Cell Surface Expression Of Mac-1 Research Articles

Protective Role of Activated Protein C against Viral Mimetic Poly(I:C)-Induced Inflammation.

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.

Regulation of neutrophil phagocytosis of Escherichia coli by antipsychotic drugs.

Antipsychotic drugs (APDs) have been used to ease the symptoms of schizophrenia. APDs have recently been reported to regulate the immune response. Our previous studies revealed that the atypical APDs risperidone and clozapine and the typical APD haloperidol can inhibit the phagocytic ability of macrophages. Our research next determined the effects of APDs on the phagocytic ability of neutrophils, which are the most abundant type of white blood cells in mammals. Here we provide evidence that clozapine and haloperidol can induce increased phagocytic uptake of Escherichia coli by differentiated HL-60 cells and by purified human neutrophils. Furthermore, clozapine and haloperidol can increase the myeloperoxidase activity and IL-8 production in neutrophils. Our results also show that clozapine can inhibit E. coli survival within differentiated HL-60 cells. Furthermore, clozapine and haloperidol are shown to enhance cell surface Mac-1 expression and the activated AKT signaling pathway in purified neutrophils exposed to E. coli. These results indicate that clozapine and haloperidol can increase the phagocytic ability of neutrophils by increasing AKT activation when cells are exposed to bacteria.

The caffeic acid in aqueous extract of Tournefortia sarmentosa enhances neutrophil phagocytosis of Escherichia coli

Tournefortia sarmentosa, a Chinese herbal medicine, is considered an antioxidant or a detoxicating agent. Recently T. sarmentosa has received attention for its effects on the immune response. Here we provide evidence that aqueous extract of T. sarmentosa can induce increased phagocytic uptake of Escherichia coli by differentiated HL-60 cells and by neutrophils. Our results also revealed that T. sarmentosa can inhibit E. coli survival within differentiated HL-60 cells. Furthermore, aqueous extract of T. sarmentosa has been shown to enhance cell surface Mac-1 expression and the activated AKT signaling pathway in E. coli-stimulated neutrophils. We also examined the effect of each constituents in aqueous extract of T. sarmentosa on phagocytic uptake of E. coli by differentiated HL-60 cells or neutrophils. Bacterial survival, cell surface Mac-1 expression, and AKT activation of neutrophils were also examined. Our results showed that caffeic acid is an important constituent in mediating aqueous extract of T. sarmentosa-induced phagocytic uptake. Taken together, these results suggest that aqueous extract of T. sarmentosa exerts effects that enhance inflammatory responses by improving phagocytic capability, inhibiting bacterial survival within cells, and increasing Mac-1 expression of neutrophils.

Immune complexes formed following the binding of anti–platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion

AbstractWe tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcγRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin.

Histoplasma capsulatum inhibits apoptosis and Mac-1 expression in leucocytes.

Histoplasma capsulatum is a fungus found intracellularly in neutrophils and peripheral blood mononuclear cells (PBMCs), suggesting that it is capable of evading damage and survives inside these cells. In this study, we report that neutrophils from H. capsulatum-infected mice, and human neutrophils and mononuclear cells exposed to H. capsulatum presented less apoptosis than those from noninfected animals or cells exposed to medium only. Moreover, cells harvested from infected animals are resistant to apoptosis induced by dexamethasone - a proapoptotic stimulant. We also show that neutrophils harvested from infected mice and PBMCs from humans exposed to the fungus had a greatly decreased Mac-1 expression. We conclude that H. capsulatum induces an antiapoptotic state on leucocytes, which correlates with decreased cell-surface Mac-1 expression. These facts may represent an escape mechanism for the fungus by delaying cell death and allowing the fungus to survive inside leucocytes.

Stimulation of P-selectin glycoprotein ligand-1 on mouse neutrophils activates beta 2-integrin mediated cell attachment to ICAM-1.

The entry of neutrophils into inflamed tissues is initiated by cell rolling on the blood vessel wall followed by arrest and transendothelial migration. Rolling is mediated by the selectins, while the two subsequent steps require activated beta 2-integrins. We have investigated whether the binding of P-selectin to mouse neutrophils could trigger the activation of beta 2-integrins. We show that cross-linking of P-selectin glycoprotein ligand-1 (PSGL-1) on mouse neutrophils with an antibody-like recombinant form of P-selectin or with monoclonal antibodies stimulated the production of reactive oxygen intermediates and enhanced neutrophil attachment to intercellular adhesion molecule 1 (ICAM-1)-expressing CHO cells. This effect was independent of whether complete antibodies or F(ab')2 fragments were used. The adhesion-stimulating effect of P-selectin could be blocked by monoclonal antibodies against PSGL-1. Increase of cell attachment was dependent on lymphocyte function-associated antigen 1 (LFA-1) and on Mac-1, since it could be blocked with antibodies against both respective integrin alpha-chains. Moreover, cell surface expression of Mac-1 increased upon cross-linking of PSGL-1. In agreement with published data, treatment of human neutrophils with P-selectin-IgG did not enhance attachment to ICAM-1. Our data suggest that ligation of PSGL-1 on mouse neutrophils, but not on human neutrophils, activates beta 2-integrin mediated cell attachment to ICAM-1.

Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.

Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.

Mac-1 (CD11b/CD18) is an oligodeoxynucleotide-binding protein.

We have studied the interactions of phosphodiester and phosphorothioate oligodeoxynucleotides with Mac-1 (CD11b/CD18; alpha M beta 2), a heparin-binding integrin found predominantly on the surface of polymorphonuclear leukocytes (PMNs), macrophages and natural killer cells. Binding of a homopolymer of thymidine occurred on both the alpha M and beta 2 subunits. Soluble fibrinogen, a natural ligand for Mac-1, was an excellent competitor of the binding of a phosphorothioate oligodeoxynucleotide to both TNF-alpha-activated and nonactivated PMNs. Upregulation of cell-surface Mac-1 expression increased cell-surface binding of oligodeoxynucleotides. Binding was inhibited by anti-Mac-1 monoclonal antibodies, and the increase in cell-surface binding was correlated with a three- to fourfold increase in internalization by PMNs. An oligodeoxynucleotide inhibited beta 2-dependent migration through Matrigel, but the production of reactive oxygen species in PMNs adherent to fibrinogen dramatically increased. Thus, our data demonstrate that Mac-1 is a cell-surface receptor for oligodeoxynucleotides that can mediate their internalization and that this binding may have important functional consequences.

The role of protein kinase C activation and inositol phosphate production in the regulation of cell-surface expression of Mac-1 by complement fragment C5a

The adhesion of leukocytes to endothelial and other cell types is an essential part of the acute inflammatory response. One means by which adherence can be increased is by activation of the CD11/CD18 family of leukocyte glycoproteins. Chemotactic peptides, lipid mediators, phorbol esters and tumour necrosis factor are all able to increase the cell surface expression of one member of this family, CD11b/CD18 or Mac-1, by an unknown signal transduction mechanism. In this report, regulation of Mac-1 expression by C5a is shown to be independent of protein kinase C (PK-C) activation. The inhibitor of PK-C, H-7, has no effect on the action of C5a and only a slight effect on phorbol ester-induced up- and down-regulation of Mac-1, at a concentration that inhibits superoxide production in response to both factors by 40%. Inositol phospholipid hydrolysis, an important pathway leading to PK-C activation, and the transient increases in cytosolic Ca 2+ associated with inositol phosphate production, are also shown to be not essential processes in C5a-stimulation of Mac-1 expression.

Effects of dexamethasone on induction of monocytic differentiation in human U‐937 cells by dimethylsulfoxide

The present studies demonstrate that dimethylsulfoxide (DMSO) treatment of human U-937 myelomonocytic leukemia cells is associated with induction of monocytic differentiation. The DMSO-induced U-937 monocytic phenotype was associated with 1) growth inhibition, 2) loss of clonogenic survival, 3) increases in alpha-naphthyl acetate esterase (NSE) staining, and 4) increases in cell surface expression of the monocyte marker Mac-1. DMSO treatment of U-937 cells was also associated with down-regulation of c-myc and c-myb gene expression as well as with increases in tumor necrosis factor (TNF) mRNA levels. The results further demonstrate that induction of U-937 monocytic differentiation by DMSO is accompanied by increases in phospholipase A2 activity. Moreover, this stimulation of phospholipase A2 was sensitive to dexamethasone. We therefore studied the effects of dexamethasone on DMSO-induced differentiation of U-937 cells. Although dexamethasone had no effect on growth inhibition or loss of clonogenic survival by DMSO, this glucocorticoid blocked increases in NSE staining and cell surface Mac-1 expression. Dexamethasone also had no effect on the down-regulation of c-myc and c-myb expression but blocked the reappearance of c-myb transcripts after 6 hr of DMSO treatment. Finally, dexamethasone inhibited DMSO-induced increases in TNF gene expression. Taken together, the results demonstrate that dexamethasone inhibits multiple characteristics, including the stimulation of phospholipase A2 activity, associated with DMSO-induced monocytic differentiation of U-937 cells.