Regulation of chemotaxis by the platelet-derived growth factor receptor-beta

Chemotaxis is an important component of wound healing, development, immunity and metastasis, yet the signalling pathways that mediate chemotaxis are poorly understood. Platelet-derived growth factor (PDGF) acts both as a mitogen and a chemoattractant. Upon stimulation, the tyrosine kinase PDGF receptor-beta (PDGFR-beta) autophosphorylates and forms a complex that includes SII2(Src homology 2)-domain-containing proteins such as the phosphatidylinositol-specific phospholipase C-gamma, Ras-GTPase-activating protein (GAP), and phosphatidylinositol-3-OH kinase. Specific tyrosine-to-phenylalanine substitutions in the PDGFR-beta can prevent binding of one SH2-domain-containing protein without affecting binding of other receptor-associated proteins. Here we use phospholipase C-gamma and PDGFR-beta mutants to map specific tyrosines involved in both positive and negative regulation of chemotaxis towards the PDGF-BB homodimer. Our results indicate that a delicate balance of migration-promoting (phospholipase C-gamma and phosphatidylinositol-3-OH kinase) and migration-suppressing (GAP) activities are recruited by the PDGFR-beta to drive chemotaxis towards PDGF-BB.     

A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium

Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA- null mutants, neither chemoattractant stimulation of intact cells nor GTPgammaS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPgammaS stimulation of adenylyl cyclase activity in piaA- lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac- piaA- double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes.     

Dissociation of chemotaxis from agonist-induced receptor internalization in a lymphocyte cell line transfected with CCR2B. Evidence that directed migration does not require rapid modulation of signaling at the receptor level

To investigate the role of the carboxyl-terminal region (52 amino acids) of the monocyte chemoattractant protein 1 receptor (CCR2B) in chemotaxis, we created a series of mutants and expressed them in a murine pre-B lymphocyte cell line. Truncation of the cytoplasmic carboxyl tail to 20 amino acids had little or no effect on chemotaxis or signal transduction, but further truncation resulted in marked functional defects. Upon incubation with monocyte chemoattractant protein 1, CCR2B underwent rapid and extensive internalization, and this was impaired progressively as the carboxyl tail was truncated from 52 to 8 amino acids. Mutation of all of the serine and threonine residues in the carboxyl tail to alanine also resulted in markedly impaired receptor internalization but did not affect signaling or chemotaxis. We conclude that the membrane-proximal portion of the cytoplasmic carboxyl tail of CCR2B is critically involved in chemotaxis and signal transduction, but neither phosphorylation of carboxyl serines or threonines nor internalization of the receptor is required for robust chemotaxis.     

Chemotactic migration triggers IL-8 generation in neutrophilic leukocytes

Neutrophils recovered from inflammatory exudates possess increased levels of IL-8, but exposure of neutrophils to chemoattractants results in only a modest stimulation of IL-8 generation. This study was undertaken to explore the hypothesis that IL-8 generation in these cells is dependent upon the process of migration. Neutrophils synthesized up to 30 times as much IL-8 during migration in response to a gradient of diverse chemoattractants than they did when stimulated directly by the attractants in the absence of a gradient. This IL-8 response was dependent on migration since it was not observed in cells exposed to concentration gradients of chemoattractants under conditions that prevented cell movement. While actinomycin-D (1 microg/ml) had little influence on the generation of IL-8 during chemotaxis, the protein synthesis inhibitor cycloheximide (10 microg/ml) markedly blunted the accumulation of cell-associated IL-8, suggesting that new protein synthesis from preexisting mRNA was responsible for the effect. Consistent with this interpretation, migrating cells incorporated over 10 times as much [3H]leucine into IL-8 as did nonmotile neutrophils exposed to chemoattractants. A substantial portion of the IL-8 generated during chemotaxis was released upon subsequent metabolic stimulation. Thus, the IL-8 synthesized during chemotaxis is uniquely positioned to exert a regulatory influence on inflammatory processes governed by neutrophilic leukocytes responding to inflammatory and infectious stimuli.     

Cytosolic free calcium and the cytoskeleton in the control of leukocyte chemotaxis

In response to a chemotactic gradient, leukocytes extravasate and chemotax toward the site of pathogen invasion. Although fundamental in the control of many leukocyte functions, the role of cytosolic free Ca2+ in chemotaxis is unclear and has been the subject of debate. Before becoming motile, the cell assumes a polarized morphology, as a result of modulation of the cytoskeleton by G protein and kinase activation. This morphology may be reinforced during chemotaxis by the intracellular redistribution of Ca2+ stores, cytoskeletal constituents, and chemoattractant receptors. Restricted subcellular distributions of signaling molecules, such as Ca2+, Ca2+/calmodulin, diacylglycerol, and protein kinase C, may also play a role in some types of leukocyte. Chemotaxis is an essential function of most cells at some stage during their development, and a deeper understanding of the molecular signaling and structural components involved will enable rational design of therapeutic strategies in a wide variety of diseases.     

