Mutational analysis of the fractalkine chemokine domain. Basic amino acid residues differentially contribute to CX3CR1 binding, signaling, and cell adhesion.

Fractalkine (FKN/CX3CL1) is a unique member of the chemokine gene family and contains a chemokine domain (CD), a mucin-like stalk, a single transmembrane region, and a short intracellular C terminus. This structural distinction affords FKN the property of mediating capture and firm adhesion of FKN receptor (CX3CR1)-expressing cells under physiological flow conditions. Shed forms of FKN also exist, and these promote chemotaxis of CX3CR1-expressing leukocytes. The goal of the present study was to identify specific residues within the FKN-CD critical for FKN-CX3CR1 interactions. Two residues were identified in the FKN-CD, namely Lys-7 and Arg-47, that are important determinants in mediating an FKN-CX3CR1 interaction. FKN-K7A and FKN-R47A mutants exhibited 30-60-fold decreases in affinity for CX3CR1 and failed to arrest efficiently CX3CR1-expressing cells under physiological flow conditions. However, these mutants had differential effects on chemotaxis of CX3CR1-expressing cells. The FKN-K7A mutant acted as an equipotent partial agonist, whereas the FKN-R47A mutant had marked decreased potency and efficacy in measures of chemotactic activity. These data identify specific structural features of the FKN-CD that are important in interactions with CX3CR1 including steady state binding, signaling, and firm adhesion of CX3CR1-expressing cells.

Inflammatory agents regulate in vivo expression of fractalkine in endothelial cells of the rat heart.

Fractalkine is distinguished structurally from other chemokines in that it contains a mucin-like stalk that tethers a CX3C chemokine module to a transmembrane-spanning region; its expression in cultured endothelial cells has been shown to be up-regulated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1). The purpose of this study was to determine whether fractalkine is expressed, in a proinflammatory agent-regulated manner, by cardiac endothelial cells in vivo. Steady state levels of fractalkine mRNA were increased in rat cardiac tissues after in vivo treatment with lipopolysaccharide (LPS), IL-1, or TNF-alpha. In situ hybridization and immunohistochemical analysis revealed that endothelial cells of the coronary vasculature and endocardium were the principal source of proinflammatory agent-inducible fractalkine, although some fractalkine immunoreactivity was also found on the myocytes. These data are the first demonstration of in vivo cardiac endothelial cell fractalkine expression and regulation by proinflammatory agents such as LPS, IL-1, or TNF-alpha. Cardiac endothelial cell-expressed fractalkine may contribute to the influx of leukocytes into the heart during inflammation.

Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia.

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.

Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis.

Chemokines are a group of pro-inflammatory peptides that mediate leukocyte migration and activation. Several members of the chemokine family have been shown to be synthesized by cells of the central nervous system (CNS). To begin to address the role of chemokine receptors in CNS physiology, we identified, by molecular cloning techniques, the rat orthologs of the chemokine receptors, CCR2, CCR3, CCR5, and CXCR4. CCR2 and CCR5 expression was detected in rat spleen, lung, kidney, thymus and macrophages; CCR5 mRNA was also detected in rat brain. Primary cultures of rat microglia expressed CCR5 mRNA that was regulated by IFN-gamma, while both cultured astrocytes and microglia were found to contain mRNA for CXCR4 and CX3CR1. Induction of experimental allergic encephalomyelitis (EAE) in the rat was accompanied by increased levels of CCR2, CCR5, CXCR4, and CX3CR1 mRNAs in the lumbar spinal cords of animals displaying clinical signs of the disease. These data identify the rat orthologs of chemokine receptors and demonstrate that brain, spinal cord, and cultured glial cells express chemokine receptors that can be regulated both in vitro and in vivo.

Cotransfection of second and third intracellular loop fragments inhibit angiotensin AT1a receptor activation of phospholipase C in HEK-293 cells.

Peptides from the intracellular regions of G protein-coupled receptors are useful probes of receptor-G protein coupling mechanisms. As a first step toward the genetic delivery of such "G protein inhibitors," we describe inhibition of angiotensin II (AII) receptor responses by expressed fragments of the second and third intracellular loops of the AT1a receptor (AT1a/i2 and AT1a/i3). Transient transfection of human embryonic kidney 293 cells with DNA encoding the rat AT1a receptor resulted in AII-dependent increases of inositol phosphates (maximum 4.5-fold). Cotransfection of AT1a/i2 and AT1a/i3 fragments raised the EC50 for AII stimulation of phospholipase C activity 5-fold (from 0.18 nM to 0.99 nM, n = 12, P < .001) and 3-fold (from 0.38 nM to 1.2 nM, n = 8, P < .002), respectively. The combined effect of AT1a/i2 and AT1a/3 was additive, and transfection of an alpha-1b adrenergic receptor third intracellular loop (alpha1b/i3) fragments also increased the EC50 for AII. Neither AT1a/i1 nor C-terminus (AT1a/Ct) constructs had significant effects on angiotensin responses. These data confirm a role for the second and third intracellular loops in angiotensin receptor responses and show the potential of this approach to blocking multiple phospholipase C-linked receptors.

Cyclooxygenase inhibition decreases nitric oxide synthase activity in human platelets.

