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.     

Rho-kinase signaling regulates pulmonary infiltration of neutrophils in abdominal sepsis via attenuation of CXC chemokine formation and Mac-1 expression on neutrophils

Excessive neutrophil infiltration is a major component in septic lung injury, although the signaling mechanisms behind pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. Herein, we hypothesized that Rho-kinase activity may play a significant role in pulmonary neutrophil recruitment and tissue damage in abdominal sepsis. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 (0.5 or 5 mg/kg) before cecal ligation and puncture (CLP). Bronchoalveolar lavage fluid 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 on neutrophils and CD40L on platelets as well as soluble CD40L and matrix metalloproteinase 9 (MMP-9) in plasma. Cecal ligation and puncture triggered significant pulmonary damage characterized by neutrophil infiltration, increased levels of CXC chemokines, and edema formation in the lung. Furthermore, CLP upregulated Mac-1 expression on neutrophils, decreased CD40L on platelets, and increased soluble CD40L and MMP-9 in the circulation. Interestingly, inhibition of Rho-kinase dose-dependently decreased CLP-induced neutrophil expression of Mac-1, formation of CXC chemokines and edema, as well as neutrophil infiltration and tissue damage in the lung. Moreover, Rho-kinase inhibition significantly reduced sepsis-provoked gene expression of CXC chemokines in alveolar macrophages. In contrast, Rho-kinase inhibition had no effect on platelet shedding of CD40L or plasma levels of MMP-9 in septic mice. In conclusion, these data demonstrate that the Rho-kinase signaling pathway plays a key role in regulating pulmonary infiltration of neutrophils and tissue injury via regulation of CXC chemokine production in the lung and Mac-1 expression on neutrophils in abdominal sepsis.     

Clinical pathogenetic types of blood-peritoneal barrier damage in abdominal sepsis

Many authors point to the peritoneum-blood barrier damage factor in the pathogenesis of abdominal sepsis and peritonitis. The study was undertaken to define the types of blood-peritoneal barrier damage, by providing evidence for a differential approach to intensive care. For this, immunochemical permeability markers and the clinical and laboratory criteria for sepsis were studied in 106 patients. The patients with generalized peritonitis from the sepsis group showed a compensated type of barrier damage. Those with septic shock exhibited its decompensated type. The described types may serve as a criterion for the differential correction of intensive care in abdominal sepsis.     

Harmful and protective roles of neutrophils in sepsis

The current studies demonstrate protective and harmful effects of neutrophils (PMN) during experimental sepsis after cecal ligation and puncture (CLP). It is known that CLP induces signaling defects in blood PMN. When PMN were depleted 12 h after CLP, there were dramatic reductions in levels of bacteremia, evidence for reduced liver and renal dysfunction, sharp reductions in serum levels of cytokines (IL-1beta, IL-6, IL-10, TNF-alpha, and IL-2), and improved survival. In contrast, PMN depletion before CLP resulted in substantial increases in bacteremia and no evidence for attenuation of liver and renal failure dysfunction. These data suggest that at the onset of sepsis, PMN are important in regulating the levels of bacteremia, whereas after the onset of sepsis, as they lose innate immune functions, their presence is associated with higher levels of bacteremia and intensified organ dysfunction.     

The role of neutrophils in severe sepsis

Neutrophils are key effectors of the innate immune response. Reduction of neutrophil migration to infection sites is associated with a poor outcome in sepsis. We have demonstrated a failure of neutrophil migration in lethal sepsis. Together with this failure, we observed more bacteria in both peritoneal exudates and blood, followed by a reduction in survival rate. Furthermore, neutrophils obtained from severe septic patients displayed a marked reduction in chemotactic response compared with neutrophils from healthy subjects. The mechanisms of neutrophil migration failure are not completely understood. However, it is known that they involve systemic Toll-like receptor activation by bacteria and/or their products and result in excessive levels of circulating cytokines/chemokines. These mediators acting together with LPS stimulate expression of iNOS that produces high amounts of NO, which in turn mediates the failure of neutrophil migration. NO reduced expression of CXCR2 on neutrophils and the levels of adhesion molecules on both endothelial cells and neutrophils. These events culminate in decreased endothelium-leukocyte interactions, diminished neutrophil chemotactic response, and neutrophil migration failure. Additionally, the NO effect, at least in part, is mediated by peroxynitrite. In this review, we summarize what is known regarding the mechanisms of neutrophil migration impairment in severe sepsis.     

