LFA-1 and MAC-1 mediate pulmonary recruitment of neutrophils and tissue damage in abdominal sepsis

Neutrophil-mediated lung damage is an insidious feature in septic patients, although the adhesive mechanisms behind pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. The aim of the present study was to define the role of lymphocyte function antigen-1 (LFA-1) and membrane-activated complex 1 (Mac-1) in septic lung injury. Pulmonary edema, bronchoalveolar infiltration of neutrophils, levels of myeloperoxidase, and CXC chemokines were determined after cecal ligation and puncture (CLP). Mice were treated with monoclonal antibodies directed against LFA-1 and Mac-1 before CLP induction. Cecal ligation and puncture induced clear-cut pulmonary damage characterized by edema formation, neutrophil infiltration, and increased levels of CXC chemokines in the lung. Notably, immunoneutralization of LFA-1 or Mac-1 decreased CLP-induced neutrophil recruitment in the bronchoalveolar space by more than 64%. Moreover, functional inhibition of LFA-1 and Mac-1 abolished CLP-induced lung damage and edema. However, formation of CXC chemokines in the lung was intact in mice pretreated with the anti-LFA-1 and anti-Mac-1 antibodies. Our data demonstrate that both LFA-1 and Mac-1 regulate pulmonary infiltration of neutrophils and lung edema associated with abdominal sepsis. Thus, these novel findings suggest that LFA-1 or Mac-1 may serve as targets to protect against lung injury in polymicrobial sepsis.     

CD44 is a physiological E-selectin ligand on neutrophils

The selectin family of adhesion molecules and their glycoconjugated ligands are essential for blood polymorphonuclear neutrophil (PMN) extravasation into inflammatory and infectious sites. However, E-selectin ligands on PMNs are not well characterized. We show here that CD44 immunopurified from G-CSF-differentiated 32D cells or from peripheral blood PMNs binds specifically to E-selectin. In contrast, CD44 extracted from bone marrow stromal or brain endothelial cell lines does not interact with E-selectin, suggesting cell-specific posttranslational modifications of CD44. PMN-derived CD44 binding activity is mediated by sialylated, alpha(1,3) fucosylated, N-linked glycans. CD44 enables slow leukocyte rolling on E-selectin expressed on inflamed endothelium in vivo and cooperates with P-selectin glycoprotein ligand-1 to recruit neutrophils into thioglycollate-induced peritonitis and staphylococcal enterotoxin A-injected skin pouch. CD44 extracted from human PMNs also binds to E-selectin. Moreover, we demonstrate that CD44 is hypofucosylated in PMNs from a patient with leukocyte adhesion deficiency type II, suggesting that it contributes to the syndrome. These findings thus suggest broader roles for CD44 in the innate immune response and uncover a potential new target for diseases in which selectins play a prominent role.     

CD44及其在肺癌研究中的进展

CD44是属于黏附分子家族的跨膜糖蛋白,具有激活淋巴细胞、参与信号传递、促进细胞黏附等多种生物学功能。CD44的异常表达与肺癌的生长、发展预后及影像学表现关系密切,本文就CD44与肺癌的关系作一综述。     

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.     

Targeting RAGE in sepsis

The receptor of advanced glycation endproducts (RAGE) is a multiligand receptor that upon activation causes sustained activation of multiple inflammatory pathways. Recent evidence, summarized in a review by Bopp and colleagues in this issue of Critical Care, has implicated RAGE as a potential therapeutic target in sepsis. Here, we discuss several open issues that need to be addressed before anti-RAGE strategies can enter the sepsis clinical trial arena.     

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.     

2型糖尿病患者红细胞CD35、CD44s和CD58分子含量的变化及临床意义

目的研究2型糖尿病(T2DM)患者红细胞补体受体1型分子(CR1,即CD35)、CD44s和CD58含量变化及临床意义。方法应用完整细胞酶免疫定量分析法对75名健康对照人及66名T2DM患者进行比较。结果T2DM患者红细胞CD35、CD58含量明显低于正常对照人群,2组差异具有非常显著意义(P<0.0001);CD44s下降不明显(P>0.05);身高和体重影响CD35含量变化。结论红细胞CD35、CD44s和CD58含量减低可能导致T2DM患者红细胞免疫功能低下,它们可能共同参与T2DM的发生发展过程。     

E—cad、CD44v6在肺癌中的表达及意义

目的研究E-cad、CD44v6在肺癌中的表达,探讨其与肺癌临床病理特征、淋巴结转移的关系.方法利用免疫组化S-P法检测65例肺癌、13例支气管黏膜不典型增生和8例正常肺组织的E-cad、CD44v6蛋白表达.结果肺癌组织的E-cad阳性率(46.2%)显著低于支气管黏膜不典型增生(84.5%)和正常肺组织(100%)(P＜0.05,P＜0.01);肺癌阳性率与淋巴结转移、临床分期呈负相关(P＜0.01,P＜0.05).在肺癌的阳性率(83.1%)显著高于不典型增生(38.5%)和正常肺组织(25%)(P＜0.01);CD44v6阳性表达与肺癌淋巴结转移、临床分期、病理分级呈正相关(P＜0.05).肺癌组织的E-cad阴性与CD44v6阳性表达有显著的相关性(P＜0.01).结论肺癌组织中的E-cad失表达、CD44v6高表达与肺癌的发生发展、淋巴结转移及预后有一定关系.     