Activity of both nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) plays an important role in the regulation of platelet function. NO has been shown to directly activate COX. This study was designed to determine whether products of the COX pathway in turn regulate NOS activity. Human platelets were incubated with aspirin, indomethacin, the selective thromboxane A2 synthase inhibitor U-63557A, or the prostaglandin H2-thromboxane A2-receptor blocker SQ-29548 for 1 h at 37 degrees C. Multiple indexes of the activity of the L-arginine-NO pathway and changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured in platelets. Both aspirin and indomethacin decreased NOS activity, measured as the conversion of L-arginine to L-citrulline and nitrite (+nitrate) formation, in platelets in a concentration-dependent fashion. Aspirin also decreased guanosine 3',5'-cyclic monophosphate accumulation in platelets. The NOS inhibitory effects of these aspirin and indomethacin effects were reversed by coincubation with the thromboxane A2 analog U-46619 or an excess of CaCl2. Incubation of COX inhibitors with platelets was associated with significant reductions in basal as well as thrombin-stimulated [Ca2+]i, and the reduction in [Ca2+]i was reversed by U-46619. Incubation of platelets with U-63557A and SQ-29548 resulted in inhibitory effects on NOS activity qualitatively similar to those of COX inhibitors. The effects of COX inhibitors or U-63557A were not associated with a change in NOS protein expression in platelets. These data suggest that NOS activity in human platelets is inhibited by COX inhibitors, mediated, at least in part, via suppression of thromboxane A2 and [Ca2+]i mobilization in platelets.

Identification of two rat genes orthologous to the human interleukin-8 receptors.

The genes encoding two functional human interleukin-8 (IL-8) receptors have been identified by molecular cloning techniques and they are members of the rhodopsin G-protein coupled receptor (GCR) superfamily. We report the molecular cloning of two rat GCR genes (rat CXCR1-like and rat CXCR2) whose conceptualized amino acid sequences are approximately 70% identical to the human IL-8 A and B receptor subtypes. The murine GRO-like peptide, macrophage inflammatory peptide-2 (MIP-2), elevates intracellular calcium levels in HEK293 cells expressing the rat CXCR2 receptor. Southern blot analysis of restriction-digested rodent and human genomic DNAs indicate that rat CXCR1-like and CXCR2 are: 1) each single copy genes in the rat genome; 2) most closely related to the human IL-8 receptor genes; and 3) orthologous to two previously identified murine genes. CXCR2 mRNA is detected in adult rat lung, spleen, and neutrophils. CXCR1-like mRNA can be detected in adult rat lung, native rat macrophages, and a rat alveolar macrophage cell line (NR8383). These data identify the rat orthologs of the human IL-8 receptors, and describe cellular and tissue targets of rat C-X-C chemokine peptides.

Trypsin induces Ca(2+)-activated Cl- currents in X. laevis oocytes.

The protease trypsin induces Ca(2+)-activated Cl- currents when applied in concentrations as low as 0.1 mg/ml to defolliculated, voltage clamped X. laevis oocytes. The response is dose-dependent and specific, as other proteases (chymotrypsin, Lys-C and Arg-C), or trypsin pretreated with soybean trypsin inhibitor, did not induce currents. Intracellular trypsin injection did not induce responses. The current does not appear to result from proteolytic activation of the endogenous receptor for lysophosphatidic acid, the only known Ca(2+)-mobilizing receptor consistently present in oocytes. These results suggest the presence on the oocyte membrane of a specific receptor for trypsin.

Responses to sphingosine-1-phosphate in X. laevis oocytes: similarities with lysophosphatidic acid signaling.

Sphingosine-1-phosphate (S1P, 50 microM) induces inward currents in Xenopus laevis oocytes voltage clamped at -70 mV. The currents are Ca(2+)-activated Cl- currents, as shown by a reversal potential of -20 mV and absence of the response after intracellular injection of ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 10 mM). The response is nearly indistinguishable from that to the related compound lysophosphatidic acid (LPA), and complete cross-desensitization occurs between LPA and S1P responses. Both the LPA and S1P responses are inhibited by suramin (2 mM) and dithiothreitol (5 mM). These responses appear mediated by a specific membrane receptor, since intracellular injection of S1P (5 microM) does not induce currents, and sphingosine and the related compounds sphingosylphosphorylcholine and N,N-dimethylsphingosine, all at 100 microM, neither induce currents nor block the response to S1P. HEK-293 and COS-1 cells respond with intracellular Ca2+ release to both 50 microM S1P and 10 microM LPA; K-562 cells do not. No cross-desensitization was noted in the responsive cells. Our findings indicate that S1P and LPA might act through the same mechanism, probably a membrane receptor.

Lysophosphatidic acid induces a pertussis toxin-sensitive Ca(2+)-activated Cl- current in Xenopus laevis oocytes.

Lysophosphatidic acid (LPA) induces a Ca(2+)-activated Cl- current in defolliculated Xenopus laevis oocytes. The response appears mediated by a specific membrane receptor, because no current is induced when related compounds [phosphatidic acid (PA), lysophosphatidylcholine (LPC), and lysophosphatidylserine (LPS)] are applied extracellularly or when LPA is injected intracellularly. Incubation in pertussis toxin prevents the response. The response is mediated by a Ca(2+)-activated Cl- current because 1) it is abolished by intracellular ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 5 mM) but not affected by changes in extracellular Ca2+ concentration and 2) the reversal potential becomes more positive at lower Cl- concentrations. Suramin (2 mM) blocks the LPA-induced current, but PA, LPS, LPC, and the platelet-activating factor antagonist WEB-2086 do not. The response is dose dependent for LPA concentrations from 10(-8) to 10(-3) M. Incubation of oocytes in LPA does not induce germinal vesicle breakdown. These findings suggest that this novel oocyte response to LPA is mediated by a specific membrane receptor linked to a pertussis toxin-sensitive G protein.