Sulfonated human immunoglobulin enhances CD16-linked CD11b expression on human neutrophils

Intravenous human immunoglobulin therapy infrequently results in excessive inflammatory responses in vivo; these effects are not fully understood. We assessed whether sulfonated human immunoglobulin (SHIG) or polyethylene glycol-treated human immunoglobulin (PHIG) enhanced expression of inflammatory receptors on peripheral blood neutrophils in vitro, such as alphaMbeta2 (CD11b/CD18) and Fc gamma receptor type III (FcgammaRIII). CD11b and CD16 expression on neutrophils was measured by fluorescence flow cytometry. Various cytokines were assessed using a highly sensitive fluorescence microsphere system. SHIG enhanced/induced CD11b expression and partial aggregations on neutrophils, but PHIG did not. No detection of aggregation IgG was observed in SHIG and PHIG. SHIG-induced CD11b expression was inhibited by treatment of corticosteroid (dexamethasone) and by anti-CD16 monoclonal antibody. Concentrations of various cytokines such as interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, RANTES, tumor necrosis factor (TNF)-alpha, and interferon (INF)-gamma in culture supernatant were not significantly changed by SHIG or PHIG. SHIG and PHIG did not enhance CD16 on neutrophils. SHIG enhanced CD16-linked CD11b expression on neutrophils in vitro. CD11b induction was inhibited by dexamethasone and by anti-CD16 antibody. These in vitro results suggest that aggregations and enhancement of CD11b on neutrophils by SHIG may induce excessive inflammatory responses in vivo.     

IL-10 does not affect oxidative burst and expression of selected surface antigen on human blood phagocytes in vitro

Cytokines play a major role in the control of inflammatory responses, participate in the regulation of blood phagocyte activities and as such are used for immunomodulatory therapy. In the present study, the influence of IL-10 on human blood phagocyte activity in the presence/absence of IL-6, IL-8 and TNF-alpha was tested in vitro. Our research analyzed the effects of cytokines on the production of reactive oxygen species measured by chemiluminescence and flow cytometry, and on the expression of surface molecules (CD11b, CD15, CD62L, CD31) measured by flow cytometry. IL-10 had no inhibitory effect on reactive oxygen species production and the expression of any examined adhesion molecule by resting or stimulated blood phagocytes within 3 h of incubation. Conversely, TNF-alpha, IL-6, and IL-8 increased reactive oxygen species production and the expression of CD11b and CD15 on both neutrophils and monocytes and decreased the expression of CD62L. These priming effects of the tested pro-inflammatory cytokines were not affected by IL-10. The obtained results suggest that IL-10 does not directly control blood phagocyte activation. These results also provide better information about the contribution of IL-6, IL-8 and TNF-alpha to the regulation of blood phagocyte-mediated inflammatory processes.     

Ligation of the FcR gamma chain-associated human osteoclast-associated receptor enhances the proinflammatory responses of human monocytes and neutrophils

We have previously described the human osteoclast associated receptor (hOSCAR), expressed in all cells of the myeloid lineage, and its immune functions. This receptor, which associates with the FcRgamma chain to transduce an activating signal, induces calcium flux in monocytes and dendritic cells, and modulates specific responses of dendritic cells. In this study, we have examined the effects of hOSCAR ligation on various proinflammatory responses of monocytes and neutrophils. Monocytes stimulated via hOSCAR ligation released IL-8/CXCL8 and other chemokines such as epithelial neutrophil-activating peptide-78/CXCL5, macrophage-derived chemokine/CCL22, and MCP-1/CCL2 and up-regulated markers involved in cell adhesion and costimulatory functions. Monocytes stimulated via hOSCAR in the absence of survival factors had an increased life span. Although the life span of neutrophils was unaffected, these cells, when stimulated via hOSCAR, rapidly released reactive oxygen intermediates, degranulated lactoferrin, myeloperoxidase, and matrix metalloproteinase-9 and also secreted IL-8/CXCL8. Neutrophils also underwent changes in cell surface molecule expression with the cleavage of CD62L and increased expression of CD11b and CD66b after 2-h stimulations. Finally, we demonstrated synergy between hOSCAR and TLR ligands on both monocytes and neutrophils, with up to 8-fold increases in cytokine secretion when hOSCAR was cross-linked in the presence of LPS or R-848. Overall, our data demonstrate that hOSCAR is a functional receptor on monocytes and neutrophils, involved in the induction of the primary proinflammatory cascade and the initiation of downstream immune responses.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