CD44 in cancer

CD44 is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines, and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival. All these biological properties are essential to the physiological activities of normal cells, but they are also associated with the pathologic activities of cancer cells. Experiments in animals have shown that targeting of CD44 by antibodies, antisense,and CD44-soluble proteins markedly reduces the malignant activities of various neoplasms, stressing the therapeutic potential of anti-CD44 agents. Furthermore, because alternative splicing and posttranslational modifications generate many different CD44 sequences, including, perhaps, tumor-specific sequences, the production of anti-CD44 tumor-specific agents may be a realistic therapeutic approach. However, in many cancers (renal cancer and non-Hodgkin's lymphomas are exceptions), a high level of CD44 expression is not always associated with an unfavorable outcome. On the contrary, in some neoplams CD44 upregulation is associated with a favorable outcome. Even worse, in many cases different research grows analyzing the same neoplastic disease reached contradictory conclusions regarding the correlation between CD44 expression and disease prognosis, possibly due to differences in methodology. These problems must be resolved before applying anti-CD44 therapy to human cancers.     

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.     

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.     

周围型非小细胞肺癌CT征象、预后与CD44v3、CD44v6表达的相关研究

目的 探讨周围型非小细胞肺癌的CT征象、预后与CD44v3、CD44v6表达的相关性.方法 经手术病理证实的46例周围型NSCLC患者进入研究.利用免疫组化SP法检测CD44v3、CD44v6在肺癌组织中的表达,分析其与肺癌CT征象及预后的关系. 结果 CD44v3、CD44v6表达与肺癌CT征象中的深分叶征、棘突征、短毛刺征及肺门、纵隔淋巴结增大密切相关(P＜0.05),与肿瘤大小、空泡征、血管集束征及胸膜凹陷征无明显相关性(P＞0.05);CD44v3、CD44v6表达与肺癌术后3年生存率密切相关(P＜0.05). 结论 深分叶征、棘突征、短毛刺征及肺门、纵隔淋巴结增大与CD44v3、CD44v6表达明显相关,提示肺癌的侵袭、转移能力较强;CD44v3、CD44v6阳性表达的肺癌患者术后生存时间更短,预后更差.     

Intravenous glutamine decreases lung and distal organ injury in an experimental model of abdominal sepsis

Introduction

The protective effect of glutamine, as a pharmacological agent against lung injury, has been reported in experimental sepsis; however, its efficacy at improving oxygenation and lung mechanics, attenuating diaphragm and distal organ injury has to be better elucidated. In the present study, we tested the hypothesis that a single early intravenous dose of glutamine was associated not only with the improvement of lung morpho-function, but also the reduction of the inflammatory process and epithelial cell apoptosis in kidney, liver, and intestine villi.

Methods

Seventy-two Wistar rats were randomly assigned into four groups. Sepsis was induced by cecal ligation and puncture surgery (CLP), while a sham operated group was used as control (C). One hour after surgery, C and CLP groups were further randomized into subgroups receiving intravenous saline (1 ml, SAL) or glutamine (0.75 g/kg, Gln). At 48 hours, animals were anesthetized, and the following parameters were measured: arterial oxygenation, pulmonary mechanics, and diaphragm, lung, kidney, liver, and small intestine villi histology. At 18 and 48 hours, Cytokine-Induced Neutrophil Chemoattractant (CINC)-1, interleukin (IL)-6 and 10 were quantified in bronchoalveolar and peritoneal lavage fluids (BALF and PLF, respectively).

Results

CLP induced: a) deterioration of lung mechanics and gas exchange; b) ultrastructural changes of lung parenchyma and diaphragm; and c) lung and distal organ epithelial cell apoptosis. Glutamine improved survival rate, oxygenation and lung mechanics, minimized pulmonary and diaphragmatic changes, attenuating lung and distal organ epithelial cell apoptosis. Glutamine increased IL-10 in peritoneal lavage fluid at 18 hours and bronchoalveolar lavage fluid at 48 hours, but decreased CINC-1 and IL-6 in BALF and PLF only at 18 hours.

Conclusions

In an experimental model of abdominal sepsis, a single intravenous dose of glutamine administered after sepsis induction may modulate the inflammatory process reducing not only the risk of lung injury, but also distal organ impairment. These results suggest that intravenous glutamine may be a potentially beneficial therapy for abdominal sepsis.     

脓毒症线粒体损伤研究进展

脓毒症(sepsis)是由病原微生物感染所致的全身炎性反应综合征,尽管目前对该疾病的病理生理有了进一步认识,其病死率仍居高不下,成为了重症医学的临床研究热点。大量研究表明严重脓毒症多器官功能障碍综合征(MODS)的发生与线粒体功能损伤关系密切。脓毒症时线粒体的损伤主要由线粒体内膜的损伤、线粒体钙超载、线粒体DNA的损伤等主要环节造成,本文就脓毒症线粒体损伤的机制、线粒体损伤后主要成分的改变及靶向治疗的研究进展进行阐述。     

Expression of CD44 variants in lung cancer and its relationship to hyaluronan binding

We examined the expression of CD44 variant forms and their binding to hyaluronan (HA) in lung cancer cell lines. There was no relationship between the level of expression of CD44 variants and HA binding in different lung cancer cell lines. The expression of CD44v6 and CD44E in some cell lines was not always associated with HA binding. There was no relationship between the tissue pathological type and CD44 expression or HA binding. Deglycosylation by neuraminidase induced CD44-HA binding in human lung cancer cell lines. Our findings suggest that the HA binding ability of CD44, which is negatively regulated by glycosylation, might be a more important factor in tumorigenesis or metastasis than the expression of CD44 variant forms.     

CD44mRNA在非小细胞肺癌中的表达及临床意义

目的探讨CD44mRNA在非小细胞肺癌（NSCLC）组织中的表达情况及其临床预后的关系。方法应用RT-PCR方法检测36例NSCLC组织和10例正常肺组织中CD44mRNA的表达，并对36例NSCLC患者进行5年以上随访。结果①36例NSCLC组织有21例CD44mRNA的过量表达，过量表达率为58．3％。②CD44mRNA在NSCLC组织中的表达与病人的年龄、性别、组织学类型、TNM分期、肿瘤大小无关，只与淋巴结转移有相关（P=0．007）。③经Kaplan-Meier生存曲线分析表明，36例NSCLC组织中CD44过量表达组生存率低于一般表达组（P=0．001）。结论CD44mRNA过量表达的NSCLC患者淋巴结转移率高，SD44RNA的表达对患者的预后和术后放疗及化疗具有指导性作用。     

A6 peptide activates CD44 adhesive activity, induces FAK and MEK phosphorylation, and inhibits the migration and metastasis of CD44-expressing cells

The A6 peptide (acetyl-KPSSPPEE-amino) has antitumor activity in the absence of significant adverse events in murine tumor models and clinical trials. A6 shares sequence homology with CD44, an adhesion receptor involved in metastasis that is also a marker of cancer stem cells and drug-resistant phenotypes. We investigated the mechanism of action of A6 by examining its effects on CD44 activity, cell migration, and metastasis. A6 inhibited the migration of a subset of ovarian and breast cancer cell lines, exhibiting IC(50) values of 5 to 110 nmol/L. The ability of A6 to inhibit migration in vitro correlated with CD44 expression. Immunopreciptation studies showed that CD44 binds A6 and that biotin-tagged A6 can be cross-linked to CD44. The binding of A6 altered the structure of CD44 such that it was no longer recognized by a monoclonal antibody to a specific epitope. Importantly, A6 potentiated the CD44-dependent adhesion of cancer cells to hyaluronic acid and activated CD44-mediated signaling, as evidenced by focal adhesion kinase and MAP/ERK kinase phosphorylation. In vivo, A6 (100 mg/kg delivered s.c. twice daily) reduced the number of lung foci generated by the i.v. injection of B16-F10 melanoma cells by 50% (P = 0.029 in an unpaired t test). We conclude that A6 potently blocks the migration of CD44-positive cells in vitro through an interaction with CD44 that alters its structure and activates CD44 to enhance ligand binding and downstream signaling. The concurrent ability of A6 to agonize the CD44 receptor suggests that CD44 activation may represent a novel strategy for inhibiting metastatic disease.     

Analysis of membrane antigens on neutrophils from patients with sepsis

The aim of the present study was to assess changes of cell membrane antigens on neutrophils in septic patients. Expression levels of neutrophil membrane antigens were measured employing a FACS calibur flow cytometer with several fluorescence-labeled monoclonal antibodies. Expression levels of the CD14 antigen were higher in patients with sepsis than in healthy individuals. In particular, the expression levels of CD14 increased in patients complicated by septic shock. Expression levels of TLR-4 were higher in patients with sepsis or septic shock than in healthy individuals. Expression levels of CD11b and CD16 were lower in patients with sepsis or septic shock than in healthy individuals and were even lower in those complicated by septic shock. Expression levels of neutrophil membrane antigens in patients with sepsis markedly changed in the acute phase. However, these levels tended to return to those of healthy individuals in the convalescing phase. Analyses of the surface antigens on neutrophils strongly involved in biological defense or tissue injury are informative for understanding the pathology of sepsis and for conducting therapy targeting neutrophils in the future.