Streptococcal M1 protein-provoked CXC chemokine formation, neutrophil recruitment and lung damage are regulated by Rho-kinase signaling

Abstract

Streptococcal toxic shock syndrome is frequently caused by Streptococcus pyogenes of the M1 serotype. The aim of this study was to determine the role of Ras-homologous (Rho)-kinase signaling in M1 protein-provoked lung damage. Male C57BL/6 mice received the Rho-kinase-specific inhibitor Y-27632 before administration of M1 protein. Edema, neutrophil accumulation and CXC chemokines were quantified in the lung 4 h after M1 protein challenge. Flow cytometry was used to determine Mac-1 expression. Quantitative RT-PCR was used to determine gene expression of CXC chemokine mRNA in alveolar macrophages. M1 protein increased neutrophil accumulation, edema and CXC chemokine formation in the lung as well as enhanced Mac-1 expression on neutrophils. Inhibition of Rho-kinase signaling significantly reduced M1 protein-provoked neutrophil accumulation and edema formation in the lung. M1 protein-triggered pulmonary production of CXC chemokine and gene expression of CXC chemokines in alveolar macrophages was decreased by Y-27632. Moreover, Rho-kinase inhibition attenuated M1 protein-induced Mac-1 expression on neutrophils. We conclude that Rho-kinase-dependent neutrophil infiltration controls pulmonary tissue damage in response to streptococcal M1 protein and that Rho-kinase signaling regulates M1 protein-induced lung recruitment of neutrophils via the formation of CXC chemokines and Mac-1 expression.

Fig. 1

Edema formation in the lung. Mice were treated with the Rho-kinase inhibitor Y-27632 (0.5 or 5 mg/kg) or vehicle (PBS) 10 min prior to M1 protein injection. Mice treated with PBS served as sham animals. One group of mice was given 5 mg/kg Y-27632 alone without M1 protein injection. Data represents mean ± SEM, * p < 0.05 versus sham and ^# p < 0.05 versus vehicle + M1 protein, n = 5.

Fig. 2

Representative hematoxylin & eosin sections of lung. a Sham animals were treated with PBS only. b They were given 5 mg/kg Y-27632 alone. Separate mice were pretreated with vehicle (PBS) (c) and 0.5 (d) or 5 mg/kg Y-27632 (e) 10 min prior to M1 protein administration. Samples were harvested 4 h after M1 protein challenge. Scale bar indicates 100 µm. f Histology score of lung injury. Data represents mean ± SEM, * p < 0.05 versus sham and ^# p < 0.05 versus vehicle + M1 protein, n = 5.

Fig. 3

Rho-kinase regulates M1 protein-induced pulmonary infiltration of neutrophils. a Lung MPO levels. b Number of BALF neutrophils in the lung. Animals were treated with Y-27632 (0.5 or 5 mg/kg) or vehicle (PBS) 10 min prior to M1 protein injection. Samples were harvested 4 h after M1 protein challenge. Mice treated with PBS served as sham animals. One group of mice was given 5 mg/kg Y-27632 alone. Data represents mean ± SEM, * p < 0.05 versus sham and ^# p < 0.05 versus vehicle + M1 protein, n = 5.

Fig. 4

Representative histogram from 5 samples: Rho-kinase regulates M1 protein-induced Mac-1 expression on neutrophils in vivo. Mac-1 expression on neutrophils in vehicle-treated (PBS) or Y-27632-treated (0.5 or 5 mg/kg) animals 4 h after M1 protein injection is shown. MFI is shown on the x-axis and cell counts on the y-axis. Data represents mean ± SEM, * p < 0.05 versus sham and ^# p < 0.05 versus vehicle + M1 protein, n = 5.

Fig. 5

Representative histogram from 5 samples: Mac-1 expression on neutrophils in vitro. Whole blood was incubated with PBS only, or M1 protein (1 µg/ml) and vehicle (PBS) or Y-27632 (10 µM). Samples were harvested 4 h after M1 protein challenge. MFI is shown on the x-axis and cell counts on the y-axis. Data represents mean ± SEM, * p < 0.05 vs. Sham and ^# p < 0.05 vs. Vehicle + M1 protein, n = 5.

Fig. 6

Rho-kinase regulates CXC chemokine formation in the lung. Animals were treated with Y-27632 (0.5 or 5 mg/kg) or vehicle (PBS) 10 min prior to M1 protein injection. Mice treated with PBS served as sham animals. One group of mice was given Y-27632 (5 mg/kg) only. a ELISA was used to quantify the levels of MIP-2 and KC in the lungs of mice 4 h after M1 protein challenge. b Quantitative RT-PCR was used to determine mRNA levels of MIP-2 and KC in alveolar macrophages 30 min after M1 protein injection. Levels of MIP-2 and KC mRNA were normalized to mRNA levels of β-actin. Data represents mean ± SEM, * p < 0.05 versus sham and ^# p < 0.05 versus vehicle + M1 protein, n = 5.

Fig. 7

Phosphorylation of p38 MAPK. Sham animals received only PBS. Mice were pretreated i.p. with vehicle (PBS) or Y-27632 (0.5 or 5 mg/kg) 10 min prior to M1 protein challenge. Samples were harvested 4 h after M1 protein challenge. a Western blot analysis of extracts from lung tissue (see Materials and Methods for details) using an anti-phospho-p38 MAPK antibody (upper) or an anti-p38 MAPK antibody (lower). b Band intensities were quantified by densitometry and the ratio of phospho-p38 MAPK expression to total p38 MAPK expression is shown. Data represents mean ± SEM, * p < 0.05 versus sham and ^# p < 0.05 versus vehicle + M1 protein, n = 3.