Induced Calcium Flux Research Articles

Adenine Nucleotides Inhibit Cytokine Generation by Human Mast Cells through a Gs-Coupled Receptor

ATP and ADP activate functionally distinct G protein-coupled purinergic (P2Y) receptors. We determined the expression and function of adenine nucleotide-specific P2Y receptors on cord blood-derived human mast cells (hMCs). Human MCs expressed mRNA encoding the ADP-specific P2Y1, P2Y12, and P2Y13 receptors; the ATP/UTP-specific P2Y2 receptor; and the ATP-selective P2Y11 receptor. ADP (0.05-50 muM) induced calcium flux that was completely blocked by a P2Y1 receptor-selective antagonist and was not cross-desensitized by ATP. Low doses of ADP induced strong phosphorylation of ERK and p38 MAPKs; higher doses stimulated eicosanoid production and exocytosis. Although MAPK phosphorylation was blocked by a combination of P2Y1- and P2Y12-selective antagonists, neither interfered with secretion responses. Unexpectedly, both ADP and ATP inhibited the generation of TNF-alpha in response to the TLR2 ligand, peptidoglycan, and blocked the production of TNF-alpha, IL-8, and MIP-1beta in response to leukotriene D(4). These effects were mimicked by two ATP analogues, adenosine 5'-O-(3-thiotriphosphate) and 2',3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), but not by adenosine. ADP, ATP, adenosine 5'-O-(3-thiotriphosphate), and 2',3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate each induced cAMP accumulation, stimulated the phosphorylation of CREB, and up-regulated the expression of inducible cAMP early repressor, a CREB-dependent inhibitor of cytokine transcription. Human MCs thus express several ADP-selective P2Y receptors and at least one G(s)-coupled ADP/ATP receptor. Nucleotides could therefore contribute to MC-dependent microvascular leakage in atherosclerosis, tissue injury, and innate immunity while simultaneously limiting the extent of subsequent inflammation by attenuating the generation of inducible cytokines by MCs.

H1 histamine receptor mediates inflammatory responses in human keratinocytes

Keratinocytes participate in initiation and amplification of T-cell-mediated skin diseases. During these disorders, histamine can be released from both residents skin cells and immigrating leukocytes, and can affect the functions of dendritic cells, monocytes, and lymphocytes. Little information is available on the effects of histamine on keratinocytes. To examine the presence of functional H1 receptors on human keratinocytes and the capacity of histamine to modulate the expression of inflammatory molecules in these cells. Primary cultures of resting and cytokine-activated keratinocytes from healthy subjects were analyzed for histamine H1 receptor expression and the production of inflammatory mediators after exposure to histamine receptor agonists and antagonists. Cultured keratinocytes constitutively expressed the H1 receptor mRNA and protein, which was not influenced by IFN-gamma, TNF-alpha, or IL-4. H1 but not H2 agonists induced calcium fluxes in keratinocytes. Treatment of keratinocytes with histamine (10 -7 to 10 -4 mol/L) or beta-histine increased the IFN-gamma-induced expression of membrane intercellular adhesion molecule 1 and MHC class I but not MHC class II molecules. Moreover, H1 stimulation promoted basal CC chemokine ligand (CCL)-5/RANTES and GM-CSF secretion and augmented IFN-gamma-induced release of the chemokines CCL2/monocyte chemoattractant protein 1, CCL5/RANTES, CCL20/macrophage inflammatory protein 3alpha, and CXC chemokine ligand 10/IFN-induced protein of 10 kd, as well as GM-CSF. Administration of the H1 antihistamine levocetirizine, but not of the H2 antihistamine cimetidine, abolished histamine-dependent expression of all of the investigated proinflammatory molecules in a dose-dependent manner (0.01-10 mumol/L). Levocetirizine at higher doses also reduced intercellular adhesion molecule 1, CCL5/RANTES, and GM-CSF release induced solely by IFN-gamma. Histamine exerts proinflammatory effects on keratinocytes via the H1 receptor, and these effects are efficiently inhibited by levocetirizine.

