Interleukin-10 is not a critical regulator of infarct healing and left ventricular remodeling

Abstract

Objective: Interleukin-10 (IL-10) exerts potent anti-inflammatory actions and modulates matrix metalloproteinase expression. We hypothesized that endogenous IL-10 may regulate infarct healing and left ventricular remodeling by promoting resolution of the post-infarction inflammatory response and by modulating extracellular matrix metabolism.

Methods: IL-10 null and wildtype (WT) mice underwent reperfused infarction protocols. We compared the healing response and remodeling-associated parameters between IL-10-/-and WT infarcts. In addition, we studied the effects of IL-10 on inflammatory gene synthesis by stimulated murine cardiac fibroblasts.

Results: Infarcted IL-10-/-mice exhibited comparable mortality rates with WT animals. Although IL-10-/-mice had higher peak tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1/CCL2 mRNA levels in the infarcted heart than WT mice, both groups demonstrated timely repression of pro-inflammatory cytokine and chemokine mRNA synthesis after 24 h of reperfusion and exhibited a similar time course of resolution of the neutrophil infiltrate. IL-10 gene disruption did not alter fibrous tissue deposition and dilative remodeling of the infarcted heart. Pre-incubation with IL-10 did not modulate the pro-inflammatory phenotype of TNF-alpha-stimulated cardiac fibroblasts, failing to inhibit chemokine mRNA synthesis. In contrast, transforming growth factor (TGF)-beta1 pre-incubation suppressed interferon-gamma-inducible protein (IP)-10/CXCL10 synthesis by cardiac fibroblasts exposed to TNF-alpha.

Conclusions: IL-10 signaling plays a non-critical role in suppression of inflammatory mediators, resolution of the inflammatory response, and fibrous tissue deposition following myocardial infarction. This may be due to the relative selectivity of IL-10-mediated anti-inflammatory actions, with respect to cell type and stimulus. Resolution of post-infarction inflammation is likely to involve multiple overlapping regulatory mechanisms controlling various pro-inflammatory pathways activated in the infarcted myocardium.

Figure 1

Neutrophil infiltration in the infarcted heart. Both WT (A) and IL-10 -/- mice (B) demonstrated intense neutrophil infiltration in the infarcted myocardium after 24h of reperfusion. Partial resolution of the neutrophilic infiltrate was noted after 72h of reperfusion in WT (C) and IL-10 null animals (D). E. Quantitative analysis shows no significant difference in neutrophil density between WT and -/- animals (scale bar=70 μm).

Figure 2

Mac-2 immunohistochemistry identifies macrophages in the infarcted mouse heart. Comparable macrophage numbers were found after 24h of reperfusion in WT (A) and IL-10 -/- (B) infarcts. The number of Mac-2 immunoreactive cells increased after 72h of reperfusion in both WT (C) and IL-10 null animals (D). E. Quantitative analysis shows comparable macrophage density in IL-10 -/- and WT infarcts (scale bar=70 μm).

Figure 3

Cytokine mRNA expression in healing myocardial infarcts. A. RPA analysis showed that IL-10 null animals had slightly higher TNF-α mRNA levels in the infarcted heart than WT mice (*p<0.05). In contrast, IL-1β (B) and IL-6 (C) mRNA expression was comparable in both groups. Both WT and IL-10 null mice exhibited timely repression of pro-inflammatory cytokine synthesis after 24-72h of reperfusion (A-C).

Figure 4

Chemokine mRNA expression in healing infarcts. A. IL-10 null mice had higher MCP-1 mRNA expression in the infarcted heart compared with WT animals. In contrast expression of the CC chemokines MIP-1α (B) and MIP-1β (C) was comparable between groups.

Figure 5

Myofibroblasts are identified in the healing infarct as extravascular α-SMA-expressing spindle-shaped cells. A. After 72 h of reperfusion dead cardiomyocytes are replaced with granulation tissue, and myofibroblasts infiltrate the infarcted area, predominantly localized in the border zone (arrows). B. IL-10 null mice show comparable myofibroblast accumulation in the infarct. C. Quantitative analysis of myofibroblast density in the infarcted myocardium (scale bar=70 μm).

Figure 6

IL-10 gene disruption does not affect left ventricular remodeling following reperfused infarction. WT (A) and IL-10 null animals (B) showed comparable dilation of the left ventricle assessed by evaluation of perfusion-fixed hearts after 7 days of reperfusion. IL-10 null and WT mice showed similar LVEDV (pNS) (C), and comparable infarct size (D) (scale bar=1mm).

Figure 7

IL-10 does not alter inflammatory activation of cardiac fibroblasts exposed to TNF-α. IL-10 (100ng/ml) stimulation for 60 min did not induce mRNA expression of the chemokines MCP-1 (A), IP-10 (B), and MIP-2 (C). In contrast, TNF-α (100ng/ml) stimulation resulted in marked chemokine upregulation. Pre-incubation with IL-10 (100ng/ml) 4h prior to TNF-α stimulation did not inhibit TNF-α -mediated chemokine upregulation. In contrast, pre-incubation with TGF-β1 (10 ng/ml) significantly decreased TNF-α-induced IP-10 synthesis (B) (**p≺0.01), but did not affect MCP-1 and MIP-2 mRNA expression. TNF-α did not induce MIP-1α and MIP-1β synthesis by isolated cardiac fibroblasts (not shown).