N-substituted 4-aminobenzamides (procainamide analogs): an assessment of multiple cellular effects concerning ion trapping.

Procainamide and related triethylamine-substituted 4-aminobenzamides, such as metoclopramide and declopramide, exert cellular effects potentially exploitable in oncology at millimolar concentrations (DNA demethylation, nuclear factor-kappaB inhibition, apoptosis) and display anti-inflammatory properties. However, these drugs induce massive cell vacuolization at similar concentrations, a response initiated by vacuolar (V-) ATPase-dependent ion trapping into and osmotic swelling of acidic organelles. We have examined whether this overlooked response might be related to the effects on cell proliferation and viability using cultured vascular smooth muscle cells and tumor-derived cell lines (Morris 7777 hepatoma, HT-1080 fibrosarcoma).

5-Lipoxygenase-activating protein homodimer in human neutrophils: evidence for a role in leukotriene biosynthesis.

FLAP (5-lipoxygenase-activating protein) is a nuclear transmembrane protein involved in the biosynthesis of LTs (leukotrienes) and other 5-LO (5-lipoxygenase) products. However, little is known about its mechanism of action. In the present study, using cross-linkers, we demonstrate that FLAP is present as a monomer and a homodimer in human PMN (polymorphonuclear cells).

Endogenous aminopeptidase N decreases the potency of peptide agonists and antagonists of the kinin B1 receptors in the rabbit aorta.

The B(1) receptor for kinins is selectively stimulated by bradykinin-related fragments lacking the C-terminal arginine, des-arginine(9)-bradykinin (des-Arg(9)-BK), and Lys-des-Arg(9)-BK. The latter peptide is the optimal agonist at the human and rabbit receptor. The B(1) receptor is inducible as a function of inflammatory conditions in the vasculature.

Signaling through CD16b in human neutrophils involves the Tec family of tyrosine kinases.

Tec kinases belong to the second largest family of nonreceptor tyrosine kinases. Although these kinases are expressed in myeloid cells, little is known about their implication in neutrophil function. We recently reported the participation of Tec kinases in the responses of human neutrophils to the bacterial peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine via G-coupled protein receptors.

Requirement of the extracellular cysteine at position six for CD40/CD40 dimer formation and CD40-induced IL-8 expression.

We recently showed that oligomerization of CD40 molecules on cell surface leads to disulfide-linked CD40/CD40 dimer formation, an event that is necessary for CD40-induced B7-2 expression in human B cells. Here, we demonstrate that CD40/CD40 dimers formation also occurs in different cell types such as T24 bladder cancer cells and CD40-transfected HEK 293 cells. Disulfide bonds mediate the formation of CD40/CD40 homodimers in CD40-activated cells.

Protein diversity is generated within the motin family of proteins by alternative pre-mRNA splicing.

The motin family of proteins is comprised of three polypeptides, angiomotin, angiomotin-like 1, and angiomotin-like 2. Angiomotin is an angiostatin-binding protein that promotes endothelial cell motility and is involved in angiogenesis. The function of human angiomotin-like-1 and angiomotin-like-2, however, remains unknown.

Potentiation of neutrophil cyclooxygenase-2 by adenosine: an early anti-inflammatory signal.

Neutrophils, which are often the first to migrate at inflamed sites, can generate leukotriene B(4) from the 5-lipoxygenase pathway and prostaglandin E(2) through the inducible cyclooxygenase-2 pathway. Adenosine, an endogenous autacoid with several anti-inflammatory properties, blocks the synthesis of leukotriene B(4) while it potentiates the cyclooxygenase-2 pathway in fMLP-treated neutrophils, following activation of the A(2A) receptor. Using the murine air pouch model of inflammation, we observed that inflammatory leukocytes from mice lacking the A(2A) receptor have less cyclooxygenase-2 induction than wild-type animals.

B cells and autoimmunity 2004: new concepts and therapeutic perspectives.