Nuclear respiratory factor 1 plays an essential role in transcriptional initiation from the hepatitis B virus x gene promoter.

The X gene of hepatitis B virus (HBV) is one of the major factors in HBV-induced hepatocarcinogenesis and is essential for the establishment of productive HBV replication in vivo. Recent studies have shown that the X gene product targets mitochondria and induces calcium flux, thereby activating Ca(+)-dependent signal transduction pathways. However, regulatory mechanisms of X gene expression have remained unclear. Previous studies had localized a minimal promoter activity to a 21-bp GC-rich sequence located 130 bp upstream of the X protein coding region and showed that there was a cellular protein bound to this DNA. Interestingly, the 21-bp sequence identified as an X gene minimal promoter does not contain any previously identified core promoter elements, such as a TATA box. To better understand the mechanisms of transcriptional initiation of the X gene, we set out to biochemically purify the binding protein(s) for the 21-bp DNA. We report here the identification of the X gene minimal promoter-binding activity as nuclear respiratory factor 1 (NRF1), a previously known transcription factor that activates the majority of nucleus-encoded mitochondrial genes and various housekeeping genes. Primer extension analyses of the X mRNAs show that mutations at the binding site specifically inactivate transcription from this promoter and that a dominant-negative NRF1 mutant and short interfering RNAs inhibit transcription from this promoter. Therefore, NRF1 specifically binds the 21-bp minimal promoter and positively contributes to transcription of the X gene. Simultaneous activation of the X gene and mitochondrial genes by NRF1 may allow the X protein to target mitochondria most efficiently.

Lysophospholipids and chemokines activate distinct signal transduction pathways in T helper 1 and T helper 2 cells

We demonstrate the expression of S1P1,3,4,5 the receptors for sphingosine 1-phosphate (S1P), and LPA1,2,3 the receptors for lysophosphatidic acid (LPA) in T helper 1 (Th1) and T helper 2 (Th2) cells. S1P and LPA induce the chemotaxis of Th1 and Th2 cells, an activity that is resistant to pertussis toxin (PTX) pretreatment in Th1, but is sensitive in Th2 cells. Also, I-TAC-induced Th1 and eotaxin-induced Th2 cell chemotaxis are blocked by PTX pretreatment. LPA but not S1P induces calcium flux response in Th1 and Th2 cells, which is due to the influx of extracellular calcium and is mediated by receptor activation, since EGTA and suramin (SUR) completely abrogate LPA-induced the release of calcium. No cross-desensitization is observed between thapsigargin (TG) and LPA in both cell types. PTX and SUR but not EGTA inhibit I-TAC- or eotaxin-induced [Ca2+]i release in Th1 and Th2 cells. Our results indicate that lysophospholipids and chemokines stimulate different signal transduction pathways.

Lysophospholipids and chemokines activate distinct signal transduction pathways in T helper 1 and T helper 2 cells

We demonstrate the expression of S1P 1,3,4,5 the receptors for sphingosine 1-phosphate (S1P), and LPA 1,2,3 the receptors for lysophosphatidic acid (LPA) in T helper 1 (Th1) and T helper 2 (Th2) cells. S1P and LPA induce the chemotaxis of Th1 and Th2 cells, an activity that is resistant to pertussis toxin (PTX) pretreatment in Th1, but is sensitive in Th2 cells. Also, I-TAC-induced Th1 and eotaxin-induced Th2 cell chemotaxis are blocked by PTX pretreatment. LPA but not S1P induces calcium flux response in Th1 and Th2 cells, which is due to the influx of extracellular calcium and is mediated by receptor activation, since EGTA and suramin (SUR) completely abrogate LPA-induced the release of calcium. No cross-desensitization is observed between thapsigargin (TG) and LPA in both cell types. PTX and SUR but not EGTA inhibit I-TAC- or eotaxin-induced [Ca 2+] i release in Th1 and Th2 cells. Our results indicate that lysophospholipids and chemokines stimulate different signal transduction pathways.

