Secretory immunoglobulin A induces human lung fibroblasts to produce inflammatory cytokines and undergo activation

Abstract

Immunoglobulin (Ig)A is the most abundant immunoglobulin in humans, and in the airway mucosa secretory IgA (sIgA) plays a pivotal role in first-line defense against invading pathogens and antigens. IgA has been reported to also have pathogenic effects, including possible worsening of the prognosis of idiopathic pulmonary fibrosis (IPF). However, the precise effects of IgA on lung fibroblasts remain unclear, and we aimed to elucidate how IgA activates human lung fibroblasts. We found that sIgA, but not monomeric IgA (mIgA), induced interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production by normal human lung fibroblasts (NHLFs) at both the protein and mRNA levels. sIgA also promoted proliferation of NHLFs and collagen gel contraction comparable to with transforming growth factor (TGF)-β, which is involved in fibrogenesis in IPF. Also, Western blot analysis and real-time quantitative polymerase chain reaction (PCR) revealed that sIgA enhanced production of α-smooth muscle actin (α-SMA) and collagen type I (Col I) by NHLFs. Flow cytometry showed that NHLFs bound sIgA, and among the known IgA receptors, NHLFs significantly expressed CD71 (transferrin receptor). Transfection of siRNA targeting CD71 partially but significantly suppressed cytokine production by NHLFs co-cultured with sIgA. Our findings suggest that sIgA may promote human lung inflammation and fibrosis by enhancing production of inflammatory or fibrogenic cytokines as well as extracellular matrix, inducing fibroblast differentiation into myofibroblasts and promoting human lung fibroblast proliferation. sIgA's enhancement of cytokine production may be due partially to its binding to CD71 or the secretory component.

Keywords: IgA; fibroblasts; fibrosis; human; lung.

Figure 1

Secretory immunoglobulin A (sIgA) up‐regulates cytokine production by normal human lung fibroblasts (NHLFs). (a,b) NHLFs were cultured in the presence and absence of sIgA or monomeric IgA (mIgA) at the indicated concentrations for 48 h. The levels of interleukin (IL)‐6, IL‐8, monocyte chemoattractant protein (MCP)‐1 and granulocyte macrophage colony‐stimulating factor (GM‐CSF) in the NHLF culture supernatants were determined by cytometric bead array (CBA). Bars represent the mean ± standard error of the mean (s.e.m.) (n = 3 independent experiments). *P < 0·05, ***P < 0·01, ****P < 0·001 versus cells without stimulus. (c,d) NHLFs were cultured in the presence and absence of sIgA or mIgA at the indicated concentrations for 4 h. The relative expression levels of mRNA for IL‐6, IL‐8, MCP‐1 and GM‐CSF were determined by real‐time polymerase chain reaction (PCR) and expressed as the mean ± s.e.m. (n = 3 independent experiments). *P < 0·05; **P < 0·01; ***P < 0·001; ****P < 0.0001 versus cells without stimulus.

Figure 2

Effect of anti‐IgA secretory component antibody (anti‐SC) treatment on cytokine production by normal human lung fibroblasts (NHLFs) with secretory immunoglobulin A (sIgA) stimulation. NHLFs were cultured for 24 h with sIgA at 0 and 100 µg/ml, followed by incubation with anti‐SC at 0, 1 and 10 µg/ml for 1 h at room temperature. The levels of interleukin (IL)‐6, IL‐8 and monocyte chemoattractant protein (MCP)‐1 in the NHLF culture supernatants were determined by cytometric bead array (CBA). Bars represent the mean ± standard error of the mean (s.e.m.) (n = 3 independent experiments). *P < 0·05; ***P < 0·001 versus cells without stimulus; n.s. = not significant.

Figure 3

Secretory immunoglobulin A (sIgA) induces proliferation of normal human lung fibroblasts (NHLFs) and collagen gel contraction by NHLFs. (a) NHLFs were cultured with sIgA or monomeric IgA (mIgA) at 100 µg/ml or transforming growth factor (TGF)‐β1 at 10 ng/ml, with bromodeoxyuridine (BrdU) for 16 h, and BrdU incorporation was analyzed using a cell proliferation enzyme‐linked immunosorbent assay (ELISA) kit. Bars represent the mean ± standard error of the mean (s.e.m.) (n = 3 independent experiments). *P < 0·05; ***P < 0·001; ****P < 0·0001 versus cells without stimulus. (b) Representative images of collagen gel contraction by NHLFs in the presence and absence of sIgA or TGF‐β1 at 24, 48 and 72 h. Data are representative of four independent experiments. (c) The gel surface areas in the presence and absence of the indicated concentrations of sIgA or TGF‐β1 at 2·5 ng/ml, at 48 h (left panel) and 72 h (right panel), were measured and analyzed using Image J software. Bars represent the mean ± standard error of mean (s.e.m.) (n = 4 independent experiments). *P < 0·05; **P < 0·01 versus cells without stimulus.

