Analysis of IL-17(+) cells in facet joints of patients with spondyloarthritis suggests that the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response

Abstract

Introduction: In this study, we analysed the number of IL-17(+) cells in facet joints, in the peripheral blood (PB) and synovial fluid (SF) of spondyloarthritis (SpA) patients and compared these results with those of patients with other rheumatic diseases and controls.

Methods: Immunohistochemical analysis of IL-17(+) cells was performed in facet joints of 33 ankylosing spondylitis (AS) patients and compared with data from 20 osteoarthritis (OA) patients. The frequency of IL-17(+)CD4(+) T cells in PB and SF of SpA patients (PB n = 30, SF n = 11), rheumatoid arthritis (RA) patients (PB n = 14, SF n = 7), OA patients (PB n = 10) and healthy controls (PB n = 12) was analysed after stimulation with Staphylococcus aureus Enterotoxin B and phorbol 12-myristate 13-acetate/ionomycin and quantified by flow cytometry.

Results: In AS facet joints, the frequency of IL-17-secreting cells was significantly higher than in samples obtained from OA patients (P < 0.001), with a slight predominance of IL-17(+) cells among the mononuclear cells (61.5% ± 14.9%) compared to cells with polysegmental nuclei. Immunofluorescence microscopy revealed that the majority of IL-17(+) cells were myeloperoxidase-positive (35.84 ± 13.06/high-power field (HPF) and CD15(+) neutrophils (24.25 ± 10.36/HPF), while CD3(+) T cells (0.51 ± 0.49/HPF) and AA-1(+) mast cells (2.28 ± 1.96/HPF) were less often IL-17-positive. The frequency of IL-17(+)CD4(+) T cells in the PB and SF of SpA patients did not differ significantly compared to RA patients, OA patients or healthy controls.

Conclusions: Our data suggest an important role for IL-17 in the inflammatory processes in AS. However, the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response.

Figure 1

In situ analysis and quantification of IL-17⁺ cells in patients with ankylosing spondylitis or osteoarthritis. (a) In situ analysis of IL-17⁺ cells in facet joints of ankylosing spondylitis (AS) (top) and osteoarthritis (OA) (bottom) patients. The specificity of IL-17 staining (top) is shown by blocking the anti-IL-17 antibody with recombinant IL-17 (rIL-17) (middle). (b) and (c) The frequency of IL-17-secreting mononuclear cells and cells with polysegmental nuclei in the bone marrow of AS facet joints was significantly higher in AS than in OA facet joints. *P < 0.001.

Figure 2

In situ immunofluorescence analysis of IL-17⁺ cells. In situ analysis of IL-17⁺ cells in facet joints of ankylosing spondylitis (AS) patients and patients with osteoarthritis (OA) by using immunofluorescence microscopy. Double-staining reveals that myeloperoxidase-positive (MPO⁺) and CD15⁺ cells are the major source of IL-17 expression. The frequency of these cells was significantly higher in AS than in OA (P < 0.05 in both cases). The population of MPO⁺ cells included mononuclear cells and cells with polysegmental nuclei. Th17 cells and mast cells are also a source for IL-17⁺ expression, both of which were significantly higher in AS patients than in OA patients (P < 0.05 in both cases).

Figure 3

Peripheral and synovial CD4⁺IL-17⁺ T cell levels in spondyloarthritis, rheumatoid arthritis and controls. Analysis of (a) peripheral blood (PB) and (b) synovial fluid (SF) CD4⁺IL-17⁺ T cells in spondyloarthritis (SpA) patients (PB n = 30, SF n = 11), rheumatoid arthritis (RA) patients (PB n = 14, SF n = 7), osteoarthritis (OA) patients (PB n = 10) and healthy controls (C) (PB n = 12). Similar levels of PB and SF CD4⁺IL-17⁺ T cells after stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin or Staphylococcus aureus Enterotoxin B (SEB) antibodies are seen when SpA patients are compared to RA patients. The frequency of PB CD4⁺IL-17⁺ T cells was only significantly lower in SpA patients than in controls when stimulated with SEB antibodies (P < 0.05).

Figure 4

CCR6 expression in CD4⁺IL-17⁺ T cells. (a) In three ankylosing spondylitis (AS) patients and three healthy controls, the expression of C-C chemokine receptor type 6 (CCR6) in CD4⁺IL-17⁺ T cells derived from peripheral blood was analysed after in vitro stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin antibodies. Percentages indicate the relative number of CCR6⁺ cells to the total number of CD4⁺IL-17⁺ T cells (top row of dot blot analysis). (b) Using an isotype control antibody after T-cell stimulation with PMA/ionomycin (bottom right) antibodies revealed no positive staining, confirming the specificity of CCR6 staining (bottom left). Without such T-cell stimulation, no CCR6⁺IL-17⁺ T cells were detected.