Adenosine up-regulates cyclooxygenase-2 in human granulocytes: impact on the balance of eicosanoid generation.

Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine's anti-inflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway.

Promotion of neutrophil adherence to human osteoblasts by microcrystals and f-Met-Leu-Phe.

Human osteoblast-like cells (hOB) stimulated by monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) microcrystals produce the neutrophil chemoattractant IL-8. We investigated whether human neutrophils can adhere to hOB and respond to hOB preactivated by MSUM, CPPD, or by f-Met-Leu-Phe (fMLP). Confluent hOB were coincubated with human blood neutrophils in the presence of MSUM, CPPD or fMLP.

Cyclic AMP-mediated inhibition of 5-lipoxygenase translocation and leukotriene biosynthesis in human neutrophils.

5-Lipoxygenase (5-LO) catalyzes the transformation of arachidonic acid to leukotrienes (LT). In stimulated human PMN, activation of 5-LO involves calcium, p38 MAP kinase (p38) phosphorylation, and translocation of 5-LO from the cytosol to nuclear membranes containing the 5-LO activating protein (FLAP). In this study, cAMP-elevating agents such as isoproterenol, prostaglandin E(2), CGS-21680 (an adenosine A(2a) receptor agonist), the type IV phosphodiesterase inhibitor RO 20-1724, the adenylate cyclase activator forskolin, and the Gs-protein activator cholera toxin all inhibited LT biosynthesis and 5-LO translocation to the nucleus in cytokine-primed human PMN stimulated with platelet-activating factor and in human PMN stimulated with the endomembrane Ca(2+)-ATPase blocker thapsigargin.

Inflammatory microcrystals alter the functional phenotype of human osteoblast-like cells in vitro: synergism with IL-1 to overexpress cyclooxygenase-2.

Chronic crystal-associated arthropathies such as gout and pseudogout can lead to local bone destruction. Because osteoblasts, which orchestrate bone remodeling via soluble factors and cell-to-cell interactions, have been described in contact with microcrystals, particularly in uratic foci of gout, we hypothesized that microcrystals of monosodium urate monohydrate (MSUM) and of calcium pyrophosphate dihydrate (CPPD) could alter osteoblastic functions. MSUM and CPPD adhered to human osteoblastic cells (hOB) in vitro and were partly phagocytized as shown by scanning electron microscopy.

Impaired protein kinase C activation/translocation in Epstein-Barr virus-infected monocytes.

Infection of human monocytes by Epstein-Barr virus (EBV) has been linked to a decrease in the production of proinflammatory mediators as well as an impairment of phagocytosis. Considering the key role of protein kinases C (PKCs) in many biological functions of monocytes, including phagocytosis, we investigated the effects of EBV on the PKC activity in infected monocytes. Our results indicate that infection of monocytes by EBV impairs both phorbol 12-myristate 13-acetate (PMA)-induced translocation of PKC isozymes alpha and beta from cytosol to membrane as well as the PKC enzymatic activity.

Chemotactic factor-induced recruitment and activation of Tec family kinases in human neutrophils. Implication of phosphatidynositol 3-kinases.

The importance of the tyrosine phosphorylation cascades in the initiation and regulation of the functional responsiveness of human neutrophils is well established. On the other hand, the link between the G protein-coupled receptors (to which the receptors for chemotactic factors belong) and the activation of tyrosine kinases is very poorly characterized. Based on previous observations indicating that the stimulation of tyrosine phosphorylation was sensitive to inhibition by the phosphatidylinositol 3-kinase inhibitor wortmannin and the recent description of pleckstrin homology domain-containing tyrosine kinases (the Tec family), we have examined the potential implication of the latter in the responses of human neutrophils to chemotactic factors.

Early events in the activation of Fc gamma RIIA in human neutrophils: stimulated insolubilization, translocation to detergent-resistant domains, and degradation of Fc gamma RIIA.

The signal transduction mechanisms associated with the ligation of FcgammaRIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of FcgammaRIIA following its cross-linking. The results obtained indicate that cross-linking of FcgammaRIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction.

ARNO but not cytohesin-1 translocation is phosphatidylinositol 3-kinase-dependent in HL-60 cells.

Cytohesin-1 and ARNO are guanine nucleotide-exchange factors (GEFs) for ADP-ribosylation factor (Arf). Here, we show that ARNO is expressed in HL-60 cells and established that granulocytic differentiation induced with Me2SO stimulated cytohesin-1 but not ARNO expression. Cytohesin-1 levels in HL-60 granulocytes were similar to those in human neutrophils.

Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates. II. A sequential lysis protocol for the analysis of tyrosine phosphorylation-dependent signalling.

In stimulated neutrophils, the majority of tyrosine phosphorylated proteins are concentrated in Triton X-100 or NP-40 insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine phosphorylation are, however, performed using the supernatants of cells lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol.

Occupancy of adenosine A2a receptors promotes fMLP-induced cyclic AMP accumulation in human neutrophils: impact on phospholipase D activity and recruitment of small GTPases to membranes.

The aim of this study was to assess in human neutrophils the implication of an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway in the inhibitory effects of A2a receptor engagement. We found that Ro20-1724, a cAMP phosphodiesterase inhibitor, in the presence of adenosine deaminase (ADA) or A2a receptor antagonists rendered transient the fMLP-induced sustained increases in cAMP levels. The role of A2a receptor stimulation was demonstrated by the ability of the A2a receptor agonist, CGS21680, to prevent ADA-mediated reduction of the persistent cAMP elevation induced by fMLP.

Involvement of zinc in the binding of Mycoplasma arthritidis-derived mitogen to the proximity of the HLA-DR binding groove regardless of histidine 81 of the beta chain.

Although our recent studies have provided the first evidence demonstrating the direct binding of Mycoplasma arthritidis-derived mitogen (MAM) to MHC class II molecules, it is not yet established how MAM interacts with these molecules. Herein, we demonstrate that MAM binds preferentially and with high affinity to HLA-DR molecules in a zinc-dependent manner. MAM's affinity (25 nM) for HLA-DR molecules is comparable to that of staphylococcal superantigens, and is slightly higher than that for murine MHC class II molecules expressed on the A20 B cell line (111 nM).

Epstein-Barr virus primes human polymorphonuclear leucocytes for the biosynthesis of leukotriene B4.

In the present study, we have investigated the effect of the short-term incubation of polymorphonuclear leucocytes (PMN) with infectious Epstein-Barr virus (EBV) on leukotriene B(4) (LTB(4)) biosynthesis. Pre-exposure of PMN to EBV led to an increased production of LTB(4) upon stimulation with either the ionophore A23187, the chemotactic peptide fMLP, or phagocytic particles (zymosan). Experiments performed with viral particles pretreated with a neutralizing antibody raised against the gp350 of the viral envelope revealed that a specific interaction between the PMN surface and the viral glycoprotein gp350 is required for the priming effect of EBV.

Trafficking of surface-linked and encapsulated liposomal antigens in macrophages: an immunocytochemical study.

Liposomal antigens are potent adjuvants of humoral and cell-mediated immunity. Although this property requires as an essential condition a physical association between the antigen and the phospholipid vehicle, the nature of the association, i.e.

Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals.

The inflammatory response in acute gouty arthritis is in large part a result of the interaction between neutrophils and monosodium urate (MSU) crystals. The tyrosine kinase Syk, which has been largely associated with the phagocytic response by Fc receptors and with spreading mediated by integrins, has been identified as one of the major proteins tyrosine-phosphorylated in human neutrophils upon stimulation by MSU crystals and is known to be mediated in part by the Fc receptor, CD16. This has led to the present examination of the implication of Syk in the activation pathways used by MSU crystals.

Anti-CD3 and concanavalin A-induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. Lack of involvement of group IV and group VI phospholipase A2 in remodeling and increased susceptibility of proliferating T cells to CoA-independent transacyclase inhibitor-induced apoptosis.

