Original Article
Clinical
Intracellular Cytokine Production in ANCA-associated Vasculitis: Low Levels of Interleukin-10 in Remission Are Associated with a Higher Relapse Rate in the Long-term Follow-up

https://doi.org/10.1016/j.arcmed.2009.04.001Get rights and content

Background and Aims

Dysregulation of cell-mediated immune response likely plays a role in the pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV), but it has not yet been fully established. The aim of this study was to assess the intracellular cytokine production in patients with AAV at different stages of the disease, in particular, in relation to the long-term prognosis.

Methods

We included 69 patients with AAV and 24 healthy controls. Using flow cytometry, the following intracellular cytokines (IC) were measured in all patients: interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 and interleukin-4 in CD3 + T cells and interleukin-10 (IL-10) and interleukin 12 (IL-12) in monocytes. Patients were then prospectively followed for a median of 43 months and cytokine production was related to the long-term prognosis.

Results

When compared to healthy controls, increased IL-12 production was observed in AAV patients, both active (p <0.01) and in remission (p <0.05). In remission, increased IFN-γ production was also found (p <0.01). IL-10 production was higher in active patients than in patients in remission (p <0.05) but did not differ from controls. Patients in remission who developed a relapse during follow-up had significantly lower IL-10 production than those without relapse (p <0.01). Results of this prospective study of IC production in AAV confirm findings of previous studies measuring circulating cytokine levels.

Conclusions

Activation of the immune system in AAV patients is noticeable even in remission. Patients with AAV display increased IL-12 production, which seems to be counterbalanced by IL-10. Low IL-10 levels in remission are associated with a higher relapse rate in the long-term follow-up.

Introduction

Anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis (AAV) is characterized by necrotizing inflammation of small blood vessels, predominantly of the respiratory tract, skin and nerves, and by pauci-immune crescentic glomerulonephritis 1, 2. Diseases including Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) are strongly associated with the presence of ANCA, which have been known as an important diagnostic tool (3) and usually—but not always—correlate with disease activity 4, 5. In WG, ANCA are typically directed against proteinase 3 (anti-PR3) and have a cytoplasmic type of immunofluorescence (c-ANCA). In MPA, the target antigen is mostly myeloperoxidase (anti-MPO) and the type of immunofluorescence is perinuclear (p-ANCA) 6, 7.

The prognosis of untreated AAV is poor. Current immunosuppressive treatment strategies help to achieve stable remission in most patients. However, in the long-term follow-up, many of the patients relapse (8). Recently, it has been shown that particularly patients with persistent anti-PR3-ANCA positivity, possibly reflecting smouldering immune activation, are at a higher risk of relapse 9, 10. The exact pathogenic mechanisms of systemic vasculitis have yet to be completely understood. Nevertheless, tremendous progress in understanding the disease pathogenesis has been made lately. Although there has been increasing evidence that ANCA might play a direct pathogenic role in triggering AAV (reviewed in References 11, 12, 13, 14), increased markers of T-cell activation (e.g., soluble interleukin-2 receptor, soluble CD30, HLA-DR or CD25) were found in AAV in numerous studies 15, 16. Moreover, contrary to B cells, activated T cells seem to persist even in remission and despite treatment 17, 18.

The cytokine production in AAV has been studied thoroughly both in in vitro studies and in serum samples and peripheral blood mononuclear cells obtained from AAV patients 19, 20, 21, 22, 23. Interestingly, some of the cytokine patterns, e.g., raised IL-6 and lower IL-10, were associated with an increased risk of relapse in previous studies 22, 23. The aim of our study was to measure the intracellular cytokine production in peripheral lymphocytes and monocytes in a cohort of patients with AAV at different stages of the disease. In a prospective follow-up, we assessed the possible relationship between specific cytokine patterns and prognosis of the patients.

Section snippets

Patients and Controls

A total of 69 patients with AAV followed up at the Department of Nephrology, General Teaching Hospital (Prague, Czech Republic) were included in this study during a 16-month period (September 2004–December 2005). The basic characteristics of the patients are summarized in Table 1.

Patients with WG, MPA and Churg-Strauss syndrome (CSS) matched the definitions for those diseases as formulated by the Chapel Hill Consensus Conference (1). Patients with WG and CSS also met the American College of

Clinical Characteristics

During the follow-up (median time: 43 months, range: 1–44 months), 28/69 patients relapsed (40.6%). The median time to relapse was 19 months (range: 2–38 months) after inclusion in the study. Relapse was observed in 19/51 patients who were examined in remission. Clinical characteristics of patients with and without relapse are summarized in Table 3. There was no significant difference in age, gender, ANCA specificity or serum creatinine levels between these two subgroups.

Eight out of 69

Interferon-γ

In our study, intracellular production of IFNγ, the hallmark cytokine of T helper cells type 1 (Th1), did not differ between active patients with AAV and healthy controls (HC) (Table 5). There was also no significant difference between patients with newly active AAV and patients with relapse (Figure 1). On the contrary, patients in remission displayed significantly higher production of IFN-γ than HC and also than patients with active disease (p <0.01, Table 5). However, no significant

Discussion

Both ANCA and T-cell-driven immune response are believed to participate in the complex pathogenic process in AAV. The examination of cytokine production in peripheral blood cells reflects the actual state of T-cell activation, T-cell functions and, eventually, also polarization towards Th1, Th2 or Th17 immune response. Our study results support the hypothesis of persistent immune system activation in remission of AAV and indicate the possible importance of ongoing immunosuppressive treatment in

Acknowledgments

Conflict of Interest Statement. No conflict of interest has been declared by any of the authors.

This study was supported by grant #MZO 00023001 (awarded by the Ministry of Health of the Czech Republic).

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