Neutrophil adherence to human endothelium in vitro occurs by CDw18 (Mo1, MAC-1/LFA-1/GP 150,95) glycoprotein-dependent and -independent mechanisms

Components of the CDw18 leukocyte surface glycoprotein complex (Mo1/LFA-1/GP 150,95 or MAC-1, LFA-1 family) are required for some adhesion-related functions of human neutrophils (PMNs). We evaluated the ability of monoclonal antibodies (MoAb) directed against specific determinants on the CDw18 glycoproteins to inhibit neutrophil adherence to cultured human endothelial cells (EC) stimulated by a variety of agonists, including thrombin and leukotriene C4, which induce the EC-dependent adhesion of PMNs. MoAb 60.3, an antibody that binds to an epitope common to the 3 heterodimer subunits of the neutrophil CDw18 complex, potently inhibited (90-100%) the rapid (5-30 minute) adherence response stimulated by N-formyl-methyionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating factor, phorbol myristate acetate, Ionophore A23187, and tumor necrosis factor. MoAbs directed against epitopes on the alpha polypeptide of the CD11b (Mol, MAC-1) heterodimer also inhibited PMN adherence to EC and to cell-free surfaces induced by these agonists. In contrast, the anti-CDw18 MoAbs had a trivial effect on maximal EC-dependent neutrophil adherence stimulated by thrombin and leukotriene C4, and incompletely inhibited PMN adherence induced by these agonists under submaximal conditions. These findings indicate that there is an alternative mechanism for neutrophil adherence, presumably resulting from molecular alterations of the EC surface, that does not require the PMN CDw18 glycoproteins. They also suggest that the inability to adhere to endothelium may not completely account for the defect in chemotaxis that is observed in vivo in neutrophils that are deficient in the CDw18 complex.     

Plasma elastase alpha 1-antitrypsin and lactoferrin in sepsis: evidence for neutrophils as mediators in fatal sepsis.

Increased vasopermeability and vasodilation, presumably the result of endothelial perturbation, are considered among the basic pathogenetic mechanisms in septic shock. Neutrophils have been implicated as a source for mediators in endothelial injury. We measured elastase-alpha 1-antitrypsin (alpha 1AT) complexes and lactoferrin as markers for release of neutrophil granule contents in plasma from patients with sepsis on admission to the Intensive Care Unit, and we delineated the relationship of neutrophil activation to other inflammatory parameters and to hemodynamic and biochemical parameters. Levels of elastase-alpha 1AT and lactoferrin significantly correlated with each other (r = 0.58; p less than 0.008), and were increased (greater than 3.33 and 5 nmol/L, respectively) in 96% and 71% of the patients, respectively. Lactoferrin, but not elastase-alpha 1AT, correlated with the number of white blood cells (r = 0.38; p = 0.008). Elastase-alpha 1 AT levels were significantly higher (p = 0.008), whereas white blood cell counts were lower (p = 0.015) in patients with shock when compared with patients without abnormal blood pressure. Both elastase-alpha 1AT and lactoferrin levels correlated with lactate levels (r = 0.33; p = 0.024 and r = 0.30; p = 0.04), suggesting a role for neutrophil activation in the pathogenesis of hypoxygenation. In addition, elastase-alpha 1AT correlated with the concentrations of interleukin 6 (IL-6) (r = 0.46; p = 0.001) and C3a (r = 0.38; p = 0.009), suggesting that cytokines and complement may contribute to the degranulation of neutrophils in sepsis. Elastase-alpha 1AT complexes were inversely related to C1-inhibitor (r = -0.33; p = 0.028) and to platelet numbers (r = -0.42; p = 0.003). Levels of elastase-alpha 1AT complexes in plasma appeared to be of prognostic significance; levels were higher in 27 patients who died than in 21 patients who survived (p = 0.01). The mortality in 27 patients with concentrations below 10 nM was 37%, whereas it was 81% in 21 patients with higher levels. The overall mortality in this study was 56%. These results provide further evidence that activation and degranulation of neutrophils, induced by multiple agonists, are involved in the development of fatal complications in patients with sepsis.     

Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro.

