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. 2008 Apr 1;111(7):3562-70.
doi: 10.1182/blood-2007-08-107664. Epub 2008 Jan 15.

Crosstalk between the alpha2beta1 integrin and c-met/HGF-R regulates innate immunity

Karissa D McCall-Culbreath  1 , Zhengzhi Li, Mary M Zutter
Affiliations

Crosstalk between the alpha2beta1 integrin and c-met/HGF-R regulates innate immunity

Karissa D McCall-Culbreath et al. Blood. .

Abstract

Data from several investigators suggest that the alpha2beta1 integrin, a receptor for collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1, endorepellin, and several viruses, is required for innate immunity and regulation of autoimmune/allergic disorders. We demonstrated that the innate immune response to Listeria monocytogenes required alpha2beta1 integrin expression by peritoneal mast cells (PMCs). Ligation of the alpha2beta1 integrin by C1q contained in immune complexes comprised of Listeria and antibody was required for PMC activation in vitro and in vivo. However, ligation of the alpha2beta1 integrin alone was insufficient to activate cytokine secretion, suggesting that one or more additional signals emanating from a coreceptor were required for PMC activation. Here, we demonstrate that C1q, but neither other complement proteins nor FcRgamma, is required for early innate immune response to Listeria. The binding of Listeria's Internalin B (InlB) to hepatocyte growth factor receptor (HGF-R)/c-met provides the costimulatory function required for PMC activation. Either HGF or Listeria InlB bound to c-met and either C1q or type I collagen bound to alpha2beta1 integrin stimulates PMC activation. These findings suggest that crosstalk between c-met and the alpha2beta1 integrin may contribute to mast-cell activation in autoimmune and inflammatory disorders.