11-Dehydro-thromboxane B2, a Stable Thromboxane Metabolite, Is a Full Agonist of Chemoattractant Receptor-homologous Molecule Expressed on TH2 Cells (CRTH2) in Human Eosinophils and Basophils

Thromboxane (TX) A(2), a cyclooxygenase-derived mediator involved in allergic responses, is rapidly converted in vivo to a stable metabolite, 11-dehydro-TXB(2), which is considered to be biologically inactive. In this study, we found that 11-dehydro-TXB(2), but not the TXA(2) analogue U46,619 or TXB(2), activated eosinophils and basophils, as assayed by flow cytometric shape change. 11-Dehydro-TXB(2) was also chemotactic for eosinophils but did not induce, nor inhibit, platelet aggregation. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) is an important chemoattractant receptor expressed by eosinophils, basophils, and TH2 lymphocytes, and prostaglandin (PG)D(2) has been shown to be its principal ligand. 11-Dehydro-TXB(2) induced calcium flux mainly from intracellular stores in eosinophils, and this response was desensitized after stimulation with PGD(2) but not other eosinophil chemoattractants. Shape change responses of eosinophils and basophils to 11-dehydro-TXB(2) were inhibited by the thromboxane (TP)/CRTH2 receptor antagonist ramatroban, but not the selective TP antagonist SQ29,548, and were insensitive to pertussis toxin. The phospholipase C inhibitor U73,122 attenuated both 11-dehydro-TXB(2)- and PGD(2)-induced shape change. 11-Dehydro-TXB(2) also induced the chemotaxis of BaF/3 cells transfected with hCRTH2 but not naive BaF/3 cells. At a threshold concentration, 11-dehydro-TXB(2) had no antagonistic effect on CRTH2-mediated responses as induced by PGD2. These data show that 11-dehydro-TXB(2) is a full agonist of the CRTH2 receptor and hence might cause CRTH2 activation in cellular contexts where PGD-synthase is not present. Given its production in the allergic lung, antagonism of the 11-dehydro-TXB(2)/CRTH2axis may be of therapeutic relevance.

Lysophosphatidic acid induces human mast cell activation in an IL-4-dependent manner

Rationale Lysophosphatidic acid (LPA) is a lipid constituent of human serum that functions as a signaling molecule through binding to 4 known G protein-coupled receptors, designated LPA1-4. LPA has previously been shown to act as a growth factor for human mast cells (hMCs) cultured under serum-free conditions. We now investigate the role of LPA in hMC activation. Methods hMCs were cultured from umbilical cord-derived mononuclear cells using serum-free medium with recombinant human stem cell factor, IL-6, IL-10, and supplemental LPA (5μM). Cells were primed with IL-4 (0.1-50 ng/mL) for up to 5 days. RT-PCR was performed to characterize LPA receptor expression. LPA-induced calcium flux was measured in Fura-2AM-loaded hMCs. For activation studies, hMCs were incubated with LPA (10-50μM) for 30 minutes to 6 hours. Histamine release, eicosanoids, and cytokine production were measured by ELISA. Kit (CD117) expression was measured by FACS analysis. Results mRNA encoding LPA1-4 are expressed by hMCs. LPA (20-50μM) induced calcium flux. LPA (10-50μM) induced MIP-1β production only in the presence of IL-4 priming. IL-4 enhanced MIP-1β generation in a dose-dependent manner, reaching 53 ± 13% (mean ± SEM, n=4) of the level produced by FcϵRI cross-link, compared with negligible levels in unprimed cells (p=0.04). In contrast, LPA did not induce hMC exocytosis or eicosanoid production. IL-4 priming significantly down-regulated Kit expression. Conclusions LPA induces cytokine generation by hMCs in an IL-4-dependent manner by a pathway that does not elicit exocytosis or eicosanoid generation.

