Myocardial TLR4 is a determinant of neutrophil infiltration after global myocardial ischemia: mediating KC and MCP-1 expression induced by extracellular HSC70

Abstract

Cardiac surgery with global myocardial ischemia-reperfusion (I/R) induces a myocardial inflammatory response that impairs cardiac recovery. Chemokines contribute to the overall myocardial inflammatory response through inducing leukocyte infiltration. Although Toll-like receptor 4 (TLR4) has an important role in postischemic myocardial injury, the relative roles of myocardial tissue and leukocyte TLR4 in leukocyte infiltration, as well as the role of TLR4 in myocardial chemokine expression, are unclear. Our recent study, in an isolated mouse heart model of global I/R, found that the 70-kDa heat shock cognate protein (HSC70) is released from cardiac cells and mediates the expression of cardiodepressant cytokines via a TLR4-dependent mechanism. In the present study, we tested the hypotheses that myocardial tissue TLR4 has a major role in mediating neutrophil infiltration and that myocardial TLR4 and extracellular HSC70 contribute to the mechanisms underlying cardiac chemokine response to global I/R. We subjected hearts isolated from TLR4-defective and TLR4-competent mice to global I/R and examined myocardial neutrophil infiltration and expression of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein-1 (MCP-1). TLR4-defective hearts exhibited reduced neutrophil infiltration regardless of the phenotypes of neutrophils perfused during reperfusion and expressed lower levels of KC and MCP-1. HSC70-specific antibody reduced myocardial expression of KC and MCP-1 after I/R. Furthermore, perfusion of HSC70 increased KC and MCP-1 expression in TLR4-competent hearts but not in TLR4-defective hearts, and HSC70 also induced the chemokine response in macrophages in a TLR4-dependent fashion. A recombinant HSC70 fragment lacking the substrate-binding domain was insufficient to induce chemokine expression in hearts and cells. This study demonstrates that myocardial tissue TLR4, rather than neutrophil TLR4, is the determinant of myocardial neutrophil infiltration after global I/R. TLR4 mediates myocardial chemokine expression, and the mechanisms involve extracellular HSC70. These results imply the HSC70-TLR4 interaction as a novel mechanism underlying the myocardial chemokine response to global I/R.

Fig. 1.

Neutrophil infiltration to the myocardium is dependent on myocardial Toll-like receptor 4 (TLR4) rather than neutrophil TLR4. A: neutrophils derived from bone marrow (left) were stained red with rat anti-mouse neutrophil marker protein, and their nuclei were stained blue with bis-benzimide. Cells appeared mature after in vitro incubation. Right: neutrophils (red) infiltrated into the space between myocytes (stained blue with wheat germ agglutinin) and were found in close proximity to resident macrophages (stained green with polyclonal anti-mouse CD68) and myocytes after perfusing ischemic heart with buffer containing neutrophils. Bar = 10 μm. B: hearts isolated from C3H/HeN (TLR4 competent) and C3H/HeJ (TLR4 defective) mice were subjected to global ischemia-reperfusion (I/R; 20 min ischemia/60 min reperfusion). After ischemia, hearts were infused with TLR4-competent or TLR4-defective neutrophils. Immunostaining demonstrates that TLR4-competent hearts exhibit greater neutrophil infiltration than TLR4-defective hearts. Perfusion of TLR4-defective neutrophils did not influence cell infiltration in TLR4-competent hearts. Data are expressed as means ± SE; n = 5 in each group. ^aP < 0.05 vs. buffer-perfused heart; ^bP < 0.05 vs. HeN heart.

Fig. 2.

Myocardial TLR4 mediates chemokine response to global I/R. Hearts isolated from C3H/HeN (TLR4 competent) and C3H/HeJ (TLR4 defective) mice were subjected to global I/R (20 min ischemia/60 min reperfusion). Control hearts (Perf) were perfused without subjecting to I/R. mRNA (A) and peptide (B) levels of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein-1 (MCP-1) after I/R are significantly lower in TLR4-defective hearts than in TLR4-competent hearts. Data are expressed as means ± SE; n = 5 in each group. ^aP < 0.05 vs. phenotype perfusion control; ^bP < 0.05 vs. HeN I/R.

Fig. 3.

Extracellular heat shock cognate protein 70 (HSC70) plays a critical role in the myocardial chemokine response following global I/R. A and B: hearts isolated from C3H/HeN (TLR4 competent) mice were subjected to global I/R (20 min ischemia/60 min reperfusion) with or without treatment with polyclonal anti-HSC70 (HSC Ab; 0.5 μg/ml) or control IgG (IgG; 0.5 μg/ml) for 10 min before ischemia and for 30 min after initiation of reperfusion. Control hearts (Perf) were perfused with perfusion buffer without subjecting to I/R. Anti-HSC70 reduced myocardial levels of KC and MCP-1 mRNA and peptides, whereas control IgG had minimal effects. Data are expressed as means ± SE; n = 6 in each group. ^aP < 0.05 vs. phenotype perfusion control; ^bP < 0.05 vs. phenotype I/R control.

Fig. 4.

Induction of myocardial chemokine response by HSC70 is TLR4 dependent and requires the peptide-binding domain. Hearts isolated from C3H/HeN (TLR4 competent) and C3H/HeJ (TLR4 defective) mice were perfused with either recombinant full-length HSC70 (rHSC; 0.5 μg/ml) or recombinant HSC70 fragment (rFrag; without the substrate-binding domain; 0.5 μg/ml) for 30 min followed by 60 min washout. Control hearts were perfused with perfusion buffer only. A and B: TLR4-competent hearts expressed KC and MCP-1 mRNA in response to HSC70 treatment, but myocardial KC and MCP-1 mRNA levels were reduced in TLR4-defective hearts. C and D: KC and MCP-1 peptide levels increased in TLR4-competent hearts treated with HSC70, but this effect of HSC70 was reduced in TLR4-defective hearts. The HSC70 fragment without the substrate-binding domain failed to upregulate mRNA or peptide expression of KC and MCP-1 (A–D). Data are expressed as means ± SE; n = 6 in each group. ^aP < 0.05 vs. phenotype control; ^bP < 0.05 vs. HeN treated with HSC70 (rHSC).

Fig. 5.

HSC70 induces macrophage expression of KC and MCP-1 through a TLR4-dependent mechanism. A: peritoneal macrophages from C3H/HeN (TLR4 competent) mice were incubated with recombinant HSC70 (rHSC; 0.5 μg/ml) or recombinant HSC70 fragment (rFrag; without the substrate-binding domain; 0.5 μg/ml) for 4 h or left untreated (control). Recombinant HSC70 induced the release of KC (A) and MCP-1 (B) in TLR4-competent cells but not in TLR4-defective cells. The recombinant HSC70 fragment had no effect on the release of KC or MCP-1. Note that recombinant HSC70 with and without polymyxin B (PolyB) had a comparable effect on cells. Data are presented as means ± SE; n = 4. ^aP < 0.05 vs. phenotype control; ^bP < 0.05 vs. HeN treated with HSC70 (rHSC).