Effects of glutamine on adhesion molecule expression and leukocyte transmigration in endothelial cells exposed to arsenic

This study evaluated whether glutamine (GLN) concentration was related to endothelial surface molecule expression and the migration of polymorphonuclear neutrophils (PMNs) through endothelial cells (ECs) stimulated by arsenic. Human umbilical vein endothelial cells (HUVECs) and PMNs were treated with different GLN concentrations (0, 300, 600 and 1000 microM) for 24 h. After that, we stimulated HUVECs for 3 h with 0.5 microM arsenic, and PMNs were allowed to transmigrate to ECs for 2 h. HUVEC surface expressions of cell adhesion molecules and integrin (CD11b) and interleukin (IL)-8 receptor expressions on PMNs were measured. The transendothelial migration of PMNs was also analyzed. The results showed that cell adhesion molecule (CAM) and integrin expressions in arsenic groups were higher than in those without arsenic. Among the arsenic groups, the expression of CAMs on ECs and CD11b, and IL-8 receptor on PMNs was lowest with 0 microM compared with the other GLN concentrations. Vascular CAM-1 on ECs and CD11b on PMN expression were higher with 300 microM than with 600 and 1000 microM GLN. IL-8 secretions from ECs and PMNs were higher with 300 muM than with 600 and 1000 microM GLN, and this was consistent with the expression of the IL-8 receptor on PMNs. Polymorphonuclear neutrophil transmigration was significantly higher with 300 muM GLN than with other GLN concentrations. These results suggest that ECs and PMNs were activated after arsenic stimulation. Cell adhesion molecule expressions on ECs and PMNs were suppressed in the absence of GLN. A low GLN concentration comparable to catabolic conditions resulted in higher adhesion molecule expression and greater transendothelial migration of neutrophils. Glutamine administration at levels similar to or higher than physiological concentrations reduced IL-8 and adhesion molecule expression; PMN transmigration was also decreased after stimulation with arsenic.     

IL-6 plays an essential role in neutrophilia under inflammation

In the present study, we explored the involvement of interleukin-6 (IL-6) in neutrophilia under inflammatory conditions. The neutrophil count in the peripheral blood was high in arthritic monkeys, and anti-IL-6 receptor antibody reduced neutrophil counts to normal levels. IL-6 injection into normal monkeys significantly increased neutrophil counts in the blood 3h after injection. The expression of cluster of differentiation (CD) 162 on circulating neutrophils was reduced by IL-6 injection. IL-6 treatment in vitro did not affect CD162 expression on neutrophils from human blood. In IL-6-treated monkeys, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in plasma were clearly elevated. IL-8 and GM-CSF treatment in vitro reduced cell-surface CD162 expression on human neutrophils, and moreover, increased soluble CD162 expression in the cell supernatant. The addition of IL-6 into human whole peripheral blood induced IL-8 production and reduced CD162 expression on neutrophils. Furthermore, IL-8 and GM-CSF augmented mRNA expression of a disintegrin and metalloprotease like domain 10 (ADAM10) in neutrophils. Knock-down of ADAM10 by siRNA in neutrophil-like HL-60 cells partially reversed the expression of CD162 reduced by GM-CSF and IL-8 on HL-60 cells. In conclusion, IL-6 induced neutrophilia and reduced CD162 expression on neutrophils in inflammation.     

Induction of CD69 activation molecule on human neutrophils by GM-CSF,IFN-gamma,and IFN-alpha

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes but it expression is induced in vitro on cells of most hematopoietic lineages, including neutrophils after stimulation with PMA or fMLP. In this study, we investigated whether CD69 expression on human neutrophils could be modulated by inflammatory or anti-inflammatory cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-alpha, TGF-beta, IFN-alpha, IFN-gamma). Resting neutrophils from healthy subjects did not express CD69 on the cell surface; moreover, a preformed intracellular pool of CD69 was not evident in these cells. CD69 was barely detectable on these cells after overnight incubation in medium while overnight incubation with GM-CSF, IFN-gamma or IFN-alpha significantly induced CD69 expression on neutrophils with GM-CSF appearing to be the most potent inducer. This induction was dependent on a new protein synthesis as it was significantly inhibited by cycloheximide (about 50% inhibition). CD69 cross-linking on GM-CSF-primed neutrophils sinergized with LPS and increased TNF-alpha production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases.     

A role for beta(2) integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response.

Growing evidence supports the idea that adhesion via beta(2) integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adherent cells. This study investigates whether beta(2) integrin-mediated adhesion of human polymorphonuclear neutrophils (PMN) has a functional impact on cytokine production. Aggregation of the beta(2) integrin Mac-1 (CD11b/CD18) by antibody cross-linking was found to induce substantial de novo synthesis of IL-8 mRNA as measured by semiquantitative RT-PCR and Northern blotting technique, respectively. Induction of IL-8 mRNA was also observed upon adhesion of PMN to immobilized fibrinogen, a functional equivalent of its clotting product fibrin that serves as a native ligand of Mac-1. Results were confirmed using PMN derived from CD18-deficient mice, which were unable to produce MIP-2 mRNA, a homologue of human IL-8, in the presence of immobilized fibrinogen. In contrast, a substantial increase of MIP-2 mRNA was observed when wild-type PMN were incubated on immobilized fibrinogen. In human PMN, ELISA technique showed that the gene activation that required tyrosine kinase activity resulted in a substantial production and secretion of biologically active IL-8 and IL-1beta. In contrast, no TNF-alpha or IL-6 production was found, revealing that beta(2) integrins mediate differential expression of proinflammatory cytokines. The biological relevance of the present findings was confirmed in an in vivo model of acute inflammation. Altogether, the present findings provide evidence for a functional link between clotting and inflammatory responses that may contribute to the recruitment and/or activation of PMN and other cells at sites of lesion.     

Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers

It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients.     

Neutrophil-activating peptide 2 and gro/melanoma growth-stimulatory activity interact with neutrophil-activating peptide 1/interleukin 8 receptors on human neutrophils

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8), neutrophil-activating peptide 2 (NAP-2), and gro/melanoma growth-stimulatory activity (gro/MGSA) are potent inflammatory cytokines with homologous structure and similar neutrophil-activating properties. Receptors on human neutrophils that interact with these peptides were studied. Analysis of 125I-NAP-1/IL-8 binding at 0-4 degrees C revealed 64,500 +/- 14,000 receptors/cell with an apparent Kd of 0.18 +/- 0.07 nM (mean +/- S.D. of six independent experiments). Unlabeled NAP-1/IL-8, NAP-2, and gro/MGSA competed with 125I-NAP-1/IL-8 for binding to human neutrophils. Competition with increasing concentrations of unlabeled NAP-2 and gro/MGSA resolved two classes of NAP-1/IL-8 binding sites: about 70% of them bound NAP-2 and gro/MGSA with high affinity (Kd: 0.34 +/- 0.2 and 0.14 +/- 0.02), while 30% were of low affinity (Kd: 100 +/- 20 and 130 +/- 10 nM). Different binding sites, however, were not apparent upon competition with unlabeled NAP-1/IL-8, suggesting that both classes of receptors have similar affinities for NAP-1/IL-8. The existence of two receptors was also suggested by ligand cross-linking and cross-desensitization experiments. Two neutrophil membrane proteins with apparent Mr of 66,000-74,000 and 42,000-46,000 became cross-linked to 125I-NAP-1/IL-8, and the labeling was decreased when excess NAP-1/IL-8, NAP-2, or gro/MGSA was present. Stimulation of neutrophils with NAP-1/IL-8 resulted in desensitization toward a subsequent challenge with NAP-2 or gro/MGSA as shown by the rise in cytosolic free calcium. By contrast, following primary stimulation with NAP-2 or gro/MGSA, responses to NAP-1/IL-8 were only moderately attenuated, supporting the existence of NAP-1/IL-8 receptors which bind NAP-2 or gro/MGSA with low affinity. In conclusion, our results demonstrate that NAP-2 and gro/MGSA act upon human neutrophils by directly interacting with two classes of receptors for NAP-1/IL-8.     

Regulation of beta2-adrenergic receptors on CD4 and CD8 positive lymphocytes by cytokines in vitro

Increasing evidence points to a close relationship between the autonomic nervous system and the immune system. To further investigate mechanisms regulating beta2-adrenergic receptor (beta2R) expression in lymphocytes, the influence of cytokines on the density of beta2R on purified CD4+ and CD8+ lymphocytes was determined in vitro. beta2R were determined by means of a radioligand binding assay with (125I)iodocyanopindolol. CD4+ and CD8+ lymphocytes were incubated with catecholamines, interleukin 1beta (IL-1beta) and interleukin 2 (IL-2) for 6-72 h. The results demonstrate declining beta2R numbers on CD4+ and CD8+ lymphocytes in vitro augmented by epinephrine. IL-1beta has no effect on beta2R expression compared to medium. However, incubation with IL-2 resulted in an up-regulation of beta2R on CD8+ lymphocytes. Thus, the study demonstrates a differential regulation of beta2R on T-lymphocyte subpopulations with CD8+ lymphocytes being more susceptible to mechanisms of beta2R modulation than CD4+ lymphocytes. The findings further strengthen the concept of a close interplay between the autonomic nervous system and the immune system.     

Reactive oxygen species up-regulate CD11b in microglia via nitric oxide: Implications for neurodegenerative diseases

Microglial activation is considered as a hallmark of several neurodegenerative disorders. During microglial activation, the expression of CD11b, the beta-integrin marker of microglia, is increased. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of reactive oxygen species (ROS) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) stimulated the expression of CD11b in mouse BV-2 microglial cells and primary microglia, the effect that was blocked by antioxidants such as N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). Furthermore, comicroinjection of either NAC or PDTC with LPS was also able to suppress LPS-stimulated expression of CD11b in striatum in vivo. Similarly, other neurotoxic molecules, such as interleukin-1beta (IL-1beta), IL-12 p40(2), fibrillar amyloid-beta (Abeta) peptides, HIV-1 gp120, and double-stranded RNA (poly(IC)), also stimulated the expression of CD11b in microglia through the involvement of ROS. Complete inhibition of LPS-stimulated expression of CD11b by catalase, induction of CD11b expression by H2O2 alone, and inhibition of superoxide-stimulated CD11b expression by catalase suggest that H2O2, but not superoxide, is in fact involved in the expression of CD11b. Interestingly, we also demonstrate that ROS stimulated the expression of CD11b after the induction of nitric oxide (NO) production and failed to stimulate CD11b when NO production was inhibited by either 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) or L-N6-(1-iminoethyl)-L-lysine (L-NIL). Taken together, these studies suggest that the up-regulation of CD11b in microglia is redox sensitive and that ROS up-regulates CD11b via NO.     

IFN-gamma and 1,25(OH)2D3 induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production, and responsiveness to contact with activated T cells.

Differentiation and maturation of monocytes are accompanied by the expression of specific surface glycoproteins, the secretion of cytokines, and the capacity to respond to ligands. These changes may be influenced by interactions with hormones, soluble lymphocytic products, or direct contact with lymphocytes. We have studied two distinct pathways in the differentiation of a human monocytic cell line, THP-1: one being induced by IFN-gamma and the other by 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In THP-1 cells, IFN-gamma induces cell surface expression of HLA-DR and CD54 and production of IL-1 beta, TNF-alpha, and IL-6. In contrast, 1,25(OH)2D3 increases cell surface expression of CD11b and CD14, but fails to stimulate cytokine production. Direct contact of THP-1 with stimulated fixed T cells markedly induces IL-1 beta, TNF-alpha, and IL-6 production by THP-1. Production is higher when THP-1 have been previously exposed to 1,25(OH)2D3 as compared to prior exposure to IFN-gamma. mAb raised against certain relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibodies to CD11a, CD11b, and CD11c, alone or in combination, only partially blocked IL-1 beta production by THP-1, whereas antibodies to CD54 and CD14 did not. Thus other unknown structures on the THP-1 cells may be involved in the induction of THP-1 cytokine production by T cell contact.     

Role of early reperfusion in the induction of adhesion molecules and cytokines in previously ischemic myocardium

Our studiesin vitro demonstrate that neutrophil mediated injury of isolated cardiac myocytes requires the presence of ICAM-1 on the surface of the myocyte and CD11b/CD18 activation on the neutrophil. In post-ischemic cardiac lymph, there is rapid appearance of C5a activity during the first hours of reperfusion. Interleukin-6 activity is present throughout the first 72 h of reperfusion and is sufficient to induce ICAM-1 on the surface of the cardiac myocyte.In situ hybridization studies suggest that ICAM-1 mRNA is found in viable myocardial cells on the edge of the myocardial infarction within 1 h of reperfusion. ICAM-1 protein expression on cardiac myocytes is seen after 6 h of reperfusion, and increases thereafter. Non-ischemic tissue demonstrates no early induction of ICAM-1 mRNA or ICAM-1 protein on myocardial cells. In our most recent experiments, we have determined that reperfusion is an absolute requirement for the early induction of myocardial ICAM-1 mRNA in previously ischemic myocardial cells. To further assess this, we have cloned and sequenced a canine interleukin-6 (IL-6) cDNA. The data suggest that early induction of IL-6 mRNA is also reperfusion dependent as it could be demonstrated in the same ischemic and reperfused segments in which ICAM-1 mRNA was found. Peak expression of IL-6 mRNA occurred much earlier than that for ICAM-1 mRNA. Similar experiments were then performed with a molecular probe for interleukin-8 (IL-8). This chemokine is a potent neutrophil stimulant and has a higher degree of specificity for neutrophils than classic chemoattractants such as C5a. The results suggest a similar pattern of induction that occurs within the first hour and is markedly, increased by reperfusion. The relationship of reperfusion to ICAM-1 and cytokine induction is discussed.     

CD11b is a calcium-dependent epitope in human neutrophils.

Human neutrophils expressing complement receptor 3 (CR3) were treated with various concentrations (0.04-10 mM) of Ca2+/Mg(2+)-chelating agent EDTA and the expression of CD11b, the CR3 alpha chain antigenic epitope, was examined using monoclonal antibodies and flow cytometry. EDTA caused a dose-dependent decrease in the reactivity of two anti-CD11b monoclonal antibodies, Leu15 and IOM1. The reduced expression of CD11b in EDTA-treated cells was partly restored by the addition of Ca2+ ions whereas the addition of Mg2+ ions had no effect on CD11b level. The expression of the CR3 beta chain epitope, CD18, was markedly decreased only by 10 mM EDTA. These results suggest that the CD11b epitope may be associated with the Ca(2+)-binding domains of CR3 alpha chain and its recognition by antibodies depends on the presence of bound Ca2+.     

Regulation of human polymorphonuclear leukocytes functions by the neuropeptide pituitary adenylate cyclase-activating polypeptide after activation of MAPKs

Anti-inflammatory activities of pituitary adenylate cyclase-activating protein (PACAP) are mediated in part through specific effects on lymphocytes and macrophages. This study shows that in human polymorphonuclear neutrophils (PMNs), PACAP acts as a proinflammatory molecule. In PMNs, vaso-intestinal peptide/PACAP receptor 1 (VPAC-1) was the only receptor found to be expressed by RT-PCR. Using VPAC-1 Ab, we found that VPAC-1 mRNA was translated into proteins. In PMNs, PACAP increases cAMP, inositol triphosphate metabolites, and calcium. It activates two of the three members of the MAPK superfamily, the ERK and the stress-activated MAPK p38. U73122, an inhibitor of phospholipase C (PLC), inhibits PACAP-induced ERK activation, whereas p38 MAPK phosphorylation was unaffected. Using specific pharmalogical inhibitors of ERK (PD098059) and p38 MAPK (SB203580), we found that PACAP-mediated calcium increase was ERK and PLC dependent and p38 independent. PACAP primes fMLP-associated calcium increase; it also primes fMLP activation of the respiratory burst as well as elastase release, these last two processes being ERK and PLC dependent and p38 MAPK independent. PACAP also increases membrane expression of CD11b and release of lactoferrin and metallo proteinase-9 (MMP-9). These effects were PLC dependent (CD 11b, lactoferrin, MMP-9), ERK dependent (CD 11b, lactoferrin, MMP-9), and p38 dependent (CD11b, lactoferrin). We conclude that PACAP is a direct PMN activator as well as an effective PMN priming agent that requires PLC, ERK, and p38 MAPK activities.     