Figure 4

Secretory immunoglobulin A (sIgA) enhances production of α‐smooth muscle actin (SMA) and collagen type 1 (Col I) by normal human lung fibroblasts (NHLFs). (a,b) NHLFs were cultured in the presence and absence of sIgA at the indicated concentrations for 4 h (α‐SMA) and 24 h (Col I). The relative expression levels of mRNA for α‐SMA and Col I were determined by real‐time polymerase chain reaction (PCR) and expressed as the mean ± standard error of the mean (s.e.m.) (n = 5 independent experiments). *P < 0·05; **P < 0·01 versus cells without stimulus. (c,d) NHLFs were cultured in the presence and absence of sIgA at 100 µg/ml for 48 h, and the cell lysates were collected to quantitate α‐SMA (c) and Col I (d) by Western blot analysis. Three independent samples from separate experiments were applied onto each gel for electrophoresis, followed by Western blot analysis. The expression of each was normalized to that of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and expressed as the mean ± s.e.m. (n = 3, 11 independent experiments). *P < 0·05; **P < 0·01.

Figure 5

Normal human lung fibroblasts (NHLFs) bind secretory immunoglobulin A (sIgA) and express a known IgA receptor, CD71, on their cell surface. (a,b) NHLFs were incubated with (solid line) and without (dashed line) IgA at the indicated concentrations for 1 h and stained with allophycocyanin (APC)‐conjugated anti‐human IgA antibody or isotype control for analysis by flow cytometry. (a) Representative histograms from three independent experiments are shown, and (b) mean fluorescence intensities (MFIs) from three experiments are expressed as the mean ± standard error of the mean (s.e.m.) (n = 3 independent experiments). *P < 0·05. (c,d) NHLFs were stained with fluorescein isothiocyanate (FITC)‐conjugated anti‐human CD71 monoclonal antibody (mAb) or isotype‐matched control antibody and analyzed by flow cytometry. (c) Representative histograms from five independent experiments are shown, and (d) MFIs from five experiments are expressed as the mean ± s.e.m. (n = 5 independent experiments). **p < 0·01. (e) NHLFs were stained with purified mouse IgG1κ isotype control (left panels) and mouse anti‐human CD71 antibody (right panels). (f,g) NHLFs were incubated with and without tumour necrosis factor (TNF)‐α or interleukin (IL)‐1β at 10 ng/ml for 48 h, and then the expression level of CD71 was analyzed by flow cytometry. (f) Representative histograms from three independent experiments are shown. The dashed lines show the cells without stimulus, and the solid lines show the cells with indicated stimuli. The gray areas show the cells with control stain. (g) MFIs from three experiments in (f) are expressed as the mean ± s.e.m. (n = 3 independent experiments). *P < 0·05; **P < 0·01. (h) NHLFs were stained with phycoerythrin (PE)‐conjugated anti‐human ASGPR1 mAb, anti‐human CD89 mAb, anti‐human CD351 mAb or isotype‐matched control antibody and analyzed by flow cytometry. Representative histograms from three independent experiments are shown. The dashed lines show the cells stained with control antibody, and the solid lines show the cells stained with specific antobody against each receptor.

Figure 6

Transfection of siRNA targeting CD71 inhibited IgA binding to normal human lung fibroblasts (NHLFs), and partly reduced up‐regulation of cytokine‐synthesis induced by sIgA. (a) NHLFs were transfected with CD71 siRNA (solid line) or control RNA (dashed line) at 10 nM for 24 h, and the expression level of CD71 on the cell surface is shown. Data are representative of three independent experiments. (b) NHLFs transfected with CD71 siRNA (black bar) or control RNA (white bar) at 10 nM for 24 h were incubated with sIgA at 100 for 1 h and then stained with anti‐human IgA antibody for analysis by flow cytometry. Mean fluorescent intensities (MFIs) are expressed as the mean ± standard error of the mean (s.e.m.) (n = 4 independent experiments); n.s. = not significant. (c) NHLFs transfected with CD71 siRNA or control RNA at 10 nM for 24 h were cultured in the presence and absence of sIgA for 24 and 48 h. The levels of interleukin (IL)‐8 and monocyte chemoattractant protein (MCP)‐1 in NHLF culture supernatants were determined by cytometric bead array (CBA). Bars represent the mean ± s.e.m. (n = 3 independent experiments). *P < 0·05; **P < 0·01.