Molecular Basis of Glycosaminoglycan Heparin Binding to the Chemokine CXCL1 Dimer

Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (K_d = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of “active” chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.     

Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells

Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.     

Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.     

CR1-mediated ATP Release by Human Red Blood Cells Promotes CR1 Clustering and Modulates the Immune Transfer Process

Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release. Our data show that CR1-mediated ATP release does not depend on Ca²⁺ or enzymes previously shown to mediate an increase in membrane deformability promoted by CR1 ligation. Furthermore, ATP release following CR1 ligation increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, and thereby enhances the binding avidity of complement-opsonized particles to the RBC CR1. Finally, we have found that RBC-derived ATP has a stimulatory effect on phagocytosis of immune-adherent immune complexes.     

Methicillin-resistant Staphylococcus aureus Bacterial Nitric-oxide Synthase Affects Antibiotic Sensitivity and Skin Abscess Development

Staphylococcus aureus infections present an enormous global health concern complicated by an alarming increase in antibiotic resistance. S. aureus is among the few bacterial species that express nitric-oxide synthase (bNOS) and thus can catalyze NO production from l-arginine. Here we generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. Loss of bNOS increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS also promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin. Surprisingly, bNOS-deficient strains gained resistance to aminoglycosides, suggesting that the role of bNOS in antibiotic susceptibility is more complex than previously observed in Bacillus species. Finally, the MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model. Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Future development of specific bNOS inhibitors could be an attractive option to simultaneously reduce MRSA pathology and enhance its susceptibility to commonly used antibiotics.     

Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34⁺ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.     

大鼠骨骼肌损伤后中性粒细胞、巨噬细胞和肌成纤维细胞数量分析研究

目的 研究大鼠骨骼肌损伤后中性粒细胞、巨噬细胞和肌成纤维细胞数量的变化情况,为今后骨骼肌损伤修复的病理学机制研究打下坚实的基础.方法 建立大鼠骨骼肌机械性损伤动物模型,随机分为伤后6h、12h、1d、3d、7d、10d、14d及正常对照组.应用免疫组织荧光染色和免疫组织化学染色,检测大鼠骨骼肌损伤后不同时间点中性粒细胞、巨噬细胞和肌成纤维细胞的数量.结果 伤后6h-12h,损伤区可见中性粒细胞和巨噬细胞浸润,中性粒细胞数量达到高峰.伤后1d,损伤区巨噬细胞数量急剧增加,迅速达到高峰,而中性粒细胞数量开始下降.伤后3d,中性粒细胞和巨噬细胞数量都显著下降.伤后7d,肌成纤维细胞开始出现.到伤后10d-14d,损伤区主要以肌成纤维细胞为主,偶见巨噬细胞.结论 大鼠骨骼肌损伤区中性粒细胞、巨噬细胞和肌成纤维细胞数量呈时间规律性变化,以期为骨骼肌损伤修复的病理学机制研究提供参考资料.     

Characterization of Endogenous Human FcγRIII by Mass Spectrometry Reveals Site,Allele and Sequence Specific Glycosylation*

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Highlights

• •Glycosylation of endogenous FcγRIII from neutrophils and matched plasma from more than 40 donors characterized at two sites involved in IgG binding.
• •Glycosylation of soluble FcγRIII glycosylation at N45 can be used to assign FcγRIIIb alleles.
• •FcγRIIIb allele specific differences in glycosylation at N162 may influence differential activity observed for primary cells.

     

Modulation of neutrophil function in host defense against disseminated Candida albicans infection in mice

Neutrophils (PMNs) constitute the main mechanism of host defense against acute invasive and disseminated candidiasis. Recent studies have demonstrated that tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) play an important role in the recruitment of PMNs at the site of invasive Candida infection. In the absence of either TNFalpha or IL-6, the course of experimental disseminated candidiasis is more severe, due to defective PMN recruitment. Treatment of mice with recombinant G-CSF (rG-CSF) leads to a significantly reduced mortality during disseminated candidiasis. The outgrowth of Candida albicans from the organs of rG-CSF-treated mice is significantly decreased. Treatment with the combination of rG-CSF and fluconazole has an additive effect on the reduction of fungal load in the organs. In subacute or chronic disseminated Candida infection, rG-CSF is less effective, indicating that neutrophil recruitment and activation are crucial in acute, life-threatening candidiasis, whereas other host defense mechanisms control the outcome of less overwhelming invasive Candida infection.