Increased peripheral T cell reactivity to microbial antigens and collagen type II in rheumatoid arthritis after treatment with soluble TNFalpha receptors

Abstract

Objective: Peripheral T cells from patients with rheumatoid arthritis (RA) are hyporesponsive when stimulated with antigen or mitogen in vitro, possibly owing to increased production of proinflammatory cytokines such as tumour necrosis factor alpha (TNFalpha). This study sought to find out if and how RA T cell reactivity is affected during treatment with etanercept (Enbrel), a soluble TNFalpha receptor.

Methods: Heparinised blood was collected from patients with RA at baseline, after four and eight weeks of etanercept treatment, and from healthy controls. After density separation spontaneous production of interferon gamma (IFNgamma), TNFalpha, interleukin 6 (IL6), and IL10 by peripheral blood mononuclear cells (PBMC) was detected by ELISPOT. For detection of T cell reactivity, PBMC were stimulated in vitro with mitogen (phytohaemagglutinin (PHA)), microbial antigens (purified protein derivative (PPD), influenza), or an autoantigen, collagen type II (CII). Supernatants were analysed for IFNgamma and IL2 content by enzyme linked immunosorbent assay (ELISA).

Results: In RA the number of cells spontaneously producing IFNgamma was significantly increased after four, but not eight weeks' treatment with etanercept. T cell reactivity, as measured by IFNgamma production to PPD, influenza, and CII was significantly increased after four and sustained after eight weeks' treatment, whereas IFNgamma production induced by PHA remained unchanged. TNFalpha production was significantly higher in patients with RA than in controls and did not change during etanercept treatment.

Conclusion: Treatment of patients with RA with etanercept may lead to increased peripheral T cell reactivity both to microbial antigens and to self antigens such as CII. These findings indicate that TNFalpha blockade may not only suppress but also stimulate certain aspects of antimicrobial immune defence and autoimmunity.

Figure 1

The number of cells spontaneously producing interferon γ (IFNγ) increases after four weeks of treatment with etanercept. The number of IFNγ producing cells/100 000 unstimulated peripheral blood mononuclear cells (PBMC) was enumerated by the ELISPOT method. PBMC from healthy controls (HC, n=17) or patients with RA (n=17) were analysed at baseline and after four and eight weeks of treatment.

Figure 2

In vitro antigen reactivity was increased after etanercept treatment. Peripheral blood mononuclear cells (PBMC) were separated from the blood of healthy controls (HC, n=17) or patients with RA (n=17) at baseline and after four and eight weeks of treatment. PBMC were incubated with the microbial derived antigen purified protein derivative (10 µg/ml) (A and B), influenza virus (C), or with collagen type II (100 µg/ml) (D). Supernatants were collected after seven days for the analysis of interferon γ (IFNγ; A, C, and D) and after three days for the analysis of interleukin 2 (IL2) (B). Cytokine measurements were performed using ELISA. Levels of cytokines obtained after incubation without further stimulation (or with acetic acid as a buffer control for collagen type II stimulation) were subtracted.

Figure 3

In vitro mitogen reactivity was increased after treatment with etanercept. Peripheral blood mononuclear cells were separated from the blood of healthy controls (HC, n=17) or patients with RA (n=17) at baseline and after four and eight weeks of treatment and incubated with phytohaemagglutinin (5 µg/ml). Supernatants were collected after three days for the analysis of interferon γ (IFNγ) (A) and interleukin 2 (IL2) (B) using ELISA. Levels of cytokine obtained after incubation without further stimulation were subtracted.