Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

Abstract

Background: Regulation of human airway smooth muscle cells (HASMC) by cytokines contributes to chemotactic factor levels and thus to inflammatory cell accumulation in lung diseases. Cytokines such as the gp130 family member Oncostatin M (OSM) can act synergistically with Th2 cytokines (IL-4 and IL-13) to modulate lung cells, however whether IL-17A responses by HASMC can be altered is not known.

Objective: To determine the effects of recombinant OSM, or other gp130 cytokines (LIF, IL-31, and IL-6) in regulating HASMC responses to IL-17A, assessing MCP-1/CCL2 and IL-6 expression and cell signaling pathways.

Methods: Cell responses of primary HASMC cultures were measured by the assessment of protein levels in supernatants (ELISA) and mRNA levels (qRT-PCR) in cell extracts. Activation of STAT, MAPK (p38) and Akt pathways were measured by immunoblot. Pharmacological agents were used to assess the effects of inhibition of these pathways.

Results: OSM but not LIF, IL-31 or IL-6 could induce detectable responses in HASMC, elevating MCP-1/CCL2, IL-6 levels and activation of STAT-1, 3, 5, p38 and Akt cell signaling pathways. OSM induced synergistic action with IL-17A enhancing MCP-1/CCL-2 and IL-6 mRNA and protein expression, but not eotaxin-1 expression, while OSM in combination with IL-4 or IL-13 synergistically induced eotaxin-1 and MCP-1/CCL2. OSM elevated steady state mRNA levels of IL-4Rα, OSMRβ and gp130, but not IL-17RA or IL-17RC. Pharmacologic inhibition of STAT3 activation using Stattic down-regulated OSM, OSM/IL-4 or OSM/IL-13, and OSM/IL-17A synergistic responses of MCP-1/CCL-2 induction, whereas, inhibitors of Akt and p38 MAPK resulted in less reduction in MCP-1/CCL2 levels. IL-6 expression was more sensitive to inhibition of p38 (using SB203580) and was affected by Stattic in response to IL-17A/OSM stimulation.

Conclusions: Oncostatin M can regulate HASMC responses alone or in synergy with IL-17A. OSM/IL-17A combinations enhance MCP-1/CCL2 and IL-6 but not eotaxin-1. Thus, OSM through STAT3 activation of HASMC may participate in inflammatory cell recruitment in inflammatory airway disease.

Figure 1

HASMC responses to OSM and IL-17A. HASMC were cultured and prepared for stimulation as described in methods, where each treatment is completed in quadruplicates within each cell line. Cells were then stimulated in medium supplemented with 2% FBS and 0–50 ng/ml of the indicated gp130 cytokines. Protein concentrations in 24-hour supernatants were determined by ELISA for (A) MCP-1/CCL-2 (Left panel) and IL-6 (Right panel). *p < 0.05; ***p < 0.001 indicates statistical significance compared to no treatment as well as other indicated cytokines using One-Way ANOVA with Bonferroni’s post-test. Panel (B) (IL-6) and (C) (MCP-1) shows 24-hour supernatants from independent cell lines derived from 5 patients, prepared as above and stimulated with the indicated amounts of OSM, IL-17A or the combination as indicated. **p < 0.01 comparing indicated cytokines combinations to either cytokine alone using One-Way ANOVA with Bonferroni’s post-test.

Figure 2

OSM stimulation with IL-17A, IL-4 or IL-13 induces MCP-1/CCL2 and IL-6. HASMC cultures were prepared and stimulated as in Figure 1 with the indicated cytokines (in quadruplicates) and concentrations of: (A) IL-17A up to 10 ng/ml and in combination with 0, 0.01, 0.1, 1, and 10 ng/ml OSM, (B) IL-4 at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM, (C) IL-13 at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM, (D) IFNγ at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM. Data shown are from one of two individual cell lines that showed identical trends. 24-hour supernatants were collected and cytokine concentrations were quantified by ELISA for MCP-1/CCL-2 (left panels) or IL-6 (right panels). *p < 0.05; **p < 0.01; ***p < 0.001 comparing indicated cytokine combinations to either cytokine alone using Two-Way ANOVA with Bonferroni’s post-test.