In this study arachidonate-phospholipid remodeling was investigated in resting and proliferating human T lymphocytes. Lymphocytes induced to proliferate with either the mitogen concanavalin A or with anti-CD3 (OKT3) in combination with interleukin 2 (OKT3/IL-2) showed a greatly accelerated rate of [3H]arachidonate-phospholipid remodeling compared with resting lymphocytes or with lymphocytes stimulated with OKT3 or IL-2 alone. The concanavalin A-stimulated cells showed a 2-fold increase in the specific activity of CoA-independent transacylase compared with unstimulated cells, indicating that this enzyme is inducible.

Evidence for a role for SAM68 in the responses of human neutrophils to ligation of CD32 and to monosodium urate crystals.

SAM68 (Src-associated in mitosis 68 kDa) is a member of the signal transduction of activator RNA novel gene family coding for proteins postulated to be involved in signal transduction and activation of RNA. It has been implicated through its phosphorylation status in the control of the transition from the G(1) to the S phases during mitosis. However, the implication and role of SAM68 in nonproliferative cells are unknown.

Characterization of cytohesin-1 monoclonal antibodies: expression in neutrophils and during granulocytic maturation of HL-60 cells.

ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in vesicular transport and the activation of phospholipase D (PLD). The conversion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchange factors such as ARNO or cytohesin-1. In order to examine the expression of ARNO and cytohesin-1 in human granulocytes, we generated specific polyclonal and monoclonal antibodies (mAbs).

Binding of Mycoplasma arthritidis-derived mitogen to human MHC class II molecules via its N terminus is modulated by invariant chain expression and its C terminus is required for T cell activation.

Mycoplasma arthritidis-derived mitogen (MAM) is considered to be a member of the super-antigen family despite the fact that there is no evidence until now indicating its binding to MHC class II molecules. To demonstrate its direct binding and to determine the regions involved in MHC class II and TCR interactions, we generated a recombinant wild-type and two truncated forms of the MAM protein. Data obtained in the course of the present investigation show that MAM binds specifically and significantly to human MHC class II molecules.

Stimulation of human neutrophils by chemotactic factors is associated with the activation of phosphatidylinositol 3-kinase gamma.

The activation of human polymorphonuclear neutrophil leukocytes (neutrophils) is associated with an increased synthesis of the highly phosphorylated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)). The aims of the present investigation were to determine whether the newly described, G protein-dependent phosphatidylinositol 3-kinase (PI3K), p110gamma, was involved in the responses to chemotactic factors interacting with G protein-coupled receptors. The presence of p110gamma in neutrophils was first established both at the protein and the mRNA level.

Infection of primary human monocytes by Epstein-Barr virus.

Previous studies have reported that infection of monocytes by viruses such as cytomegalovirus and human immunodeficiency virus weakens host natural immunity. In the present study, we demonstrated the capability of Epstein-Barr virus (EBV) to infect and replicate in freshly isolated human monocytes. Using electron microscopy analysis, we observed the presence of EBV virions in the cytoplasm and nuclei of approximately 20% of monocytes.

Adenosine receptor occupancy suppresses chemoattractant-induced phospholipase D activity by diminishing membrane recruitment of small GTPases.

Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)-induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation.

Modulation of human neutrophil responses to CD32 cross-linking by serine/threonine phosphatase inhibitors: cross-talk between serine/threonine and tyrosine phosphorylation.

The interplay between serine/threonine and tyrosine phosphorylation was studied in human neutrophils. The direct effects of calyculin and okadaic acid, potent inhibitors of PP1 and PP2A serine/threonine phosphatases, on the patterns of neutrophil phosphorylation, and their effects on the responses of neutrophils to CD32 cross-linking were monitored. After a 2-min incubation with 10-6 M calyculin, a transient tyrosine phosphorylation of a subset of proteins, among which Cbl and Syk, was observed.

Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes.

We focus on the mechanisms of regulation of phospholipase D (PLD) activity. Three agonists known to stimulate PLD activity, fMet-Leu-Phe (fMLP), phorbol 12-myristate 13-acetate (PMA) and V4+-OOH, induced a differential translocation of ADP-ribosylation factor (ARF), RhoA, and protein kinase Calpha (PKCalpha), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V4+-OOH only elicited RhoA translocation and PMA induced ARF and PKCalpha translocation.

CD40 associates with the MHC class II molecules on human B cells.

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell-cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos.