Higher levels of interleukin IL-17 and antigen-specific IL-17 responses in pulmonary sarcoidosis patients with Löfgren's syndrome

Abstract

Sarcoidosis is a granulomatous disorder of unknown aetiology. The presence of Mycobacterium tuberculosis catalase-peroxidase (mKatG) in sarcoidosis tissue has been reported. T helper type 1 (Th1) responses against mKatG have previously been observed. However, little is known about interleukin (IL)-17 and Th17 responses in sarcoidosis. Here, we investigated the levels of IL-17 and frequencies of IL-17-producing cells responding to mKatG in sarcoidosis patients with different prognosis. Peripheral blood and bronchoalveolar lavage (BAL) cells were obtained from sarcoidosis patients with or without Löfgren's syndrome (often associated with spontaneous recovery), and also stratified according to human leucocyte antigen (HLA) type. Cells producing IL-17 and interferon (IFN)-γ after stimulation with mKatG were enumerated by enzyme-linked immunospot (ELISPOT). The level of IL-17 in the BAL fluid of sarcoidosis patients and healthy controls was measured by quantitative immuno-polymerase chain reaction (qIPCR). We also performed flow cytometry and immunohistochemistry for further characterization of IL-17 expression. Patients with Löfgren's syndrome had a higher frequency of IL-17-producing cells responding to mKatG in BAL fluid compared to patients without Löfgren's syndrome (P < 0·05). The HLA-DR3(+) sarcoidosis patients with Löfgren's syndrome (known to have a particularly good prognosis) also had a clearly higher level of IL-17 in BAL fluid compared to healthy controls and sarcoidosis patients without Löfgren's syndrome (P < 0·01) and (P < 0·05), respectively. No such difference between patient groups was observed with regard to IFN-γ and not with regard to either cytokine in peripheral blood. These findings suggest that IL-17-producing cells may be a useful biomarker for the prognosis of sarcoidosis and play a role in the spontaneous recovery typical of patients with Löfgren's syndrome.

Keywords: IL-17; T cells; cytokines; inflammation; sarcoidosis.

Fig. 1

Enzyme-linked immunospot assay assay for detection of interleukin (IL)-17- and interferon (IFN)-γ-producing cells in bronchoalveolar lavage (BAL) samples. (a) IL-17- and IFN-γ-producing cells in bronchoalveolar lavage (BAL) samples from patients with and without Löfgren's syndrome after stimulation with Mycobacterium tuberculosis catalase-peroxidase (mKatG), PHA or anti-CD3. (b) Paired analysis of IL-17- and IFN-γ-producing cells reacting to mKatG in BAL samples from patients with or without Löfgren's syndrome. (c) Correlation of neutrophil percentage and number of mKatG-reactive IL-17-producing cells in BAL fluid of sarcoidosis patients with Löfgren's syndrome. Graphs show mean numbers of spots per 10⁶ cells per well after deduction of background. The correlations were analysed using Spearman's rank test (r = 0·84; P < 0·01; *P < 0·05; ***P < 0·001).

Fig. 2

Enzyme-linked immunospot assay for evaluation of interleukin (IL)-17- and interferon (IFN)-γ-producing cells in response to Mycobacterium tuberculosis catalase-peroxidase (mKatG) in peripheral blood mononuclear cells (PBMCs). Results for cytokine-producing cells in PBMCs from purified protein derivative (PPD)^– (open symbols) and PPD⁺ (filled symbols) healthy controls and sarcoidosis patients with and without Löfgren's syndrome are shown. Graphs show mean numbers of spots from 10⁶ cells per well after deduction of background.

Fig. 3

Paired analysis of bronchoalveolar lavage (BAL) and blood cytokine responses to Mycobacterium tuberculosis catalase-peroxidase (mKatG) detected with enzyme-linked immunospot for interleukin (IL)-17 and interferon (IFN)-γ production. Results for sarcoidosis patients with or without Löfgren's syndrome are presented in separate graphs. Graphs show mean numbers of spots from 10⁶ cells per well after deduction of background. *P < 0·05.

Fig. 4

(a) Interleukin (IL)-17 levels in bronchoalveolar lavage (BAL) fluid determined by immuno-polymerase chain reaction (IPCR) in samples from healthy controls (HC), sarcoidosis patients without Löfgren's syndrome (non-Löf) and with Löfgren's syndrome. Sarcoidosis patients with Löfgren's syndrome were divided into two subgroups; human leucocyte antigen (HLA)-DR3⁺ and HLA-DR3^–. (b) Comparison of IL-17 levels between. DR3⁺ Löfgren's patients and DR3^– non-Löfgren's patients. *P < 0·05; **P < 0·01.

Fig. 5

a, Flow cytometry plots of in-vitro-stimulated bronchoalveolar lavage (BAL) fluid cells of a patient with Löfgren's syndrome. Successive gating was performed on lymphocytes, CD3⁺, CD4⁺ and CD8⁺ T cells. Cytokine expression in CD4⁺ T cells in response to medium alone, purified protein derivative (PPD) and Staphylococcus enterotoxin A (SEA)/S. enterotoxin B (SEB) were evaluated by intracellular staining for detection of interferon (IFN-)γ and interleukin (IL)-17. (b) Paired analysis show frequencies of CD4⁺ and CD8⁺ T cells expressing either IFNγ, IL-17 or both cytokines simultaneously in response to superantigen (SEA/SEB) stimulation. Samples were from BAL (b; top row) and blood (b; bottom row) of sarcoidosis patients with Löfgren's syndrome (filled symbols) and sarcoidosis patients without Löfgren's syndrome (open symbols). Note different scales on axes.

Fig. 6

Immunohistochemical analysis of interferon (IL)-17 in lung tissue. (a) Sample from granuloma of a sarcoidosis patient with Löfgren's syndrome. (b) Granuloma of a sarcoidosis patient without Löfgren's syndrome. (c) Respiratory bronchiole from healthy control and (d), non-granulomatous alveolar section from a sarcoidosis patient with Löfgren's syndrome. (e–h) Contiguous corresponding slides were stained with rabbit immunoglobulin (Ig)G as a negative control, respectively. IL-17-producing cells are indicated by arrows. Magnification: ×400.