Figure 3

OSM and other gp130 cytokine stimulation and HASMC responses. (A-C) HASMC cultures were prepared and stimulated as in Figure 1 with the indicated cytokines and concentrations of 10 ng/ml for each. Data shown are from one of two individual cell lines that showed identical trends. 24-hour culture supernatants were assessed by ELISA for levels of MCP-1/CCL-2 (A), IL-6 (B) and eotaxin-1 (C). ***p < 0.001 indicates statistical significance comparing indicated cytokine combinations to either cytokine alone using One-Way ANOVA with Bonferroni’s post-test. (D) HASMC cultures were stimulated with OSM, LIF, IL-11, IL-31 or IL-6 (10 ng/ml) for 20 minutes and cell lysates were prepared for immunoblots as described in methods. The lysates were probed for p-Y-STAT1, STAT1, p-Y-STAT3, STAT3, p-Y- STAT5, STAT5, p-Y-STAT6, p-Ser-Akt, p-T/Y-p38, p-T/Y-JNK and Actin as indicated. (E) Quantitative analysis of band intensity using densitometry (ImageJ) corrected to Actin or total protein for each probe and expressed as fold change relative to control (unstimulated).

Figure 4

Regulation of chemokine RNA in HASMC by cytokines. HASMC were plated and prepared for RNA analysis as in methods after 6 hours (A) and 18 hours (B) of stimulation with OSM at 2 ng/ml and/or the indicated cytokines at 5 ng/ml. RNA was prepared and analyzed by qRT-PCR using probes for the cytokines as indicated in methods. Levels are expressed as fold change relative to unstimulated (control) and corrected to β-Actin as an endogenous reference control.

Figure 5

Receptor chain mRNA levels in stimulated HASMC. HASMC were plated and prepared for RNA analysis as described in methods after 6 hours of stimulation with the indicated cytokines at 5 ng/ml or 2 ng/ml OSM (n = 2). RNA was prepared and analyzed by qRT-PCR using probes for the receptor subunits IL-17RA, IL-17RC, OSMRβ, gp130/IL-6ST and IL-4Rα, as indicated. Levels are expressed as fold change relative to unstimulated (control) and corrected to β-Actin as an endogenous reference control.

Figure 6

STAT3, p38 (MAPK) and Akt activation in HASMC and effects of inhibition on HASMC responses. (A) HASMC cultures were prepared as previously outlined for immunoblots of lysates of cells stimulated for 20 minutes with 5 ng/ml of IL-4, IL-13 and IL-17A (and/or 1 ng/ml of OSM). Total cell lysates from 3 different cell lines were probed for p-Y-STAT3, STAT3, p-Y-STAT6, STAT6, p-S-Akt, Akt, p-T/Y-p38, and total p38 as indicated (one representative cell line shown). (B) Quantification of band intensity using densitometry (ImageJ) corrected to total protein for each probe and expressed as the average (3 cell lines) fold change relative to control (un-stimulated) *p <0.05 using one-tail t-test. (C). HASMC cultures were prepared as previous for immunoblots of lysates of cells stimulated for 20 minutes with 1 ng/ml OSM, with or without pre-incubation with 1.25, or 2.5 or 5 uM Stattic or its vehicle DMSO (Veh). The lysates were probed for p-Y-STAT1, total STAT1, p-Y-STAT3, p-Y-STAT5, p-T/Y-p38, total p38, p-S-Akt and actin as indicated.

Figure 7

Effects of STAT3 pharmacologic inhibition on HASMC responses. HASMC cultures were prepared and stimulated, as previous, with the indicated cytokines IL-4, IL-13, or IL-17A at 5 ng/ml in the absence or presence of 1 ng/ml OSM. Parallel cultures were stimulated in the presence of either (A) 2.5 uM Stattic (black bars), (B) 10 uM SB203580 (dashed lines) or (C) 5 uM Akt X (white bars). Cell lines were stimulated for 18 hours and cell culture supernatants were collected and assessed by ELISA for levels of MCP-1/CCL-2 (left panel) and IL-6 (right panels). *p < 0.05; **p < 0.01, ***p < 0.001 comparing presence and absence of the indicated inhibitor in each cytokine treatment with 1 ng/ml OSM using Two-Way ANOVA with Bonferroni’s post-test.