The chymase mouse mast cell protease 4 degrades TNF, limits inflammation, and promotes survival in a model of sepsis

Abstract

Mouse mast cell protease 4 (mMCP-4), the mouse counterpart of human mast cell chymase, is thought to have proinflammatory effects in innate or adaptive immune responses associated with mast cell activation. However, human chymase can degrade the proinflammatory cytokine TNF, a mediator that can be produced by mast cells and many other cell types. We found that mMCP-4 can reduce levels of mouse mast cell-derived TNF in vitro through degradation of transmembrane and soluble TNF. We assessed the effects of interactions between mMCP-4 and TNF in vivo by analyzing the features of a classic model of polymicrobial sepsis, cecal ligation and puncture (CLP), in C57BL/6J-mMCP-4-deficient mice versus C57BL/6J wild-type mice, and in C57BL/6J-Kit(W-sh/W-sh) mice containing adoptively transferred mast cells that were either wild type or lacked mMCP-4, TNF, or both mediators. The mMCP-4-deficient mice exhibited increased levels of intraperitoneal TNF, higher numbers of peritoneal neutrophils, and increased acute kidney injury after CLP, and also had significantly higher mortality after this procedure. Our findings support the conclusion that mMCP-4 can enhance survival after CLP at least in part by limiting detrimental effects of TNF, and suggest that mast cell chymase may represent an important negative regulator of TNF in vivo.

Figure 1

mMCP-4 can enhance mouse survival after CLP. A: Survival after CLP (50% ligation; single puncture with a 22-gauge needle) in Mcpt4^+/+ mice (n = 14) and Mcpt4^−/− mice (n = 15). *P < 0.0001 versus Mcpt4^−/− mice. B: Representative images of kidneys at baseline and at 18 to 24 hours after CLP in Mcpt4^+/+ and Mcpt4^−/− mice. At baseline, the Mcpt4^−/− kidneys have minimal tubular lesions with intratubular apical laminated hyaline material as indicated by H&E staining (arrow) and mildly irregular PAS staining of the brush border (arrow), whereas corresponding sections from Mcpt4^+/+ kidneys appear normal. At 18 to 24 hours after CLP, the sections of Mcpt4^+/+ kidney exhibit mild proximal renal tubular swelling and apical blebbing under H&E staining (arrow) with some vacuolation (not shown), as well as mild irregular PAS staining and ectasia of affected tubules (arrow). In Mcpt-4^−/− kidney, CLP resulted in moderate multifocal acute renal tubular necrosis with intraluminal sloughing of hyalinized necrotic debris as indicated by H&E staining (arrow), occasional apoptotic cells (not shown), and a mild increase in neutrophils within renal vasculature as indicated by both H&E and PAS staining (arrowheads). Degenerate epithelial cells contain numerous PAS⁺ cytoplasmic droplets (arrow). Insets are digitally magnified portions of the same section, corresponding to the boxed region. C: Scoring of acute kidney injury in H&E-stained sections at baseline and at 18 to 24 hours after CLP in Mcpt4^+/+ mice (n = 3 or 4 per group) and Mcpt4^−/− mice (n = 3 to 10 per group). D: Blood urea nitrogen (BUN) levels at baseline and at 18 to 24 hours in Mcpt4^+/+ mice (n = 9 or 10 per group) and Mcpt4^−/− mice (n = 9 or 10 per group). Data were pooled from three independent experiments, each of which gave similar results. In C and D, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001. n.s., not significant (P > 0.05). Scale bar = 50 μm. Original magnification: ×400 (H&E); ×600 (PAS).

Figure 2

mMCP-4-deficient mice have increased numbers of intraperitoneal neutrophils after CLP. Numbers of neutrophils [polymorphonuclear leukocytes (PMN)] (A) and macrophages (B) in the live cell population were analyzed by flow cytometry (Gr-1^high/F4/80⁻ cells for PMN and Gr-1^high/F4/80⁺ for macrophages) in the peritoneal lavage fluid at baseline, and at 6, 18 to 24, and 48 hours after CLP, in Mcpt4^+/+ mice (n = 6 to 15 per group) and Mcpt4^−/− mice (n = 5 to 20 per group). Data were pooled from three independent experiments, each of which gave similar results. *P < 0.05; n.s. = not significant (P > 0.05).

Figure 3

mMCP-4-deficient mice have increased amounts of intraperitoneal TNF after CLP. Amounts of TNF (A), IL-6 (B), and IFN-γ (C) in the peritoneal lavage fluid at baseline, and at 6, 18 to 24, and 48 hours after CLP were quantified in Mcpt4^+/+ mice (n = 3 to 15 per group) and Mcpt4^−/− mice (n = 3 to 20 per group). Data were pooled from three independent experiments, each of which gave similar results. n.s. = not significant (P > 0.05).

Figure 4

mMCP-4 can limit the amounts of TNF generated by mast cells through degradation of proTNF and sTNF. A: TNF production by Mcpt4^+/+ and Mcpt4^−/− BMCMCs. Cells (1 × 10⁵/100 μL) were sensitized with IgE mAb to DNP (2 μg/mL) overnight at 37°C and then were challenged with DNP-HSA (10 ng/mL) for 18 hours at 37°C. ***P < 0.001. B and C: Lysates of peritoneal mast cells (PMCs) isolated from WT (Mcpt4^+/+) or mMCP-4-deficient (Mcpt4^−/−) mice were incubated with mouse recombinant sTNF (B) or proTNF in the presence or absence of an inhibitor of TACE (TAPI-1) (C) for 1.5 hours at 37°C and subjected to Western blot analysis. proTNF (C) was incubated with mouse recombinant TACE in the presence or absence of TAPI-1 to show that this inhibitor can significantly impair TACE ability to generate sTNF from proTNF under the conditions examined. Data were pooled from three independent experiments, each of which gave similar results (A) or are representative of two independent experiments, each of which gave similar results (B and C). n = 3 per group (A).

Figure 5

In the absence of mMCP-4, mast cell-derived TNF can contribute to increased amounts of intraperitoneal TNF and numbers of peritoneal neutrophils, and decreased survival, after CLP. A: Survival after CLP (50% ligation; single puncture with a 22-gauge needle) in Mcpt4^+/+ BMCMCs→Kit^W-sh/W-sh (n = 19), Mcpt4^−/− BMCMCs→Kit^W-sh/W-sh (n = 24), Tnf^−/− BMCMCs→Kit^W-sh/W-sh (n = 12), and Mcpt4^−/−Tnf^−/− BMCMCs→Kit^W-sh/W-sh (n = 12). Survival for Mcpt4^−/− BMCMCs→Kit^W-sh/W-sh mice was significantly reduced, compared with each of the other three types. *P < 0.02; ^†P < 0.005; ^‡P < 0.0005. B–D: Change in rectal temperature (B), amounts of TNF (C), and numbers of neutrophils [polymorphonuclear leukocytes (PMN) in the live cell population analyzed by flow cytometry; Gr-1^high/F4/80⁻ cells] (D) in the peritoneal lavage fluid at 18 to 24 hours after CLP in Mcpt4^+/+ BMCMCs→Kit^W-sh/W-sh (n = 10 or 11), Mcpt4^−/− BMCMCs→Kit^W-sh/W-sh (n = 10 or 11), Tnf^−/− BMCMCs→Kit^W-sh/W-sh (n = 8 to 10), and Mcpt4^−/−Tnf^−/− BMCMCs→Kit^W-sh/W-sh (n = 5 to 7). Data were pooled from three independent experiments, each of which gave similar results. *P < 0.05, **P < 0.01; n.s., not significant (P > 0.05).