Recently, studies of B-cell physiology have continued to provide new and surprising insights into the nature of autoimmunity, highlighting novel potential immunointervention strategies. The meeting on 'B cells and autoimmunity: new concepts and therapeutic perspectives' brought together basic scientists and clinicians with research interests in a range of autoimmune diseases. Recent advances in different facets of B-cell biology were discussed in the prospect of understanding autoimmunity, and significant advances in our understanding of the mechanisms that regulate the autoimmune response at the B cell-level were described.

HLA-DR signaling inhibits Fas-mediated apoptosis in A375 melanoma cells.

Although melanocytes are devoid of the human major histocompatibility complex class II (HLA II) molecules, melanomas often display constitutive expression of these molecules, particularly HLA-DR. This constitutive expression of HLA-DR molecules is associated with tumor progression and poor prognosis but the molecular basis for this association remains poorly understood. Within the hypothesis of a role in immune escape, we analyzed the regulation of Fas-mediated apoptosis by HLA-DR signaling in the HLA-DR-positive malignant melanoma cell line A375.

Independent regulation of prostaglandins and monocyte chemoattractant protein-1 by interleukin-1beta and hCG in human endometrial cells.

Background: Inflammatory mediators such as prostaglandins (PG), chemokines, cytokines and their interactions regulate reproductive functions. The relationship between PG and monocyte chemoattractant protein-1 (MCP-1) has not been elucidated in human endometrium. The presence of hCG receptors in the human endometrium suggests that this embryonic signal may exert a local function in this tissue.

Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3.

We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58953Da and an estimated isoelectric point of 5.78.

Recruitment of the cross-linked opsonic receptor CD32A (FcgammaRIIA) to high-density detergent-resistant membrane domains in human neutrophils.

We have previously shown that CD32A (or FcgammaRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed.

Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-8.

A novel mammalian plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase), named NTPDase8, has been cloned and characterized. Analysis of cDNA reveals an open reading frame of 1491 base pairs encoding a protein of 497 amino acid residues with an estimated molecular mass of 54650 Da and a predicted isoelectric point of 5.94.

Delineation of the HLA-DR region and the residues involved in the association with the cytoskeleton.

Whereas the association of major histocompatibility complex (MHC) class II molecules with the cytoskeleton and their recruitment into lipid rafts play a critical role during cognate T/antigen-presenting cell interactions, MHC class II-induced signals, regions, and residues involved in their association and recruitment have not yet been fully deciphered. In this study, we show that oligomerization of HLA-DR molecules induces their association with the cytoskeleton and their recruitment into lipid rafts. The association of oligomerized HLA-DR molecules with the cytoskeleton and their recruitment into lipid rafts occur independently.

Histamine-induced inhibition of leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP.

1. Histamine is generally regarded as a pro-inflammatory mediator in diseases such as allergy and asthma. A growing number of studies, however, suggest that this autacoid is also involved in the downregulation of human polymorphonuclear leukocyte (PMN) functions and inflammatory responses through activation of the Gs-coupled histamine H(2) receptor.

CD40/CD40 homodimers are required for CD40-induced phosphatidylinositol 3-kinase-dependent expression of B7.2 by human B lymphocytes.

Preformed CD40/CD40 homodimers were initially observed on human Burkitt lymphoma cell lines, normal B cells, and transitional bladder carcinoma cell lines. However, the nature and the biological relevance of these homodimers have not yet been investigated. In the present study, we demonstrated that Epstein-Barr virus-transformed B cells and CD40-transfected HEK 293 cells constitutively expressed disulfide-linked CD40/CD40 homodimers at low levels.

[Major histocompatibility complex (MHC) class II: are lipid rafts the missing link?].

Aside from their crucial roles in the presentation of nominal antigen to CD4+ T cells and susceptibility to autoimmune diseases, substantial evidences suggest that MHC class II molecules act as signal transducer receptors as well. The signals transmitted affect diverse biological functions. Paradoxically, the cytoplasmic and transmembrane domains of these molecules are devoid of classic signaling motifs.

