Antibodies to proteinase 3 prime human oral, lung, and kidney epithelial cells to secrete proinflammatory cytokines upon stimulation with agonists to various Toll-like receptors, NOD1, and NOD2

Abstract

Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies, the detection of which in serum can be used in the diagnosis of Wegener's granulomatosis (WG). Proteinase 3 (PR3) is a major target antigen of ANCA in WG patients, and the interaction of PR3 ANCA with leukocytes causes a debilitating autoimmune disease. The first signs and symptoms in WG patients are observed in the oral cavity, lungs, and kidneys. Human epithelial cells generally do not secrete proinflammatory cytokines upon stimulation with pathogen-associated molecular patterns (PAMPs). In this study, anti-PR3 antibodies (Abs) and PR3 ANCA-containing sera from WG patients endowed human oral, lung, and kidney epithelial cells with responsiveness to PAMPs in terms of the production of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1, and tumor necrosis factor alpha. Protease-activated receptor-2 (PAR-2) agonist peptides mimicked the priming effects of PR3 ANCA against PAMPs. Furthermore, the anti-PR3 Ab-mediated cell activation was significantly abolished by RNA interference targeting PAR-2 and NF-kappaB. This is the first report of priming effects of anti-PR3 Abs (PR3 ANCA) on epithelial cells. The results suggest that anti-PR3 Abs (PR3 ANCA) prime human epithelial cells to produce cytokines upon stimulation with various PAMPs, and these mechanisms may be involved in severe chronic inflammation in WG.

FIG. 1.

Human oral, lung, and kidney epithelial cells did not secrete IL-8 in response to synthetic PAMPs. Oral epithelial HSC-2, lung epithelial A549, and kidney epithelial Caki-1 cells were stimulated with FSL-1 (1 nM), poly(I-C) (10 μg/ml), lipid A (10 ng/ml), poly(U) (10 μg/ml), CpG DNA (10 nM), FK156 (100 μg/ml), FK565 (100 μg/ml), MDP (100 μg/ml), TNF-α (10 ng/ml), or IL-1α (10 ng/ml) for 24 h in triplicate. Human TNF-α and IL-1α were used as positive controls. The levels of IL-8 in the culture supernatants were determined by ELISAs. Data are expressed as mean values ± standard deviations (SD). *, P < 0.01 versus results for cells stimulated with medium alone. The results presented are representative of three different experiments demonstrating similar results.

FIG. 2.

Human oral, lung, and kidney epithelial cells preincubated with anti-PR3 Abs secreted IL-8 in response to synthetic PAMPs. Oral epithelial HSC-2, lung epithelial A549, and kidney epithelial Caki-1 cells were preincubated for 6 h with anti-PR3 Abs (1 μg/ml) or with an equal amount of an isotype-matched immunoglobulin G (IgG) Ab. Subsequently, the cells were stimulated with FSL-1 (1 nM), poly(I-C) (10 μg/ml), lipid A (10 ng/ml), poly(U) (10 μg/ml), CpG DNA (10 nM), FK156 (100 μg/ml), FK565 (100 μg/ml), or MDP (100 μg/ml) for 24 h in triplicate. The levels of IL-8 in the culture supernatants were determined by ELISAs. Data are expressed as mean values ± SD. *, significantly different from results for cells in the respective cultures incubated with control IgG (P < 0.01). The results presented are representative of three different experiments demonstrating similar results.

FIG. 3.

Human oral, lung, and kidney epithelial cells preincubated with anti-PR3 Abs secreted IL-6, MCP-1, and TNF-α in response to synthetic PAMPs. Oral epithelial HSC-2, lung epithelial A549, and kidney epithelial Caki-1 cells were preincubated for 6 h with anti-PR3 Abs (1 μg/ml) or with an equal amount of an isotype-matched IgG Ab. Subsequently, the cells were stimulated with FSL-1 (1 nM), poly(I-C) (10 μg/ml), lipid A (10 ng/ml), poly(U) (10 μg/ml), CpG DNA (10 nM), FK156 (100 μg/ml), FK565 (100 μg/ml), or MDP (100 μg/ml) for 24 h in triplicate. The levels of IL-6, MCP-1, and TNF-α in the culture supernatants were determined by ELISAs. Data are expressed as mean values ± SD. *, significantly different from results for cells in the respective cultures incubated with control IgG (P < 0.01). The results presented are representative of three different experiments demonstrating similar results.

FIG. 4.

Human oral, lung, and kidney epithelial cells preincubated with PR3 ANCA-containing sera secreted IL-8 in response to synthetic PAMPs. Oral epithelial HSC-2, lung epithelial A549, and kidney epithelial Caki-1 cells were preincubated for 6 h with 1:100 dilutions of PR3 ANCA-containing sera from WG patients or with equal amounts of normal serum. Subsequently, the cells were stimulated with FSL-1 (1 nM), poly(I-C) (10 μg/ml), lipid A (10 ng/ml), poly(U) (10 μg/ml), CpG DNA (10 nM), FK156 (100 μg/ml), FK565 (100 μg/ml), or MDP (100 μg/ml) for 24 h in triplicate. The levels of IL-8 in the culture supernatants were determined by ELISAs. Data are expressed as mean values ± SD. *, significantly different from results for cells in the respective cultures incubated with normal serum (P < 0.01). The results presented are representative of three different experiments demonstrating similar results.

FIG. 5.

Human oral, lung, and kidney epithelial cells preincubated with PAR-2AP secreted IL-8 in response to synthetic PAMPs. Oral epithelial HSC-2, lung epithelial A549, and kidney epithelial Caki-1 cells were preincubated for 6 h with PAR-2AP (10 nM) or with an equal amount of control peptides. Subsequently, the cells were stimulated with FSL-1 (1 nM), poly(I-C) (10 μg/ml), lipid A (10 ng/ml), poly(U) (10 μg/ml), CpG DNA (10 nM), FK156 (100 μg/ml), FK565 (100 μg/ml), or MDP (100 μg/ml) for 24 h in triplicate. The levels of IL-8 in the culture supernatants were determined by ELISAs. Data are expressed as mean values ± SD. *, significantly different from results for cells in the respective cultures incubated with control peptides (P < 0.01). The results presented are representative of three different experiments demonstrating similar results.

FIG. 6.

The priming effect of anti-PR3 Abs occurred in a PAR-2- and NF-κB p65-dependent manner. Oral epithelial HSC-2, lung A549, and kidney Caki-1 cells were transfected with PAR-2-, NF-κB p65-, or lamin A/C-specific siRNA. After 24 h, the transfected cells were preincubated for 6 h with anti-PR3 Abs (1 μg/ml). Subsequently, the cells were stimulated with FSL-1 (1 nM), poly(I-C) (10 μg/ml), lipid A (10 ng/ml), poly(U) (10 μg/ml), CpG DNA (10 nM), FK156 (100 μg/ml), FK565 (100 μg/ml), or MDP (100 μg/ml) for 24 h in triplicate. The levels of IL-8 in the culture supernatants were determined by an ELISA. Data are expressed as mean values ± SD. * and #, values differed significantly from those obtained with medium alone and from those obtained for respective cultures stimulated with the indicated ligands, respectively (P < 0.01). The results presented are representative of three different experiments demonstrating similar results.