Synergism between platelet-activating factor-like phospholipids and peroxisome proliferator-activated receptor gamma agonists generated during low density lipoprotein oxidation that induces lipid body formation in leukocytes.

Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.

Lysophosphatidic acid induces human natural killer cell chemotaxis and intracellular calcium mobilization.

We have investigated the effects of the bioactive lipid lysophosphatidic acid (LPA) on several functions of activated human natural killer (NK) cells. Flow cytometric and immunoblot analyses show that these cells express LPA(1, )LPA(2) and LPA(3). LPA but not its precursor phosphatidic acid (PA) induces the chemotaxis of NK cells, an activity that is inhibited by prior treatment of the cells with pertussis toxin (PTX). In addition, LPA induces the mobilization of intracellular calcium, an effect that is markedly inhibited by PTX, but is not inhibited by the addition of EGTA. PA also induces calcium flux in NK cells, but with much lower efficacy than LPA. Cross-desensitization experiments demonstrate that LPA and PA utilize different receptors. Moreover, LPA or PA but not sphingosine 1-phosphate, enhances IFN-gamma secretion by activated NK cells. Our results may shed some light on the findings that activated NK cells are found at the sites of tumor growth.

Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1

AbstractComplement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34+ cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell–derived factor 1 (SDF-1)–dependent chemotaxis of human CD34+ cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1–dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)–mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1+ cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF–CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

LIR9, an immunoglobulin-superfamily–activating receptor, is expressed as a transmembrane and as a secreted molecule

LIRs are immunoglobulinlike receptors that have activating and inhibitory functions in leukocytes. Here we report the identification of the first LIR family member, LIR9, expressed as a membrane-bound receptor and as a secreted molecule. We identified 4 different forms of LIR9, 2 of which encode transmembrane molecules and 2 encode secreted molecules. The transmembrane forms of LIR9 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region that is likely to mediate its association with another coreceptor. LIR9 is mostly expressed in myeloid cells, including monocytes and neutrophils. Cross-linking of LIR9 on the surfaces of monocytes induces calcium flux and secretion of the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and IL-6, indicating that LIR9 could play a role in triggering innate immune responses.

A Novel Assay to Measure the Calcium Flux in Human Basophils: Effects of Chemokines and Nerve Growth Factor

The calcium flux in human basophils was measured by flow cytometry. Peripheral blood mononuclear cells were labeled with anti-CD123 and anti-HLA-DR antibodies, loaded with fluo-3 acetoxymethyl ester (2 µmol/l) in the presence of probenecid (2.5 µmol/l) and Pluronic F-127 (0.02%) for 20 min, and equilibrated with Ca²⁺ (1.8 mmol/l) and Mg²⁺ (1 mmol/l) for 5 min. The levels of intracellular free calcium were monitored as changes in fluorescence. Cross-linking of surface IgE on basophils with anti-IgE antibodies caused effective calcium flux in atopic, but not in healthy, donors. Concentration-dependent responses to monocyte chemoattractant protein 1 (MCP-1), eotaxin, macrophage inflammatory protein 1 alpha (MIP-1α), and C5a (0.3–10 nmol/l) were observed in all subjects, with a rank order of potency of C5a = MCP-1 > eotaxin > MIP-1α. In contrast, the rank order of potency in causing basophil shape change (i.e., increase in forward scatter) was eotaxin > C5a > MCP-1 > MIP-1α. Nerve growth factor (NGF; 15 nmol/l) did not induce calcium flux in basophils, and pretreatment of cells with a low concentration of NGF (0.3 nmol/l), which has previously been shown to prime basophils for mediator release, had no effect on the calcium response to subsequent stimulation with C5a. We conclude that calcium mobilization is differentially involved in signaling to chemoattractants in basophils and that it is correlated with the agonist’s efficacy to induce mediator release. The data also suggest that priming of basophil responses by NGF does not rely on enhanced calcium mobilization.