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Figures

Figure 1
Figure 1
Wild-type mice, but not α2β1 integrin-deficient mice, display increased levels of IL-1β, IL-6, and TNF-α in response to Listeria infection. Wild-type and α2β1 integrin-deficient mice were infected with 5 × 104 Listeria intraperitoneally. At the indicated time points after infection, the concentration of IL-1β (A), IL-6 (B), TNF-α (C), and the percentage of neutrophils (D) were determined. Shown is a combination of 3 experiments (mean ± SEM) with each time point representing 3 mice.
Figure 2
Figure 2
Mast-cell activation by Listeria immune complex does not require FcRγ. (A) A proposed model is a 2-site, 2-receptor model in which concurrent activation of the α2β1 integrin and secondary receptor (complement receptor, FcRγ, Listeria receptor) stimulates mast-cell activation. Adapted from Edelson et al with permission. (B) Purified PMCs (2 × 104) from either WT (WT) mice or from mice lacking FcRγ (FcRγ−/−) were assayed for adhesion to a matrix consisting of (1) Listeria plus anti-Listeria antibody alone, (2) Listeria, anti-Listeria antibody and 50% murine serum, or (3) type I collagen in the presence or absence of 1 mM EDTA. (C) Purified PMCs (5 × 104) from WT and FcRγ−/− mice were incubated for 1 hour with a washed suspension of Listeria, anti-Listeria antibody, and 50% murine serum. Supernatants were collected and analyzed for IL-6 production by ELISA. All adhesion and activation experiments were carried out in the presence of 2 mM MgCl2. Results are mean plus or minus SEM from triplicate wells of a single experiment and represent 1 of at least 3 experiments demonstrating similar results. (D,E) WT and FcRγ−/− mice were infected for 1 or 6 hours with 5 × 104 Listeria intraperitoneally. At the indicated time points, the percentage of PMNs and concentration of IL-6 in the peritoneal fluid were determined. Shown is a representative example of at least 3 experiments (mean ± SEM), all carried out in triplicate.
Figure 3
Figure 3
C1q, but not other complement components, supplies stimulatory signal for mast-cell activation by Listeria-immune complex. (A) Purified PMCs (2 × 104 cells/well) isolated from WT (WT) mice were assayed for adhesion to a matrix consisting of Listeria plus anti-Listeria antibody, Listeria, anti-Listeria antibody alone, and 50% murine serum obtained from either WT mice or mice deficient in the complement components, C1q, C3, C4, C5, or factor B (C1q−/−, C3−/−, C4−/−, C5−/−, FB−/−) or type I collagen. (B) Purified PMCs (5 × 104) from WT and mice were incubated for 1 hour with a washed suspension of Listeria, anti-Listeria antibody, and 50% murine serum from either WT or mice deficient in the complement components C1q, C3, C4, C5, and factor B. Supernatants were collected and analyzed for IL-6 production by ELISA. All adhesion and activation experiments were carried out in the presence of 2 mM MgCl2. Results are mean plus or minus SEM from triplicate wells of a single experiment and represent 1 of at least 3 experiments demonstrating similar results. (C,D) WT and C1q−/− mice were infected for 1 or 6 hours with 5 × 104 Listeria intraperitoneally. At the indicated time points, the percentage PMN and IL-6 in the peritoneal fluid were determined. Shown is representative of at least 3 experiments (mean ± SEM), carried out in triplicate. Statistics were performed using unpaired Student t test (***P < .001).
Figure 4
Figure 4
HGF-R/c-met is the receptor providing the costimulatory signal for mast-cell activation. (A) Purified PMCs (5 × 104) isolated from WT mice were incubated for 1 hour with a washed suspension of Listeria and anti-Listeria antibody alone (LA), Listeria, anti-Listeria antibody plus 50% serum from WT mice (LAS), latex beads coated with bovine serum albumin (BSA) plus anti-BSA antibody alone (BA), BSA plus anti-BSA antibody and 50% WT murine serum (BAS), BSA plus anti-BSA antibody and 50% WT murine serum (BAS) with either LPS (100 ng/mL), Pam3Cys (100 μg/mL), Listeria (108), heat-killed Listeria (108 organisms), or LPS (100 ng/mL), Pam3Cys (100 μg/mL), Listeria (108), heat-killed Listeria (108 organisms) alone. Supernatants were collected and analyzed for IL-6 production by ELISA. (B) Representative flow cytometric histograms of PMCs stained with c-met PMCs from WT (panel A) and α2β1 integrin-deficient (KO, panel B) were stained with phycoerythrin–anti–c-kit and APC–anti–c-met and assessed by flow cytometry. Mast cells were identified as c-kithigh–staining cells and represented 1% to 3% of resident peritoneal cells in both WT and KO mice. (C) Purified PMCs isolated from WT mice were pretreated with inhibitory antibodies toward E-cadherin, c-met, or irrelevant control antibody for 1 hour before stimulation with a washed suspension of Listeria, anti-Listeria antibody alone, or Listeria, anti-Listeria antibody, and 50% WT murine serum. Supernatants were collected and analyzed for IL-6 production by ELISA. (D) Purified PMCs (5 × 104) isolated from WT mice were incubated with Listeria and anti-Listeria antibody alone (LA), Listeria, and anti-Listeria antibody plus 50% serum from WT mice (LAS), latex beads coated with BSA, plus anti-BSA antibody alone (BA), or latex beads coated with BSA, anti-BSA antibody, plus 50% serum (BAS) plus or minus the addition of Listeria (108) or HGF (2 mg/mL), or Listeria (108), or HGF (2 mg/mL) alone. Supernatants were collected and analyzed for the concentration of IL-6 by ELISA. (E) Purified PMCs (5 × 104) isolated from WT mice were allowed to adhere to a matrix of Listeria and anti-Listeria antibody (LA), Listeria, anti-Listeria antibody plus serum (LAS), type I collagen (25 mg/mL), C1q (25 mg/mL), or tissue culture plastic (No Matrix) with or without either Listeria (108) or HGF (2 mg/mL). Supernatants were collected and analyzed for IL-6 production by ELISA. All experiments were carried out in the presence of 2 mM MgCl2. Results are presented as means plus or minus SEM from triplicate wells of a single experiment and represent 1 of at least 3 experiments demonstrating similar results. The P values were determined by unpaired Student t test (*P < .05, **P < .01).
Figure 5
Figure 5
Internalin B is required for mast cell–mediated innate and adaptive immune responses. (A) Purified PMCs (5 × 104) from WT and α2-null mice were incubated for 1 hour with a washed suspension of WT Listeria EGD, Listeria ΔInlA (L(ΔA)), or Listeria (L(ΔB)), and anti-Listeria antibody plus or minus 50% murine serum. Supernatants were collected and assayed for IL-6 by ELISA. (B,C) WT and α2-null mice were infected for 1 or 6 hours with 5 × 104 Listeria (EGD), Listeria (ΔInlA), or Listeria (ΔInlB) intraperitoneally. At the indicated time points, the percentage PMN and IL-6 in the peritoneal fluid were determined. Shown is representative of 2 experiments (mean ± SEM), with each point representing 5 or 6 mice.
Figure 6
Figure 6
Predicted model of mast-cell activation through c-met and the α2β1 integrin. Mast cell stimulation through c-met and the α2β1 integrin results in cross talk between the 2 receptors, resulting in the activation of the mast cell leading to release of the pro-inflammatory cytokine, IL-6.
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