like to thank our colleagues for collecting blood samples from the rheumatology clinics, and Mrs Sue Dorward and Miss Sue Haynes for typing the manuscript. This study was supported by Arthritis Research Campaign grant no. C0559 and ICAC grant no. P0526.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1.  Emery P, Panayi GS, Nouri AM. Interleukin-2 reverses deficient cell-mediated immune responses in rheumatoid arthritis. Clin Exp Immunol 1984;57:123–9.[Medline]
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Submitted 30 September 1998; revised version accepted 1 June 1999.
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