Platelet-Derived CCL5 Regulates CXC Chemokine Formation and Neutrophil Recruitment in Acute Experimental Colitis.

Accumulating data suggest that platelets not only regulate thrombosis and haemostasis but also inflammatory processes. Platelets contain numerous potent pro-inflammatory compounds, including the chemokines CCL5 and CXCL4, although their role in acute colitis remains elusive. The aim of this study is to examine the role of platelets and platelet-derived chemokines in acute colitis. Acute colitis is induced in female Balb/c mice by administration of 5% dextran sodium sulfate (DSS) for 5 days. Animals receive a platelet-depleting, anti-CCL5, anti-CXCL4, or a control antibody prior to DSS challenge. Colonic tissue is collected for quantification of myeloperoxidase (MPO) activity, CXCL5, CXCL2, interleukin-6 (IL-6), and CCL5 levels as well as morphological analyses. Platelet depletion reduce tissue damage and clinical disease activity index in DSS-exposed animals. Platelet depletion not only reduces levels of CXCL2 and CXCL5 but also levels of CCL5 in the inflamed colon. Immunoneutralization of CCL5 but not CXCL4 reduces tissue damage, CXC chemokine expression, and neutrophil recruitment in DSS-treated animals. These findings show that platelets play a key role in acute colitis by regulating CXC chemokine generation, neutrophil infiltration, and tissue damage in the colon. Moreover, our results suggest that platelet-derived CCL5 is an important link between platelet activation and neutrophil recruitment in acute colitis.

STAT3-dependent CXC chemokine formation and neutrophil migration in streptococcal M1 protein-induced acute lung inflammation.

Streptococcus pyogenes cause infections ranging from mild pharyngitis to severe streptococcal toxic shock syndrome (STSS). The M1 serotype of Streptococcus pyogenes is most frequently associated with STSS. Herein, it was hypothesized that STAT3 signaling might be involved in M1 protein-evoked lung inflammation. The STAT3 inhibitor, S3I-201, was administered to male C57Bl/6 mice before iv challenge with M1 protein. Bronchoalveolar fluid and lung tissue were harvested for quantification of STAT3 activity, neutrophil recruitment, edema, and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Levels of IL-6 and HMGB1 were determined in plasma. CXCL2-induced neutrophil chemotaxis was studied in vitro. Administration of S3I-201 markedly reduced M1 protein-provoked STAT3 activity, neutrophil recruitment, edema formation, and inflammatory changes in the lung. In addition, M1 protein significantly increased Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Treatment with S3I-201 had no effect on M1 protein-induced expression of Mac-1 on neutrophils. In contrast, inhibition of STAT3 activity greatly reduced M1 protein-induced formation of CXC chemokines in the lung. Interestingly, STAT3 inhibition markedly decreased plasma levels of IL-6 and HMGB1 in animals exposed to M1 protein. Moreover, we found that S3I-201 abolished CXCL2-induced neutrophil migration in vitro. In conclusion, these novel findings indicate that STAT3 signaling plays a key role in mediating CXC chemokine production and neutrophil infiltration in M1 protein-induced acute lung inflammation.

Streptococcal M1 protein triggers chemokine formation, neutrophil infiltration, and lung injury in an NFAT-dependent manner.

Streptococcus pyogenes of the M1 serotype can cause STSS, which is associated with significant morbidity and mortality. The purpose of the present study was to examine the role of NFAT signaling in M1 protein-induced lung injury. NFAT-luc mice were treated with the NFAT inhibitor A-285222 before administration of the M1 protein. Neutrophil infiltration, edema, and CXC chemokines were quantified in the lung, 4 h after challenge with the M1 protein. Flow cytometry was used to determine Mac-1 expression. Challenge with the M1 protein increased NFAT-dependent transcriptional activity in the lung, spleen, and liver in NFAT-luc mice. Administration of the NFAT inhibitor A-285222 abolished M1 protein-evoked NFAT activation in the lung, spleen, and liver. M1 protein challenge induced neutrophil recruitment, edema, and CXC chemokine production in the lung, as well as up-regulation of Mac-1 on circulating neutrophils. Inhibition of NFAT activity attenuated M1 protein-induced neutrophil infiltration by 77% and edema formation by 50% in the lung. Moreover, administration of A-285222 reduced M1 protein-evoked pulmonary formation of CXC chemokine >80%. In addition, NFAT inhibition decreased M1 protein-triggered Mac-1 up-regulation on neutrophils. These findings indicate that NFAT signaling controls pulmonary infiltration of neutrophils in response to streptococcal M1 protein via formation of CXC chemokines and neutrophil expression of Mac-1. Thus, the targeting of NFAT activity might be a useful way to ameliorate lung injury in streptococcal infections.

Human thrombin-derived host defense peptides inhibit neutrophil recruitment and tissue injury in severe acute pancreatitis.

Severe acute pancreatitis (AP) is characterized by leukocyte infiltration and tissue injury. Herein, we wanted to examine the potential effects of thrombin-derived host defense peptides (TDPs) in severe AP. Pancreatitis was provoked by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of l-arginine in C57BL/6 mice. Animals were treated with the TDPs GKY20 and GKY25 or a control peptide WFF25 30 min before induction of AP. TDPs reduced blood amylase levels, neutrophil infiltration, hemorrhage, necrosis, and edema formation in the inflamed pancreas. Treatment with TDPs markedly attenuated the taurocholate-induced increase in plasma levels of CXCL2 and interleukin-6. Moreover, administration of TDPs decreased histone 3, histone 4, and myeloperoxidase levels in the pancreas in response to taurocholate challenge. Interestingly, administration of TDPs abolished neutrophil expression of Mac-1 in mice with pancreatitis. In addition, TDPs inhibited CXCL2-induced chemotaxis of isolated neutrophils in vitro. Fluorescent-labeled TDP was found to directly bind to isolated neutrophils. Finally, a beneficial effect of TDPs was confirmed in l-arginine-induced pancreatitis. Our novel results demonstrate that TDPs exert protective effects against pathological inflammation and tissue damage in AP. These findings suggest that TDPs might be useful in the management of patients with severe AP.

Ras regulates alveolar macrophage formation of CXC chemokines and neutrophil activation in streptococcal M1 protein-induced lung injury.

Streptococcal toxic shock syndrome (STSS) is associated with a high mortality rate. The M1 serotype of Streptococcus pyogenes is most frequently associated with STSS. Herein, we examined the role of Ras signaling in M1 protein-induced lung injury. Male C57BL/6 mice received the Ras inhibitor (farnesylthiosalicylic acid, FTS) prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Quantitative RT-PCR was used to determine gene expression of CXC chemokines in alveolar macrophages. Administration of FTS reduced M1 protein-induced neutrophil recruitment, edema formation and tissue damage in the lung. M1 protein challenge increased Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Inhibition of Ras activity decreased M1 protein-induced expression of Mac-1 on neutrophils and secretion of CXC chemokines in the lung. Moreover, FTS abolished M1 protein-provoked gene expression of CXC chemokines in alveolar macrophages. Ras inhibition decreased chemokine-mediated neutrophil migration in vitro. Taken together, our novel findings indicate that Ras signaling is a potent regulator of CXC chemokine formation and neutrophil infiltration in the lung. Thus, inhibition of Ras activity might be a useful way to antagonize streptococcal M1 protein-triggered acute lung injury.

Targeting CD162 protects against streptococcal M1 protein-evoked neutrophil recruitment and lung injury.

Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome and acute lung damage. CD162 is an adhesion molecule that has been reported to mediate neutrophil recruitment in acute inflammatory reactions. In this study, the purpose was to investigate the role of CD162 in M1 protein-provoked lung injury. Male C57BL/6 mice were treated with monoclonal antibody directed against CD162 or a control antibody before M1 protein challenge. Edema, neutrophil infiltration, and CXC chemokines were determined in the lung, 4 h after M1 protein administration. Fluorescence intravital microscopy was used to analyze leukocyte-endothelium interactions in the pulmonary microcirculation. Inhibition of CD162 reduced M1 protein-provoked accumulation of neutrophils, edema, and CXC chemokine formation in the lung by >54%. Moreover, immunoneutralization of CD162 abolished leukocyte rolling and firm adhesion in pulmonary venules of M1 protein-treated animals. In addition, inhibition of CD162 decreased M1 protein-induced capillary trapping of leukocytes in the lung microvasculature and improved microvascular perfusion in the lungs of M1 protein-treated animals. Our findings suggest that CD162 plays an important role in M1 protein-induced lung damage by regulating leukocyte rolling in pulmonary venules. Consequently, inhibition of CD162 attenuates M1 protein-evoked leukocyte adhesion and extravasation in the lung. Thus, our results suggest that targeting the CD162 might pave the way for novel opportunities to protect against pulmonary damage in streptococcal infections.

Targeting Rac1 signaling inhibits streptococcal M1 protein-induced CXC chemokine formation, neutrophil infiltration and lung injury.

Infections with Streptococcus pyogenes exhibit a wide spectrum of infections ranging from mild pharyngitis to severe Streptococcal toxic shock syndrome (STSS). The M1 serotype of Streptococcus pyogenes is most commonly associated with STSS. In the present study, we hypothesized that Rac1 signaling might regulate M1 protein-induced lung injury. We studied the effect of a Rac1 inhibitor (NSC23766) on M1 protein-provoked pulmonary injury. Male C57BL/6 mice received NSC23766 prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Quantitative RT-PCR was used to determine gene expression of CXC chemokines in alveolar macrophages. Treatment with NSC23766 decreased M1 protein-induced neutrophil infiltration, edema formation and tissue injury in the lung. M1 protein challenge markedly enhanced Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Inhibition of Rac1 activity had no effect on M1 protein-induced expression of Mac-1 on neutrophils. However, Rac1 inhibition markedly decreased M1 protein-evoked formation of CXC chemokines in the lung. Moreover, NSC23766 completely inhibited M1 protein-provoked gene expression of CXC chemokines in alveolar macrophages. We conclude that these novel results suggest that Rac1 signaling is a significant regulator of neutrophil infiltration and CXC chemokine production in the lung. Thus, targeting Rac1 activity might be a potent strategy to attenuate streptococcal M1 protein-triggered acute lung damage.

Geranylgeranyl transferase regulates streptococcal M1 protein-induced CXC chemokine formation and neutrophil recruitment in the lung.

Streptococcal toxic shock syndrome is most frequently associated with Streptococcus pyogenes of the M1 serotype. Simvastatin protects against M1 protein-induced acute lung damage, although downstream mechanisms remain elusive. Herein, we hypothesized that geranylgeranylation might regulate proinflammatory effects in M1 protein-induced lung injury. Male C57BL/6 mice received the geranylgeranyl transferase inhibitor, GGTI-2133, before M1 protein injection. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema, and CXC chemokine formation. Mac-1 expression on neutrophils was quantified by use of flow cytometry. Quantitative reverse transcriptase-polymerase chain reaction was used to determine gene expression of CXC chemokines in alveolar macrophages. GGTI-2133 reduced M1 protein-provoked infiltration of neutrophils, edema, and tissue injury in the lung. Inhibition of geranylgeranyl transferase had no effect on M1 protein-evoked upregulation of Mac-1 on neutrophils. However, geranylgeranyl transferase inhibition completely inhibited pulmonary formation of CXC chemokines in mice exposed to M1 protein. Notably, GGTI-2133 abolished M1 protein-induced gene expression of CXC chemokines in alveolar macrophages. These novel findings indicate that geranylgeranyl transferase is an important regulator of neutrophil recruitment and CXC chemokine production in the lung. Thus, targeting geranylgeranyl transferase might be a potent way to ameliorate streptococcal M1 protein-triggered acute lung injury.

Streptococcal m1 protein triggers farnesyltransferase-dependent formation of CXC chemokines in alveolar macrophages and neutrophil infiltration of the lungs.

The M1 serotype of Streptococcus pyogenes plays an important role in streptococcal toxic shock syndrome. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been shown to inhibit streptococcal M1 protein-induced acute lung damage, although downstream mechanisms remain elusive. Protein isoprenylation, such as farnesylation and geranylgeranylation, has been suggested to regulate anti-inflammatory effects exerted by statins. Here, we examined the effect of a farnesyltransferase inhibitor (FTI-277) on M1 protein-triggered lung inflammation. Male C57BL/6 mice were treated with FTI-277 prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema, and CXC chemokine formation. Flow cytometry was used to determine Mac-1 expression on neutrophils. The gene expression of CXC chemokines was determined in alveolar macrophages by using quantitative reverse transcription (RT)-PCR. We found that the administration of FTI-277 markedly decreased M1 protein-induced accumulation of neutrophils, edema formation, and tissue damage in the lung. Notably, inhibition of farnesyltransferase abolished M1 protein-evoked production of CXC chemokines in the lung and gene expression of CXC chemokines in alveolar macrophages. Moreover, FTI-277 completely inhibited chemokine-induced neutrophil migration in vitro. However, farnesyltransferase inhibition had no effect on M1 protein-induced expression of Mac-1 on neutrophils. Our findings suggest that farnesyltransferase is a potent regulator of CXC chemokine formation in alveolar macrophages and that inhibition of farnesyltransferase not only reduces neutrophil recruitment but also attenuates acute lung injury provoked by streptococcal M1 protein. We conclude that farnesyltransferase activity is a potential target in order to attenuate acute lung damage in streptococcal infections.

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

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.

p38 Mitogen-activated protein kinase signaling regulates streptococcal M1 protein-induced neutrophil activation and lung injury.

M1 serotype of Streptococcus pyogenes can cause STSS and acute lung damage. Herein, the purpose was to define the role of p38 MAPK signaling in M1 protein-induced pulmonary injury. Male C57BL/6 mice were treated with specific p38 MAPK inhibitors (SB 239063 and SKF 86002) prior to M1 protein challenge. Edema, neutrophil infiltration, and CXC chemokines were determined in the lung, 4 h after M1 protein administration. Flow cytometry was used to determine Mac-1 expression. Phosphorylation and activity of p38 MAPK were determined by immunoprecipitation and Western blot. IVM was used to analyze leukocyte-endothelium interactions in the pulmonary microcirculation. M1 protein challenge increased phosphorylation and activity of p38 MAPK in the lung, which was inhibited by SB 239063 and SKF 86002. Inhibition of p38 MAPK activity decreased M1 protein-induced infiltration of neutrophils, edema, and CXC chemokine formation in the lung, as well as Mac-1 up-regulation on neutrophils. IVM showed that p38 MAPK inhibition reduced leukocyte rolling and adhesion in the pulmonary microvasculature of M1 protein-treated mice. Our results indicate that p38 MAPK signaling regulates neutrophil infiltration in acute lung injury induced by streptococcal M1 protein. Moreover, p38 MAPK activity controls CXC chemokine formation in the lung, as well as neutrophil expression of Mac-1 and recruitment in the pulmonary microvasculature. In conclusion, these findings suggest that targeting the p38 MAPK signaling pathway may open new opportunities to protect against lung injury in streptococcal infections.

Simvastatin regulates CXC chemokine formation in streptococcal M1 protein-induced neutrophil infiltration in the lung.

Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome and acute lung injury. Statins exert beneficial effects in septic patients although the mechanisms remain elusive. This study examined effects of simvastatin on M1 protein-provoked pulmonary inflammation and tissue injury. Male C57BL/6 mice were pretreated with simvastatin or a CXCR2 antagonist before M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for determination of neutrophil infiltration, formation of edema, and CXC chemokines. Flow cytometry was used to determine Mac-1 expression on neutrophils. Gene expression of CXC chemokines was determined in alveolar macrophages by using quantitative RT-PCR. M1 protein challenge caused massive infiltration of neutrophils, edema formation, and production of CXC chemokines in the lung as well as upregulation of Mac-1 on circulating neutrophils. Simvastatin reduced M1 protein-induced infiltration of neutrophils and edema in the lung. In addition, M1 protein-induced Mac-1 expression on neutrophils was abolished by simvastatin. Furthermore, simvastatin markedly decreased pulmonary formation of CXC chemokines and gene expression of CXC chemokines in alveolar macrophages. Moreover, the CXCR2 antagonist reduced M1 protein-induced neutrophil expression of Mac-1 and accumulation of neutrophils as well as edema formation in the lung. These novel findings indicate that simvastatin is a powerful inhibitor of neutrophil infiltration in acute lung damage triggered by streptococcal M1 protein. The inhibitory effect of simvastatin on M1 protein-induced neutrophil recruitment appears related to reduced pulmonary generation of CXC chemokines. Thus, simvastatin may be a useful tool to ameliorate acute lung injury in streptococcal infections.

Simvastatin antagonizes CD40L secretion, CXC chemokine formation, and pulmonary infiltration of neutrophils in abdominal sepsis.

Statins have been reported to exert anti-inflammatory actions and protect against septic organ dysfunction. Herein, we hypothesized that simvastatin may attenuate neutrophil activation and lung damage in abdominal sepsis. Male C57BL/6 mice were pretreated with simvastatin (0.5 or 10 mg/kg) before CLP. In separate groups, mice received an anti-CD40L antibody or a CXCR2 antagonist (SB225002) prior to CLP. BALF and lung tissue were harvested for analysis of neutrophil infiltration, as well as edema and CXC chemokine formation. Blood was collected for analysis of Mac-1 and CD40L expression on neutrophils and platelets, as well as soluble CD40L in plasma. Simvastatin decreased CLP-induced neutrophil infiltration and edema formation in the lung. Moreover, Mac-1 expression increased on septic neutrophils, which was significantly attenuated by simvastatin. Inhibition of CD40L reduced CLP-induced up-regulation of Mac-1 on neutrophils. Simvastatin prevented CD40L shedding from the surface of platelets and reduced circulating levels of CD40L in septic mice. CXC chemokine-induced migration of neutrophils in vitro was decreased greatly by simvastatin. Moreover, simvastatin abolished CLP-evoked formation of CXC chemokines in the lung, and a CXCR2 antagonist attenuated pulmonary accumulation of neutrophils. Our data suggest that the inhibitory effect of simvastatin on pulmonary accumulation of neutrophils may be related to a reduction of CD40L secretion into the circulation, as well as a decrease in CXC chemokine formation in the lung. Thus, these protective mechanisms help to explain the beneficial actions exerted by statins, such as simvastatin, in sepsis.

Streptococcal M1 protein-induced lung injury is independent of platelets in mice.

Streptococcus pyogenes of the M1 serotype is frequently associated with severe streptococcal infections. M1 protein challenge can cause widespread microthrombosis, suggesting a role of platelets in streptococcal sepsis. Herein, we hypothesized that platelets may play a role in M1 protein-induced lung inflammation and injury. M1 protein was injected intravenously in C57Bl/6 mice. For platelet and neutrophil depletion, an anti-GP1bα antibody and an anti-Gr-1 antibody, respectively, were administered before M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for analysis of neutrophil infiltration, edema, and macrophage inflammatory protein 2 (MIP-2) formation. Blood was collected for analysis of membrane-activated complex 1 (Mac-1) and CD40 ligand (CD40L) expression on neutrophils and platelets as well as soluble CD40L in plasma. M1 protein caused significant pulmonary damage characterized by neutrophil infiltration, increased formation of edema and MIP-2 in the lung, and enhanced Mac-1 expression on neutrophils. However, M1 protein challenge had no effect on platelet surface expression of CD40L or soluble CD40L levels in plasma. Interestingly, platelet depletion had no influence on M1 protein-induced neutrophil recruitment, MIP-2 production, and tissue damage in the lung or Mac-1 expression on neutrophils. Moreover, we observed that M1 protein could bind to neutrophils but not to platelets. On the other hand, neutrophil depletion abolished M1 protein-induced edema formation and tissue damage in the lung. Our data suggest that neutrophils but not platelets are involved in the pathophysiology of M1 protein-provoked pulmonary damage. Thus, neutrophils may constitute a key target in infections caused by S. pyogenes of the M1 serotype.