Functional expression of IgG-Fc receptors in human airway smooth muscle cells

Abstract

IgE-Fc receptors and IgG-Fc receptors are expressed on hematopoietic cells, but some evidence suggests that these receptors are also found on nonhematopoietic cells, including human airway smooth muscle (hASM) cells. Our study characterizes the expression of IgE-Fc receptors (FcεRI/CD23) and IgG-Fc receptors (FcγRs-I, -II, and -III) in cultured hASM cells by flow cytometry and Western blotting, and the functional activity of receptors was determined through quantification of cell proliferation and released cytokines. Expression of Fc receptor-linked intracellular signaling proteins and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 and p38(MAPK) in hASM cells was examined by Western blotting. Expression of FcεRI and CD23 was not detectable in hASM cells. However, FcγRI and FcγRII were shown to be expressed on these cells. Specific antibodies, validated using transfected cell lines, revealed that the inhibitory IgG receptor, FcγRIIb, was the most abundant Fc receptor subtype expressed. Although cross-linking FcγR with heat-aggregated γ globulin (HAGG) did not induce detectable cell stimulation, pretreating hASM cells with HAGG significantly inhibited IL-1α-induced increases in cytokine levels and basic fibroblast growth factor-induced cell proliferation. This inhibitory effect of HAGG was abrogated by preincubation of cells with an anti-FcγRIIb antigen-binding fragment (Fab). Expression of proteins involved in the canonical FcγRIIb inhibitory signaling pathway was established in hASM cells. Pretreatment of hASM cells with HAGG significantly inhibited IL-1α- and basic fibroblast growth factor-induced extracellular signal-regulated kinase 1/2 and p38(MAPK) phosphorylation. This study identifies functional expression of FcγRIIb in hASM cells, with the potential to suppress their remodeling and immunomodulatory roles.

Figure 1.

No detectable IgE-Fc receptor (FcεR) I and CD23 expression on primary human airway smooth muscle (hASM) cells. Positive control human mast cell line (HMC)–1α cells showed expression of FcεRI (A) and low expression of CD23 (B). Whereas CD49b was strongly expressed in hASM cells (C), no expression of FcεRI (D) or CD23 (E) was observed, which was confirmed by hIgE labeling (F). Results shown are representative of experiments conducted using eight different primary hASM cell cultures. (G) The mRNA of the three subunits of FcεRI (αβγ) and CD23 were detected in peripheral blood leukocytes (PBLs), whereas a low-level expression of mRNA for FcRγ and CD23, but not FcεRI α or β subunits, was detected in hASM cells (n = 6).

Figure 2.

Expression of FcγRI and FcγRIIb in hASM cells. There was low expression of FcγRI (A), moderate expression of FcγRII (B), and no detectable FcγRIII (C) on hASM cells. Using the anti-FcγRIIa–selective monoclonal antibodies (mAb) IV.3 (D) and anti-FcγRIIb–specific mAb 4F5 (E), FcγRIIb was shown to be the FcγRII isoform expressed. This expression pattern was confirmed by Western blotting (F). Results shown are representative of experiments conducted using five to eight different hASM cell cultures.

Figure 3.

Expression of FcγRII in ASM in situ by immunohistochemistry. FcγRII immunoreactivity was observed in the smooth muscle cells of human airway sections (C). An anti–smooth muscle α-actin mAb was used to clearly identify the ASM (B), whereas the isotype control mAb showed no immunostaining (A). The findings are representative of results generated from three donors without asthma.

Figure 4.

FcγR aggregation inhibits stimulus-induced hASM cell activation. Treatment of hASM cells with heat-aggregated γ globulin (HAGG) inhibits IL-1α–induced IL-8 and IL-6 release (A), and basic fibroblast growth factor (bFGF)-induced cell proliferation (B). Cytokine level or cell numbers are normalized to the mean percentage (±SEM) of the IL-1α–only responses (IL-8, 71.5 ± 21.2 ng/ml; IL-6, 12.0 ± 3.4 ng/ml; eotaxin, 409.8 ± 66.8 pg/ml), or bFGF-only cell number per milliliter (1.4 × 10⁵ ± 0.1 × 10⁵ cells). Student's t test was used to compare the HAGG pretreated groups to the IL-1α– or bFGF-only group (**P < 0.01; ***P < 0.001). Using linear regression analysis, the inhibitory effects were directly correlated to the cell expression of FcγRIIb (C) (*P < 0.05; n = 6–7).

Figure 5.

Blockade of FcγRIIb inhibits HAGG activity on IL-1α–induced responses in hASM cells. Treating hASM cells with antigen-binding fragment (Fab) X63, Fab IV.3, and/or HAGG did not change the basal IL-8 production. Whereas HAGG significantly inhibited IL-1α–induced response, pretreating the cells with Fab X63, but not Fab IV.3, blocked this inhibitory effect. Experiments were conducted using one immortalized hASM cell line (and repeated three times), and on one primary hASM cell culture. IL-8 level is normalized to the mean percentage (±SEM) of the IL-1α–only responses (62.8 ± 19.1 ng/ml). Differences were identified using one-way repeated measurement ANOVA and Bonferroni's multiple comparison test (**P < 0.01 compared with IL-1α treatment alone; ^##P < 0.01 compared with HAGG + IL-1α treatment).

Figure 6.

Expression of FcγRIIb-associated signaling proteins in hASM cells. Lysates of positive control HMC-1α cells (lane 1) and hASM cultures were analyzed by Western blotting after 24-hour serum starvation. To avoid possible ambiguity introduced by repetitive stripping and reprobing of blots, results from the Src tyrosine kinase Lyn, spleen tyrosine kinase (Syk), and downstream of tyrosine kinase (Dok)-1 were generated on separate blots. In addition, Lyn and Dok-1 results are a composite of two separate experiments. Results shown for Src homology (SH)2–containing inositol phosphatase (SHIP)–1 and protein tyrosine phosphatase SHP–1 are from the same immunoblot. The expression of β-actin was used as a loading control throughout, with all immunoblots showing equivalent protein loading. The blot for β-actin shown was generated after reprobing of the SHIP-1/SHP-1 blot. Representative results of five (from a total of eight) independent hASM cultures are shown.

Figure 7.

FcγR aggregation inhibits stimulus-induced mitogen-activated protein kinase (MAPK) phosphorylation in hASM cells. Pretreating hASM cells with HAGG significantly inhibited bFGF (300 pM)-induced p38^MAPK (A) and extracellular signal–regulated kinase (ERK) 1/2 phosphorylation (C), and also inhibited IL-1α (0.01 ng/ml)–induced ERK1/2 (D), but not p38^MAPK phosphorylation (B). Differences were identified using one-way repeated measurement ANOVA and Bonferroni's multiple comparison test (*P < 0.05; **P < 0.01; ***P < 0.001 compared with IL-1α treatment alone or bFGF treatment alone).