Milladur Rahman

Platelets support pulmonary recruitment of neutrophils in abdominal sepsis

Objective: Recent findings indicate that platelets not only regulate thrombosis and hemostasis but may also be involved in proinflammatory activities. Herein, we hypothesized that platelets may play a role in sepsis by activating and priming circulating neutrophils for subsequent recruitment into the lung. Design: Prospective experimental study. Setting: University Hospital Research Unit. Subject: Male C57BL/6 mice. Interventions: Lung edema, bronchoalveolar infiltration of neutrophils, levels of myeloperoxidase, expression and function of membrane-activated complex-1 (Mac-1) on neutrophils and the CXC chemokines, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant were determined after cecal ligation and puncture (CLP). Mice received a platelet-depleting antibody as well as antibodies directed against P-selectin glycoprotein-ligand-1 and Mac-1 before CLP induction. Measurements and Main Results: CLP caused significant pulmonary damage characterized by neutrophil infiltration, increased levels of CXC chemokines, and edema formation in the lung. Furthermore, CLP up-regulated Mac-1 expression on neutrophils and increased the number of neutrophils binding platelets in the circulation. Interestingly, depletion of platelets reduced CLP-induced edema and neutrophil recruitment in the bronchoalveolar space by >60%. Furthermore, depletion of platelets reduced Mac-1 expression on neutrophils. On the other hand, inhibition of P-selectin glycoprotein-ligand-1 abolished CLP-induced neutrophil–platelet aggregation but had no effect on neutrophil expression of Mac-1. Conclusions: These data demonstrate that platelets play a key role in regulating infiltration of neutrophils and edema formation in the lung via upregulation of Mac-1 in abdominal sepsis.

Platelet-Derived CD40L (CD154) Mediates Neutrophil Upregulation of Mac-1 and Recruitment in Septic Lung Injury.

Objective:To define the role of CD40L in abdominal sepsis. Background:Platelets promote sepsis-induced pulmonary recruitment of neutrophils. However, the identity of the platelet-derived molecule regulating neutrophil infiltration is not known. The hypothesis of the present study was that platelet-derived CD40L might be responsible for platelet-mediated activation and accumulation of neutrophils in sepsis. Methods:Wild-type C57BL/6 mice and CD40L gene-deficient mice were exposed to cecal ligation and puncture (CLP). Lung edema, bronchoalveolar neutrophils, CD40L and macrophage inflammatory protein-2 (MIP-2) plasma levels, myeloperoxidase activity and Mac-1 expression were determined up to 24 hours after CLP induction. For platelet depletion was an anti-GP1bα antibody administered before CLP. Results:Plasma levels of soluble CD40L increased and surface expression of CD40L on platelets decreased in CLP mice. Platelet depletion reduced CLP-induced CD40L levels by 90%. CLP-provoked Mac-1 expression on neutrophils was abolished in CD40L-deficient mice. Interestingly, CLP-induced edema and myeloperoxidase activity in the lung as well as neutrophil infiltration in the broncoalveolar space were markedly reduced in mice lacking CD40L. In vitro experiments showed that CD40L was not capable of directly increasing Mac-1 levels on neutrophils. Instead, CLP-induced plasma levels of MIP-2 were significantly reduced in CD40L-deficient mice and inhibition of the MIP-2 receptor (CXCR2) decreased Mac-1 expression on neutrophils in septic animals. Conclusions:CD40L derived from platelets is a potent activator of neutrophils and mediates sepsis-induced neutrophil recruitment and lung edema. The neutrophil activating mechanism of CD40L is indirect and mediated via MIP-2 formation and CXCR2 signaling. Targeting CD40L may be an effective approach to limit pulmonary damage in abdominal sepsis.

Pro-inflammatory role of neutrophil extracellular traps in abdominal sepsis.

Excessive neutrophil activation is a major component in septic lung injury. Neutrophil-derived DNA may form extracellular traps in response to bacterial invasions. The aim of the present study was to investigate the potential role of neutrophil extracellular traps (NETs) in septic lung injury. Male C57BL/6 mice were treated with recombinant human (rh)DNAse (5 mg/kg) after cecal ligation and puncture (CLP). Extracellular DNA was stained by Sytox green, and NET formation was quantified by confocal microscopy and cell-free DNA in plasma, peritoneal cavity, and lung. Blood, peritoneal fluid, and lung tissue were harvested for analysis of neutrophil infiltration, NET levels, tissue injury, as well as CXC chemokine and cytokine formation. We observed that CLP caused increased formation of NETs in plasma, peritoneal cavity, and lung. Administration of rhDNAse not only eliminated NET formation in plasma, peritoneal cavity, and bronchoalveolar space but also reduced lung edema and tissue damage 24 h after CLP induction. Moreover, treatment with rhDNAse decreased CLP-induced formation of CXC chemokines, IL-6, and high-mobility group box 1 (HMGB1) in plasma, as well as CXC chemokines and IL-6 in the lung. In vitro, we found that neutrophil-derived NETs had the capacity to stimulate secretion of CXCL2, TNF-α, and HMGB1 from alveolar macrophages. Taken together, our findings show that NETs regulate pulmonary infiltration of neutrophils and tissue injury via formation of proinflammatory compounds in abdominal sepsis. Thus we conclude that NETs exert a proinflammatory role in septic lung injury.

P-selectin glycoprotein-ligand-1 regulates pulmonary recruitment of neutrophils in a platelet-independent manner in abdominal sepsis

Background and purpose:  Neutrophil‐mediated lung injury is an insidious feature in sepsis although the mechanisms regulating pulmonary recruitment of neutrophils remain elusive. Here, we investigated the role of P‐selectin glycoprotein‐ligand‐1 (PSGL‐1) in sepsis‐induced neutrophil recruitment and tissue injury in the lung.

Neutrophil Extracellular Traps Induce Trypsin Activation, Inflammation, and Tissue Damage in Mice With Severe Acute Pancreatitis.

BACKGROUND & AIMS Neutrophils are involved in the development of acute pancreatitis (AP), but it is not clear how neutrophil-induced tissue damage is regulated. In addition to secreting antimicrobial compounds, activated neutrophils eliminate invading microorganisms by expelling nuclear DNA and histones to form extracellular web-like structures called neutrophil extracellular traps (NETs). However, NETs have been reported to contribute to organ dysfunction in patients with infectious diseases. We investigated whether NETs contribute to the development of AP in mice. METHODS AP was induced in C57BL/6 mice by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of L-arginine. Pancreata were collected and extracellular DNA was detected by Sytox green staining, levels of CXC chemokines, histones, and cytokines also were measured. Cell-free DNA was quantified in plasma samples. Signal transducer and activator of transcription 3 phosphorylation and trypsin activation were analyzed in isolated acinar cells. NETs were depleted by administration of DNase I to mice. Plasma was obtained from healthy individuals (controls) and patients with severe AP. RESULTS Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice and increased levels of cell-free DNA in plasma. Neutrophil depletion prevented taurocholate-induced deposition of NETs in the pancreas. Administration of DNase I to mice reduced neutrophil infiltration and tissue damage in the inflamed pancreas and lung, and decreased levels of blood amylase, macrophage inflammatory protein-2, interleukin 6, and high-mobility groups protein 1. In mice given taurocholate, DNase I administration also reduced expression of integrin α M (macrophage-1 antigen) on circulating neutrophils. Similar results occurred in mice with L-arginine-induced AP. Addition of NETs and histones to acinar cells induced formation of trypsin and activation of signal transducer and activator of transcription 3; these processes were blocked by polysialic acid. Patients with severe AP had increased plasma levels of NET components compared with controls. CONCLUSIONS NETs form in the pancreata of mice during the development of AP, and NET levels are increased in plasma from patients with AP, compared with controls. NETs regulate organ inflammation and injury in mice with AP, and might be targeted to reduce pancreatic tissue damage and inflammation in patients.

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.

Platelet shedding of CD40L is regulated by matrix metalloproteinase-9 in abdominal sepsis.

Platelet‐derived CD40L is known to regulate neutrophil recruitment and lung damage in sepsis. However, the mechanism regulating shedding of CD40L from activated platelets is not known. We hypothesized that matrix metalloproteinase (MMP)‐9 might cleave surface‐expressed CD40L and regulate pulmonary accumulation of neutrophils in sepsis.

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.

Targeting CD44 Expressed on Neutrophils Inhibits Lung Damage in Abdominal Sepsis.

Neutrophil infiltration is an insidious feature in septic lung injury, although the specific adhesive mechanisms regulating pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. The aim of this present study was to define the role of CD44 in sepsis-induced neutrophil infiltration and lung damage. Mice were treated with a monoclonal antibody against CD44 before cecal ligation and puncture (CLP) induction. Edema formation, bronchoalveolar accumulation of neutrophils, myeloperoxidase activity, and macrophage inflammatory protein 2 (MIP-2) levels in the lung were determined after CLP. Expression of Mac-1 and CD44 on neutrophils was quantified by using flow cytometry. In separate experiments, fluorescent-labeled neutrophils coincubated with an anti-CD44 antibody were adoptively transferred to CLP mice. Cecal ligation and puncture triggered clear-cut lung damage characterized by edema formation, neutrophil infiltration, and increased levels of MIP-2 in the lung. Notably, immunoneutralization of CD44 reduced CLP-induced pulmonary accumulation of neutrophils. In addition, functional inhibition of CD44 decreased CLP-induced lung damage and edema. However, formation of MIP-2 in the lung and neutrophil expression of Mac-1 were intact in septic mice pretreated with the anti-CD44 antibody. Adoptive transfer experiments revealed that neutrophil rather than lung CD44 mediates neutrophil accumulation in septic lung injury. Moreover, administration of hyaluronidase had no effect on CLP-induced neutrophil recruitment and tissue damage in the lung. Our data demonstrate that CD44 contributes to pulmonary infiltration of neutrophils and lung damage associated with abdominal sepsis. Thus, these novel findings suggest that CD44 may serve as a target to protect against lung injury in polymicrobial sepsis.

Role of platelets in experimental acute pancreatitis.

Platelets not only control thrombosis and haemostasis but may also regulate inflammatory processes. Acute pancreatitis (AP) is characterized by changes in both coagulation and proinflammatory activities. The role of platelets in AP is not yet known.