The motilin pharmacophore in CHO cells expressing the human motilin receptor

We performed a structure–activity study with the human motilin receptor, which was recently cloned from thyroid tissue. N-terminal fragments, Ala-analogs of motilin, and motilides were tested in a cell line that expresses the cloned human motilin receptor and apoaequorin. Full potency to induce calcium fluxes was obtained with N-terminal fragments of 14 amino acids. Motilin fragments 1–14 in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala showed the largest reduction in potency. Only motilides with an enol configuration had markedly higher potencies compared to erythromycin A. The potencies to induce Ca 2+ fluxes correlated strongly with rabbit binding and contractility data, suggesting that the cloned receptor is indeed the motilin receptor, responsible for contractile effects. Conservation of the motilin pharmacophore in evolution indicates an important physiological role of motilin.

The role of rat cytokine-induced neutrophil chemoattractants (CINCs) in inflammation

Rat cytokine-induced neutrophil chemoattractants (CINCs) are the members of the CXC chemokine family. Four neutrophil chemokines, CINC-1, CINC-2 alpha, CINC-2 beta and CINC-3, were purified from the conditioned medium of granulation-tissue culture. CINC-2 alpha and CINC-2 beta differ only in the sequence of three carboxy-terminal residues and are produced by alternative splicing. CINC-3 had neutrophil chemotactic activity similar to that of CINC-1 and CINC-2, but induced greater calcium mobilization than CINC-1 and CINC-2. CINC-1, -2 and -3 induced calcium flux in CXCR2-transfected HEK293 cells. In addition, anti-CXCR2 serum inhibited neutrophil chemotactic activities of the three types of CINCs almost completely. These results indicate that rat CXCR2 is a unique receptor for CINC-1, -2 and -3. CINCs induced calcium mobilization through pertussis toxin-insensitive G-protein but induced chemotaxis through pertussis toxin-sensitive G-protein. CINC-1/-2 and CINC-3 may stimulate both G-proteins with distinct efficiency. The concentration of CINC-1 increased transiently in rat air pouch/lipopolysaccharide inflammation, whereas the CINC-2 level increased linearly. The number of infiltrated cells increased up to 8 h. The increase in cell number was correlated with the total concentration of CINC-1 and CINC-2. Northern blot analyses and enzyme-linked immunosorbent assay showed that CINC expression was very low in rat macrophages without stimulation and increased after lipopolysaccharide stimulation. These data suggest that CINCs are expressed by inflammatory cells such as macrophages at a site of inflammation and play important roles in neutrophil infiltration.

The endogenous opioid spinorphin blocks fMet-Leu-Phe-induced neutrophil chemotaxis by acting as a specific antagonist at the N-formylpeptide receptor subtype FPR.

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), first isolated from bovine spinal cord, whose sequence matches a conserved region of beta-hemoglobin. Also referred to as LVV-hemorphin-4 and a member of the nonclassical opioid hemorphin family, spinorphin inhibits enkephalin-degrading enzymes and is analgesic. Recently, spinorphin was reported to block neutrophil activation induced by the chemotactic N-formylpeptide N-formylmethionylleucylphenylalanine (fMLF), suggesting a potential role as an endogenous negative regulator of inflammation. Here we use both gain- and loss-of-function genetic tests to identify the specific mechanism of spinorphin action on neutrophils. Spinorphin induced calcium flux in normal mouse neutrophils, but was inactive in neutrophils from mice genetically deficient in the fMLF receptor subtype FPR (N-formylpeptide receptor). Consistent with this, spinorphin induced calcium flux in human embryonic kidney 293 cells transfected with mouse FPR, but had no effect on cells expressing the closely related fMLF receptor subtype FPR2. Despite acting as a calcium-mobilizing agonist at FPR, spinorphin was a weak chemotactic agonist and effectively blocked neutrophil chemotaxis induced by fMLF at concentrations selective for FPR. Spinorphin did not affect mouse neutrophil chemotaxis induced by concentrations of fMLF that selectively activate FPR2. Thus, spinorphin blocks fMLF-induced neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR.