The adherence of human neutrophils to human umbilical vein endothelial cells (HUVEC) is partially dependent on the CD11/CD18 family of glycoproteins on the neutrophil and ICAM-1 on the HUVEC. The CD18 heterodimer involved in this adherence was evaluated in vitro using subunit-specific monoclonal antibodies (MAbs). The adherence of unstimulated neutrophils to IL-1-stimulated HUVEC was significantly inhibited by anti-CD11a but not CD11b MAbs, while the adherence of fMLP-stimulated neutrophils was significantly inhibited by both anti-CD11a and -CD11b. Anti-CD11a, but not anti-CD11b MAbs, reduced the adherence of unstimulated neutrophils on purified ICAM-1 to the same low level untreated neutrophils exhibited on a control protein, glycophorin. Stimulation with fMLP significantly increased neutrophil attachment to purified ICAM-1, but not to the control protein. Anti-CD11b MAbs reduced this chemotactically augmented adherence to that of unstimulated neutrophils, and in combination with anti-CD11a MAbs reduced adherence to that on the control protein. The results in this report indicate that unstimulated neutrophils exhibit LFA-1-dependent attachment to ICAM-1, and chemotactic stimulation enhances the attachment of human neutrophils to ICAM-1 by a Mac-1-dependent process.     

A3 and P2Y2 receptors control the recruitment of neutrophils to the lungs in a mouse model of sepsis

We have recently shown that A3 adenosine receptors and P2Y2 purinergic receptors play an important role in neutrophil chemotaxis. Chemotaxis of neutrophils to sites of infections is critical for immune defense. However, excessive accumulation of neutrophils in the lungs can cause acute lung tissue damage. Here we assessed the role of A3 and P2Y2 receptors in neutrophil sequestration to the lungs in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) using adult male C57BL/6J mice (wild type [WT]), homozygous A3 receptor knockout (A3KO) mice, and P2Y2 receptor knockout (P2Y2KO) mice. Animals were killed 2, 4, 6, or 8 h after CLP, and peritoneal lavage fluid and blood were collected. Lungs were removed, and neutrophil infiltration was evaluated using elastase as a marker. Leukocyte and bacterial counts in peritoneal lavage fluid and blood samples were determined. Survival after sepsis was determined in a separate group. Leukocyte counts in the peritoneum were lower in A3KO and P2Y2KO mice than in WT mice. Conversely, initial leukocyte counts in the peripheral blood were higher in KO mice than in WT mice. Neutrophil sequestration to the lungs reached a maximum 2 h after CLP and remained significantly higher in WT mice compared with A3KO and P2Y2KO mice (P < 0.001). Survival after 24 h was significantly lower in WT mice (37.5%) than in A3KO or P2Y2KO mice (82.5%; P < 0.05). These data suggest that A3 and P2Y2 receptors are involved in the influx of neutrophils into the lungs after sepsis. Thus, pharmaceutical approaches that target these receptors might be useful to control acute lung tissue injury in sepsis.     

结缔组织疾病患者心脏损害与肺动脉高压的相关性分析

目的探讨结缔组织疾病(Connective tissue disense,CTD)患者心脏损害与肺动脉高压(pulmonary arterial hyper-tension,PAH)并发的相关性。方法 193例CTD患者中合并PAH者44例(PAH组),149例无PAH(非PAH组),首先对相关因素采用单因素(性别、心包积液、心律失常、劳力性呼吸困难)非条件logistic回归分析,将单因素分析有意义的变量采用多因素非条件logistic回归分析。结果 PAH组性别、心包积液、心律失常、劳力性呼吸困难与非PAH组相比,差异均有统计学意义(P<0.05);Logistic回归显示心包积液(P=0.019)、心律失常(P=0.037)、劳力性呼吸困难(P=0.043)与PAH独立相关。结论 CTD患者心包积液、心律失常、劳力性呼吸困难与PAH发生、发展有明显的相关性。     

Intra-abdominal sepsis alters tumor necrosis factor-alpha and interleukin-1 beta binding to human neutrophils.

OBJECTIVE: To determine the effects of intraabdominal sepsis on polymorphonuclear leukocyte tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) receptor expression. DESIGN: Prospective, randomized comparison between patients undergoing elective colon surgery vs. patients with intra-abdominal sepsis. SETTING: Tertiary-care center with all patients with intra-abdominal sepsis in a surgical ICU environment. PATIENTS: Group 1 (n = 7) represents control patients who underwent elective colon surgery without intra-abdominal sepsis. Group 2 (n = 10) represents patients with intra-abdominal sepsis. MEASUREMENTS AND MAIN RESULTS: Polymorphonuclear leukocyte TNF-alpha and IL-1 beta receptor expression +/- stimulation of the oxidative burst was measured using 125I TNF-alpha and 125I IL-1 beta. Superoxide anion production and candicidal activity were measured in the presence of TNF-alpha and IL-1 beta. Group 2 patients expressed fewer TNF-alpha and IL-1 beta receptors on their cell surface, and stimulation of oxidative burst reduced TNF-alpha and IL-1 beta receptor expression in group 2 more than in group 1. Diminished TNF-alpha and IL-1 beta binding reduced superoxide anion production by group 2 polymorphonuclear leukocytes. Decreased TNF-alpha binding but not IL-1 beta, reduced polymorphonuclear leukocyte candicidal activity by group 2 polymorphonuclear leukocytes. CONCLUSIONS: a) Intra-abdominal sepsis reduces polymorphonuclear leukocyte TNF-alpha and IL-1 beta receptor expression. b) Expression of these surface receptors is altered by stimulation of the polymorphonuclear leukocyte oxidative burst. c) Diminished TNF-alpha and IL-1 beta receptor expression is associated with functional impairments in polymorphonuclear leukocyte activity.     

Immature circulating neutrophils in sepsis have impaired phagocytosis and calcium signaling

Patients with sepsis commonly develop leukocytosis, which is presumed to reflect a host response to infection. Effective phagocytosis by neutrophils is crucial in the clearance of invading microbes. However, efficacy of phagocytosis in sepsis is controversial. We hypothesized that host phagocytic capacity in sepsis can be affected by immature neutrophils that are released into the circulation. Circulating neutrophils were evaluated in 16 patients with severe sepsis and 5 healthy donors. Immature neutrophils were identified by the cell morphology. Phagocytosis was evaluated by micromanipulation technique and simultaneous cytosolic-free Ca2+ imaging. Leukocytosis was present in 12 of 16 patients. Nine of the 12 patients with leukocytosis and 3 of 4 patients with normal white blood cell counts had increased circulating immature neutrophils (mean, 39.3% +/- 20.7%; normal 相似文献   

C1q governs deposition of circulating immune complexes and leukocyte Fcgamma receptors mediate subsequent neutrophil recruitment

Inflammation induced by circulating immunoglobulin G-immune complexes (ICs) characterizes many immune-mediated diseases. In this work, the molecular requirements for the deposition of circulating ICs and subsequent acute leukocyte recruitment in mice were elucidated. We show that after intravenous injection, preformed soluble ICs are rapidly deposited in the postcapillary venules of the cremaster microcirculation, secondary to increased vascular permeability. This deposition is dependent on complement C1q. IC deposition is associated with leukocyte recruitment. Leukocyte rolling, which is mediated by P-selectin in the exteriorized cremaster muscle, is not further increased in response to ICs. In contrast, leukocyte rolling velocity is significantly decreased and leukocyte adhesion is significantly increased in the presence of ICs. The IC-mediated slow leukocyte rolling velocity and subsequent adhesion and emigration are dependent on Fcgamma receptors (FcgammaRs), particularly FcgammaRIII, with complement C3 and C5 having no detectable role. These studies suggest a regulatory mechanism of IC deposition and leukocyte trafficking in IC-mediated inflammation requiring C1q and FcgammaRs in sequential, noninteracting roles.     

Differential choice of intensive care in abdominal sepsis

The purpose of the case study was substantiation of a differential intensive care in abdominal sepsis with respect to its clinical-and-pathogenetic variations. Retro- and prospective clinical-and-biochemical parameters were investigated in 60 patients with disseminated peritonitis and abdominal sepsis. The patients were shared between 3 groups with respect to their clinical-and-laboratory findings. Clinical-and-pathogenetic types were defined for the clinical course of abdominal sepsis (AS). It was found as necessary to add enteral detoxication to the combined intensive care scheme in type 1 AS. A single-stage intravenous perftoran infusion plus enteral detoxication are required in type 2 AS. And in type 3 AS, the therapeutic scheme should be expanded through intravenous infusion of perftoran with subsequent enteral detoxication.     

可溶性髓系细胞触发受体-1对腹部创伤并发脓毒症的诊断价值

目的 探讨腹部创伤患者腹腔引流液可溶性髓系细胞触发受体-1(sTREM-1)的表达及其对腹部创伤后脓毒症的诊断价值.方法 将本院ICU收治的65例腹部创伤患者作为病例组,18例胃大部切除术(择期手术)后患者作为对照组,分别检测两组患者入ICU后0、24、48、72 h腹腔引流液sTREM-1、血清sTREM-1、前降钙素(PCT)和C反应蛋白(CRP)浓度,评价上述指标对创伤后脓毒症的早期诊断价值.结果 创伤组腹腔引流液sTREM-1、血清sTREM-1、PCT和CRP浓度明显高于对照组(P<0.05).创伤组中并发脓毒症组腹腔引流液sTREM-1、血清sTREM-1、PCT和CRP浓度明显高于未并发脓毒症组(P<0.05),腹腔引流液sTREM-1诊断创伤后脓毒症的敏感性82.5%,特异性84.0%,ROC曲线下面积0.885,明显优于血清sTREM-1、PCT及CRP(P<0.05).结论 腹腔引流液sTREM-1对腹部创伤后脓毒症的早期诊断有重要价值.     

Functional activity of neutrophils in patients with abdominal typhus

Cytochemistry and cytofluorometry were employed to study the main components of the intraleukocyte microbicide system of neutrophils in 52 patients with typhoid fever. During the disease there was a consistent decrease in the content of cationic protein and in the activity of leukocyte myeloperoxidase depending on the stage and severity of the pathological process. At the same time an increase in the glycogen content and activation of acid and alkaline phosphatases of leukocytes with a maximum at the disease height were detected. Addition of the complications aggravated cytochemical alterations in the cell and led to the delay in their elimination. Profound qualitative and quantitative changes in the activity of intracellular components with a visible clinical recovery of the patients pointed to an unfavourable prognosis.     

Hepatic phosphofructokinase-1 activity and fructose 2,6-bisphosphate levels in patients with abdominal sepsis.

1. In sepsis various processes of carbohydrate metabolism, such as hepatic gluconeogenesis and glycolysis, are altered. Phosphofructokinase-1, a key glycolytic enzyme, is controlled in the long term via regulation of synthesis and degradation of the protein itself, while in the short term it is regulated by allosteric effectors, such as fructose 2,6-bisphosphate (the most potent). In the present study hepatic phosphofructokinase-1 activity as well as phosphofructokinase-2 activity and the concentration of fructose 2,6-bisphosphate were assayed to determine if they might contribute to the derangement of carbohydrate metabolism seen commonly in sepsis. 2. The levels of glycogen and fructose 2,6-bisphosphate and the activity of phosphofructokinase-1 and phosphofructokinase-2 were determined in hepatic biopsies obtained at laparotomy from six patients with and seven patients without abdominal septic foci. 3. A significant increase in plasma lactate concentration was observed in the septic patients, whereas no significant differences in tissue glycogen content or plasma glucose concentration were seen between the groups. 4. No significant change in plasma insulin concentration was observed. However, levels of the counter-regulatory hormones (glucagon, cortisol and adrenaline) were elevated in the septic patients. 5. A 60% decrease in hepatic phosphofructokinase-1 activity was seen in the septic patients. However, no significant changes in hepatic phosphofructokinase-2 activity and fructose 2,6-bisphosphate content were observed in the septic patients. 6. The present results demonstrate that the decrease in hepatic phosphofructokinase-1 activity occurring in sepsis does not appear to reflect alterations in the concentration of fructose 2,6-bisphosphate.     

Platelets,endothelium and shear join forces to mislead neutrophils in sepsis

Neutrophils are circulating leukocytes with great cytotoxic potential, responsible for the first combat against invading pathogens. Their accumulation in tissues must be highly controlled so that the number of neutrophils delivered to the affected site is sufficient to control infection with minimum injury to the surrounding healthy tissue. In sepsis, neutrophil migration is dysregulated - resulting in insufficient delivery of neutrophils to the infectious site and massive neutrophil accumulation in uninfected organs. This dysregulation has the potential to cause inappropriate tissue injury that may explain the multiple organ dysfunction observed in severe sepsis. A better understanding of the mechanisms that contribute to this process is fundamental to design therapeutic strategies to circumvent tissue injury and organ dysfunction in sepsis.