The effect of IL-1beta on the expression of inflammatory cytokines and their receptors in human chondrocytes

Cytokines released at sites of inflammation and infection can alter the normal processes of cartilage turnover, resulting in pathologic destruction or formation. Interleukin (IL)-1beta plays a central role in the pathophysiology of cartilage damage and degradation in arthritis. In the present study, we examined the effect of IL-1beta on the expression of IL-1beta, IL-6, IL-8, IL-11, tumor necrosis factor-alpha (TNF-alpha), and their receptors in human chondrocytes. The cells were cultured either with or without 100 U/ml of IL-1beta for up to 28 days. The level of expression of the cytokines and their receptors was estimated by determining mRNA levels using real-time PCR or by determining protein levels using ELISA. The expression of IL-1beta, IL-8, and TNF-alpha markedly increased in the presence of IL-1beta after day 14 of culture. The expression of IL-6 and IL-11 increased greatly in the presence of IL-1beta on day 1 and after day 14 of culture. The expression of IL-1beta, IL-8, IL-11, and TNF-alpha receptors significantly decreased in the presence of IL-1beta after day 14 of culture, whereas the expression of IL-6 receptor significantly increased. The expression of these cytokines, except for IL-6, decreased with the addition of human IL-1 receptor antagonist. These results suggest that IL-1beta promotes the resolution system of cartilage matrix turnover through an increase in inflammatory cytokine production by chondrocytes and that it also may promote the autocrine action of IL-6 through an increase in IL-6 receptor expression in the cells.     

Intracellular localization of a leukocyte adhesion glycoprotein family in the tertiary granules of human neutrophils

Stimulation of human neutrophils by the chemoattractant formyl-methionyl-leucyl-phenylalanine or the calcium ionophore A23187 induced increments in the cell surface expression of the alpha subunits of Mo1 (CD11b) and gp150, 95 (CD11c) and their common beta subunit (CD18). These increases showed a good correlation with the extracellular release of gelatinase, a marker for tertiary granules in human neutrophils. Conversely, the cell surface expression of the alpha subunit of the lymphocyte function-associated antigen-1 or LFA-1 (CD11a) was not altered upon cell activation. By subcellular fractionation and immunoprecipitation studies, we have found that CD11b, CD11c and CD18 molecules were mainly localized in the membranes of tertiary granules, which were resolved from the specific and azurophilic granules as well as from the plasma membrane fraction.     

The effect of inhibition of leukotriene synthesis on the activity of interleukin-8 and granulocyte-macrophage colony-stimulating factor

The cytokines interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the extracellular release of arachidonate metabolites from ionophore-stimulated neutrophils by 145 +/- 10% (mean +/- S.E.M., n = 13) and 182 +/- 11% (n = 16), respectively. To determine whether enhanced leukotriene production mediates the effects of these cytokines on neutrophil activity, two different specific arachidonate 5-lipoxygenase (5-LO) inhibitors, piriprost and MK-886, were used to inhibit leukotriene synthesis. Neither inhibitor affected the upregulation of CD11b beta(2)-integrin expression or priming of superoxide generation stimulated by IL-8 and GM-CSF. It is concluded that leukotrienes do not mediate either the direct or priming effects of these cytokines and that these classes of anti-inflammatory drugs are therefore unlikely to inhibit the effects of IL-8 and GM-CSF on neutrophil activation.