Gp120-Induced Bob/GPR15 Activation: A Possible Cause of Human Immunodeficiency Virus Enteropathy

Human immunodeficiency virus (HIV)-infected patients often develop malabsorption and increased intestinal permeability with diarrhea, called HIV enteropathy, even without enteric opportunistic infections. HIV gp120-induced calcium signaling, microtubule loss, and physiological changes resembling HIV enteropathy were previously found in the HT-29 intestinal cell line. How gp120 caused these changes was unclear. We show that the HIV co-receptor Bob/GPR15, unlike CCR5 and CXCR4, is abundant at the basal surface of small intestinal epithelium. The gp120-induced effects on HT-29 cells were inhibited by anti-Bob neutralizing antibodies, the selective G protein inhibitor pertussis toxin, and the phospholipase inhibitor U73122, but not neutralizing antibodies to CXCR4. Gp120 strains that induced signaling in HT-29 cells also induced calcium fluxes in Bob-transfected Ghost (3) cells, whereas gp120 strains not activating HT-29 cells also did not activate Bob-transfected cells. Bob is the first HIV co-receptor shown to be abundantly expressed on the basolateral surface of intestinal epithelium. Although Bob is an inefficient infection-inducing co-receptor, it mediates viral strain-specific gp120-induced calcium signaling at low, physiologically reasonable gp120 concentrations, up to 10,000-fold lower gp120 concentrations than the principal co-receptors. Gp120-induced Bob activation is a plausible cause of HIV enteropathy.

Detection of anaphylatoxin receptors on CD83+ dendritic cells derived from human skin.

Dendritic cells (DC) are recruited to sites of inflammation for the initiation of immune responses. As the anaphylatoxins C5a and C3a are important mediators of inflammation, we investigated the expression of their receptors (C3aR and C5aR) on human DC. DC were isolated from human skin or generated from purified blood monocytes and were identified by their expression of CD1a or CD83. Freshly isolated or cultured dermal CD1a+ and CD83+ DC bound anti-C5aR and anti-C3aR monoclonal antibodies (mAbs), as detected by flow cytometry. C5a induced calcium fluxes in dermal CD1a+ and CD83+ DC, which could be inhibited by C17/5, an anti-C5a mAb. C3a did not induce calcium fluxes in these cells. Anaphylatoxin receptor expression was down-regulated on dermal DC by adding tumour necrosis factor-alpha (TNF-alpha) to the culture medium. On CD1a+ CD83- cells generated from isolated blood monocytes by culture with 6.25 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 125 U/ml of interleukin-4 (IL-4), expression of both C5aR and C3aR was observed. In these cells, both C5a and C3a induced calcium fluxes. After addition of TNF-alpha to the culture medium, the majority of the CD1a+ cells expressed CD83+. These cells - expressing a phenotype of 'mature DC' - down-regulated the expression of the anaphylatoxin receptors and lost their reactivity to the respective ligands. Our results demonstrate the expression of the anaphylatoxin receptors C5aR and C3aR on human skin-derived DC and blood-derived cells expressing the DC-associated membrane molecule, CD1a. Furthermore, the expression of anaphylatoxin receptors on CD83+ dermal DC is indicative of an intermediate stage of maturation of these cells, which was not observed on in vitro-differentiated CD83+ cells.