Human neutrophils as a source of nociceptin: a novel link between pain and inflammation.

Nociceptin is a neuropeptide sharing sequence homology with classical opioid peptides but with a distinct pharmacological profile. Through activation of its receptor, NociR, nociceptin has been linked with several physiological functions in the central nervous system including memory, locomotion, and processing of pain signals. Recently, peripheral blood neutrophils (PMNs) were demonstrated to express a functional NociR, a result suggesting that additional functions of the neuropeptide remain to be elucidated.

Identification of an autoantigen on the surface of apoptotic human T cells as a new protein interacting with inflammatory group IIA phospholipase A2.

One of the most studied secreted phospholipases A2 (sPLA2), the group IIA sPLA2, is found at high levels in inflammatory fluids of patients with autoimmune diseases. A characteristic of group IIA sPLA2 is its preference for negatively charged phospholipids, which become exposed on the extracellular leaflet of apoptotic cell membranes. We recently showed that low molecular weight heparan sulfate proteoglycans (HSPGs) and uncharacterized detergent-insoluble binding site(s) contribute to the enhanced binding of human group IIA PLA2 (hGIIA) to apoptotic human T cells.

Chemotactic factor-induced recruitment and activation of Tec family kinases in human neutrophils. II. Effects of LFM-A13, a specific Btk inhibitor.

Tyrosine phosphorylation events play major roles in the initiation and regulation of several functional responses of human neutrophils stimulated by chemotactic factors such as the bacterially derived tripeptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). However, the links between the G protein-coupled receptors, the activation of the tyrosine kinases, and the initiation of neutrophil functional responses remain unclear. In the present study we assessed the effects of a Btk inhibitor, leflunomide metabolite analog (LFM-A13), on neutrophils.

Zinc-binding sites in the N terminus of Mycoplasma arthritidis-derived mitogen permit the dimer formation required for high affinity binding to HLA-DR and for T cell activation.

Zinc-dependent superantigens can be divided into two subfamilies based on how they use zinc ions for interactions with major histocompatibility complex (MHC) class II molecules. Members of the first subfamily use zinc ions for interactions with histidine 81 on the beta-chain of MHC class II molecules, whereas members of the second subfamily use zinc ions for dimer formation. The zinc-binding motif is located in the C terminus of the molecule in both subfamilies.

Signaling through HLA-DR induces PKC beta-dependent B cell death outside rafts.

Signals through HLA-DR molecules contribute to optimal activation of antigen-presenting cells (APC) during T cell/APC interactions participating in the generation of productive interactions, and to the induction of APC death, which has been postulated to play a role in the termination of the immune response. To understand how these molecules accommodate both cellular responses, we studied the not yet well-defined signaling events and the biochemical requirements for HLA-DR-mediated death. We demonstrate that in B cells the HLA-DR-activated protein kinase C (PKC) beta is required for HLA-DR-mediated death whereas the HLA-DR-activated Src family of PTK is redundant.

Arachidonic acid activates phospholipase D in human neutrophils; essential role of endogenous leukotriene B4 and inhibition by adenosine A2A receptor engagement.

We report in human neutrophils (PMN) that phospholipase D (PLD) was stimulated by micromolar concentrations of arachidonic acid (AA) and nanomolar concentrations of leukotriene B(4) (LTB(4)), and eicosapentaenoic acid was inactive. The stimulatory effect of AA occurred only when adenosine was eliminated from PMN suspensions or when PMN were incubated with adenosine A(2A) receptor antagonists. The mechanism of AA-induced PLD activation was investigated.

Lipid raft-dependent and -independent signaling through HLA-DR molecules.

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses.

Immunoblotting and sequential lysis protocols for the analysis of tyrosine phosphorylation-dependent signaling.

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 that are lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol.