Alveolar macrophage cathelicidin deficiency in severe sarcoidosis

Abstract

Background: Dysfunctional immune responses characterize sarcoidosis, but the status of cathelicidin, a potent immunoregulatory and antimicrobial molecule, has not been established in clinical disease activity.

Methods: Alveolar macrophage cathelicidin expression was determined in biopsy-proven sarcoidosis patients classified clinically as 'severe' (requiring systemic treatment) or 'non-severe' (never requiring treatment). Bronchoalveolar lavage (BAL) cells from sarcoidosis patients and healthy controls were analyzed for mRNA expression of cathelicidin, vitamin D receptor (VDR) and the VDR coactivator steroid receptor coactivator-3 (SRC3) by quantitative PCR. Cathelicidin-derived peptide LL-37 was determined by immunocytochemistry. Serum calcidiol (25-hydroxyvitamin D2; vitD2) and calcitriol (1,25-dihydroxyvitamin D3; vitD3) were quantified.

Results: The results indicated reduced BAL cell expression of cathelicidin and SRC3 in severe but not non-severe sarcoidosis compared to controls. Serum levels of biologically active vitD3 in both severe and non-severe patients were within the control range even though vitD2 levels in both groups were below the recommended level (30 ng/ml). Sarcoidosis and control alveolar macrophages were studied in vitro to determine cathelicidin responses to vitD3 and tumor necrosis factor-α (TNFα), a vitD3 antagonist elevated in active sarcoidosis. Alveolar macrophage cathelicidin was stimulated by vitD3 but repressed by TNFα, which also repressed SRC3.

Conclusions: These findings suggest that TNFα-mediated repression of SRC3 contributes to alveolar macrophage cathelicidin deficiency in severe sarcoidosis despite healthy vitD3 levels. Deficiency of cathelicidin, a multifunctional regulator of immune cells and proinflammatory cytokines, may impede resolution of inflammation in the lungs of patients with severe sarcoidosis.

Fig. 1

Deficiency in cathelicidin mRNA expression correlates with clinical disease severity of sarcoidosis. a Samples of cDNA derived from BAL cells of sarcoidosis patients with severe (n = 16) or non-severe (n = 12) disease and healthy controls (n = 21) were evaluated for cathelicidin expression by quantitative PCR. Data represent the relative fold change in cathelicidin expression after subtraction of raw GAPDH cycle values and normalization to healthy control threshold cycle values. Means and SEMs are shown. * p < 0.05. b, c Immunocytochemistry of alveolar macrophage cytospins illustrates cathelicidin peptide LL-37 levels in a healthy control (b) and reduced cathelicidin peptide LL-37 in a severe sarcoidosis patient (c) (n = 2). d Cathelicidin mRNA expression is also significantly decreased in sarcoidosis pulmonary epithelial cells. Means and SEMs are shown. Controls: n = 6; severe sarcoidosis patients: n = 4; non-severe sarcoidosis patients: n = 2. * p < 0.05.

Fig. 2

Circulating vitD2 levels parallel disease severity in sarcoidosis. VitD2 (a) and vitD3 (b) levels were quantified in sera from patients with severe (n = 11) and non-severe (n = 10) sarcoidosis. Medians are shown, and dotted lines indicate the recommended normal levels of vitD2 (a) and the normal range of vitD3 (b) are provided. * p < 0.05.

Fig. 3

Cathelicidin expression is upregulated by vitD3 and downregulated by TNFα in sarcoidosis and healthy control alveolar macrophages. a Quantitative PCR analysis ofcDNA samples from sarcoidosis and healthy control alveolar macrophages cultured for 24 h in medium with or without vitD3 (10 nM). Values for cathelicidin mRNA were determined after normalization to GAPDH. Data are expressed as the fold change in cathelicidin mRNA expression relative to untreated cells (medium alone). n = 5 per group. * p < 0.05. b Cathelicidin-derived LL-37 peptide synthesis in supernatant fluids from sarcoidosis and healthy control alveolar macrophages cultured in vitro with or without vitD3. Data are normalized to untreated (medium) cultured cells. Controls: n = 4; sarcoidosis patients: n = 3. * p < 0.05. c A dot plot illustrates repression of alveolar macrophage cathelicidin mRNA expression by TNFα (250–500 units/ml) in sarcoidosis patients (n = 3) and healthy controls (n = 5). * p < 0.05.

Fig. 4

Expression of SRC3 is repressed by TNFα and decreased in severe sarcoidosis. a Quantitative PCR analysis of SRC3 expression was performed in cDNA samples from healthy control and sarcoidosis alveolar macrophages cultured for 24 h in medium with or without TNFα (500 units/ml). Means and SEMs are given. Controls: n = 4; sarcoidosis patients: n = 2. * p < 0.05. b SRC3 mRNA expression was quantitated in BAL cells derived from healthy controls (n = 3) and patients with severe (n = 4) or non-severe (n = 3) sarcoidosis. Data were normalized to healthy controls. * p < 0.05. c Intrinsic alveolar macrophage SRC3 and cathelicidin mRNA values show significant correlation by regression analysis. ΔCt = Change in threshold cycle values. d, e Immunocytochemistry illustrates marked SRC3 protein expression in alveolar macrophages from a healthy control (d) and reduced SRC3 in a patient with severe sarcoidosis (e) (n = 2).