The CGM1a (CEACAM3/CD66d)-mediated Phagocytic Pathway of Neisseria gonorrhoeae Expressing Opacity Proteins Is Also the Pathway to Cell Death

Phagocytosis of Opa+ Neisseria gonorrhoeae (gonococcus, GC) by neutrophils is in part dependent on the interaction of Opa proteins with CGM1a (CEACAM3/CD66d) antigens, a neutrophil-specific receptor. However, the signaling pathways leading to phagocytosis have not been characterized. Here we show that interaction of OpaI bacteria with neutrophils or CGM1a-transfected DT40 cells induces calcium flux, which correlates with phagocytosis of bacteria. We identified an immunoreceptor tyrosine-based activation motif (ITAM) in CGM1a, and showed that the ability of CGM1a to transduce signals and mediate phagocytosis was abolished by mutation of the ITAM tyrosines. We also demonstrated that CGM1a-ITAM-mediated bacterial phagocytosis is dependent on Syk and phospholipase C activity in DT40 cells. Unexpectedly, the activation of the CGM1a-ITAM phagocytic pathway by Opa+ GC results in induction of cell death.

Role of Coagulation Inhibitors in Inflammation

It is becoming increasingly clear that coagulation augments inflammation and that anticoagulants, particularly natural anticoagulants, can limit the coagulation induced increases in the inflammatory response. The latter control mechanisms appear to involve not only the inhibition of the coagulation proteases, but interactions with the cells that either generate anti-inflammatory substances, such as prostacyclin, or limit cell activation. Recent studies have demonstrated a variety of mechanisms by which coagulation, particularly the generation of thrombin, factor Xa and the tissue factor-factor VIIa complex, can augment acute inflammatory responses. Many of these responses are due to the activation of one or more of the protease activated receptors. Activation of these receptors on endothelium can lead to the expression of adhesion molecules and platelet activating factor, thereby facilitating leukocyte activation. Therefore, anticoagulants that inhibit any of these factors would be expected to dampen the inflammatory response. The three major natural anticoagulant mechanisms seem to exert a further inhibition of these processes by impacting cellular responses. Antithrombin has been shown in vitro to increase prostacyclin responses and activated protein C has been shown to inhibit a variety of cellular responses including endotoxin induced calcium fluxes in monocytes and the nuclear translocation of NFKB, a key step in the generation of the inflammatory response. In some, but not all, in vivo models, these natural anticoagulants have been able to inhibit endotoxin/E. coli-mediated leukocyte activation and to diminish cytokine elaboration (TNF, IL-6 and IL-8). Phase III clinical studies for treatment of patients with severe sepsis have been completed for APC, which was successful (1), and for antithrombin, which was not (2). A phase III trial with tissue factor pathway inhibitor is in progress. In this review, the mechanisms by which the different natural anticoagulants are thought to function will be reviewed.

Biological significance of the expression of HIV-related chemokine coreceptors (CCR5 and CXCR4) and their ligands by human hematopoietic cell lines.

The aim of this study was to learn more about the role of the HIV-related chemokine-chemokine receptor axes in human hematopoiesis. To address this issue we phenotyped 35 selected hematopoietic cell lines for the expression of CD4, CXCR4 and CCR5. We next evaluated the functionality of these chemokine receptors by calcium flux and chemotaxis assays, and by the ability of SDF-1, MIP-1alpha, MIP-1beta and RANTES to influence the growth of the cells expressing CXCR4 and/or CCR5. Lastly, we examined whether human hematopoietic cell lines may secrete some HIV-related chemokines, and whether endogenously secreted chemokines might interfere with the infectability. of hematopoietic cells by X4 and R5 HIV strains. These results demonstrate that: (1) HIV-related receptors are widely expressed on human hematopoietic cell lines; (2) stimulation of CXCR4 by SDF-1 induces calcium flux and chemotaxis in several hematopoietic cell lines more efficiently than stimulation of CCR5 by receptor-specific beta-chemokines; (3) chemokines do not regulate proliferation of the hematopoietic cells; and finally (4) infectability of the hematopoietic cells by HIV-1 may be auto-modulated by endogenously secreted chemokines. These data shed more light on the role of HIV-related chemokine-chemokine receptors axes in human hematopoiesis and interaction of hematopoietic cells with HIV.