Methylation of FrzCD defines a discrete step in the developmental program of Myxococcus xanthus

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.     

Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression

Initiation of Myxococcus xanthus multicellular development requires integration of information concerning the cells' nutrient status and density. A gain-of-function mutation, sasB7, that bypasses both the starvation and high cell density requirements for developmental expression of the 4521 reporter gene, maps to the sasS gene. The wild-type sasS gene was cloned and sequenced. This gene is predicted to encode a sensor histidine protein kinase that appears to be a key element in the transduction of starvation and cell density inputs. The sasS null mutants express 4521 at a basal level, form defective fruiting bodies, and exhibit reduced sporulation efficiencies. These data indicate that the wild-type sasS gene product functions as a positive regulator of 4521 expression and participates in M. xanthus development. The N terminus of SasS is predicted to contain two transmembrane domains that would locate the protein to the cytoplasmic membrane. The sasB7 mutation, an E139K missense mutation, maps to the predicted N-terminal periplasmic region. The C terminus of SasS contains all of the conserved residues typical of the sensor histidine protein kinases. SasS is predicted to be the sensor protein in a two-component system that integrates information required for M. xanthus developmental gene expression.     

Contact stimulation of Tgl and type IV pili in Myxococcus xanthus

Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl+ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.     

Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils

At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP > C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.     

Pituitary adenylate cyclase-activating polypeptide (PACAP38) modulates lymphocyte and macrophage functions: stimulation of adherence and opposite effect on mobility

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) in a concentration range from 10(-13) to 10(-6) M were studied, in vitro, on two functions of peritoneal rat lymphocytes and macrophages: adherence and mobility (spontaneous and chemotaxis). The results show that PACAP38 raised the adherence of the two cell types, increased the mobility of macrophages and decreased the mobility of lymphocytes. The maximal effects were observed at 10(-10) M in macrophages and at 10(-9) M in lymphocytes. Moreover, incubation with increasing concentrations of phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, resulted in a progressive enhancement of adherence and chemotaxis of both macrophages and lymphocytes. In contrast, retinal, a PKC inhibitor, significantly decreased these capacities. Incubation of macrophages with both PMA and PACAP38 did not have a synergistic effect on chemotaxis and adherence whereas, with lymphocytes, adherence was increased and chemotaxis was partially decreased. On the other hand, incubation with forskolin (an enhancer of intracellular cyclic AMP [cAMP] levels) caused inhibition and stimulation of chemotaxis and adherence, respectively, in both cell types. PACAP38 prevented the inhibitory effect of forskolin on chemotaxis of macrophages but not of lymphocytes, whereas the simultaneous presence of PACAP38 and forskolin was synergistic for adherence of both peritoneal cells. In addition, PACAP38 was chemoattractant for macrophages but not for lymphocytes. Furthermore, a VIP receptor antagonist was able to partially reverse the modulatory effects of PACAP38 on lymphocytes, but not on macrophages. These data suggest that PACAP38 exerts its action through the binding to type I PACAP receptors and PKC activation in macrophages and through the elevation of intracellular cAMP levels by binding to type II PACAP receptors in lymphocytes. The present work reveals an additional link between neuropeptides and the immune system and suggests that the peptide PACAP modulates the immunological function of macrophages and lymphocytes.     

Are growth factors chemotactic agents?

We report the first direct observation of chemotaxis in slowly moving malignant cells. Two sarcoma cell lines of different metastatic potential were used. In a direct-viewing chemotaxis chamber with two concentric wells, the pooled trajectories of highly malignant rat T15 cells were strongly biased toward the outer well which contained platelet-derived and insulin-like growth factors. In individual experiments, however, the trajectories of the T15 cells showed a significant directional bias which, depending on the cell distribution, sometimes deviated by as much as 170 degrees from the gradient direction. Cells of a less malignant rat line, K2, did not respond to the gradient but a strong K2 response appeared if T15 cells were placed in the outer well along with the growth factors. We conclude that stimulated T15 cells release a chemoattractant, for both T15 and K2 cells, which overrides any chemoattractive effect of the growth factors. These results call into question whether growth factors are ever directly chemotactic in this system and demonstrate the need for direct observation in determining whether any substance is a direct chemoattractant.     

Phosphorylation of chemoattractant receptors is not essential for chemotaxis or termination of G-protein-mediated responses

In several G-protein-coupled signaling systems, ligand-induced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss of ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that for chemoattractant receptors agonist-induced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation.     

Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils

Reports that interleukin-8 (IL-8) induces the infiltration of neutrophils followed by T-cells into injection sites led us to postulate that by stimulation of neutrophil degranulation IL-8 may cause the release of factors with chemoattractant activity for T-lymphocytes. Extracts of human neutrophil granules were chromatographed to isolate and purify T-lymphocyte chemoattractant factors. Two major peaks of T-cell chemotactic activity were purified by C18 reversed phase high pressure liquid chromatography (HPLC). The first peak was resolved further by C4 reversed phase HPLC and yielded an active fraction shown by NH2-terminal amino acid sequence analysis to contain defensins HNP-1, HNP-2, and HNP-3. Purified defensins HNP-1 and HNP-2 (kindly provided by Dr. R. I. Lehrer, UCLA) were also potent chemoattractants for human T-cells, while HNP-3 was inactive. The second peak of T-cell chemoattractant activity was also further purified to homogeneity by C4 reversed phase HPLC and identified by NH2-terminal sequence analysis as CAP37/azurocidin, a protein with sequence homology to serine proteases. 0.1 100 ng of defensins and 1.0 100 ng/ml CAP37 were able to stimulate in vitro T-cell chemotaxis. Neutrophil activating factors, i.e. IL-8, phorbol 12-myristate 13-acetate/ionomycin, and formylmethionylleucylphenylalanine each induced the release of CAP37 and defensins from neutrophil granules. Subcutaneous administration of defensins or CAP37/azurocidin into BALB/c mice resulted in a moderate neutrophil and mononuclear cell infiltrate by 4 h, which was greater by 24 h at the site of injection. Additionally, subcutaneous injection of defensins into chimeric huPBL-SCID mice resulted in significant infiltration by human CD3+ cells within 4 h. These results identify the antimicrobial proteins, CAP37/azurocidin and defensins HNP-1 and HNP-2, as potent neutrophil-derived chemoattractants for T-cells. These proteins represent primordial antimicrobial peptides which may have evolved into acute inflammatory cell-derived signals that mobilize immunocompetent T-cells and other inflammatory cells.     

Platelet agonists enhance the import of phosphatidylethanolamine into human platelets

It is unknown whether the endocytosis-independent transfer of phospholipids from lipoproteins to platelets is regulated by platelet agonists such as thrombin. The movements of the choline phospholipids phosphatidylcholine and sphingomyelin (labeled with either 14C or the fluorescent pyrenedecanoic acid) between low density lipoproteins and platelets were unaffected by thrombin (0.5 unit/ml). In contrast, thrombin accelerated the import of diacyl phosphatidylethanolamine (PE) and alkenylacyl phosphatidylethanolamine into platelets by about 4-fold. Similarly, thrombin receptor-activating peptide (15 microM), collagen (10 microgram/ml), and ADP (10 microM) enhanced PE uptake. High density lipoprotein particles and egg phosphatidylcholine vesicles were also donors for stimulation of platelet PE import. Part of the [14C]arachidonic acid-labeled PE transferred from low density lipoprotein to platelets activated by thrombin and collagen was metabolized to 14C-eicosanoids. Inhibitors of protein kinase C partially prevented thrombin-induced [14C]PE uptake, while direct activators of protein kinase C increased incorporation of [14C]PE into platelets. Proteinaceous factor(s) recovered in the extracellular medium from ADP- and thrombin-activated platelet suspensions were found to accelerate the transfer of pyrenedecanoic acid-labeled PE between donor and acceptor lipid vesicles. The stimulation of import of ethanolamine phospholipids led to a 2-fold enhancement of the prothrombinase activity of thrombin-activated platelets. Our study demonstrates that physiological platelet stimuli increase specifically the transfer of ethanolamine phospholipids from lipoproteins to platelets through a secretion-dependent mechanism. This might contribute to the increase of procoagulant activity of stimulated platelets.     

Multistep navigation and the combinatorial control of leukocyte chemotaxis

Cells migrating within tissues may encounter multiple chemoattractant signals in complex spatial and temporal patterns. To understand leukocyte navigation in such settings, we have explored the migratory behavior of neutrophils in model scenarios where they are presented with two chemoattractant sources in various configurations. We show that, over a wide range of conditions, neutrophils can migrate "down" a local chemoattractant gradient in response to a distant gradient of a different chemoattractant. Furthermore, cells can chemotax effectively to a secondary distant agonist after migrating up a primary gradient into a saturating, nonorienting concentration of an initial attractant. Together, these observations suggest the potential for cells' step-by-step navigation from one gradient to another in complex chemoattractant fields. The importance of such sequential navigation is confirmed here in a model system in which neutrophil homing to a defined domain (a) requires serial responses to agonists presented in a defined spatial array, and (b) is a function of both the agonist combination and the sequence in which gradients are encountered. We propose a multistep model of chemoattractant-directed migration, which requires that leukocytes display multiple chemoattractant receptors for successful homing and provides for combinatorial determination of microenvironmental localization.     

Neonatal piglet model of intraaortic balloon pumping: improved efficacy using echocardiographic timing

Secretoneurin (SN), a 33-amino acid neuropeptide, is derived from secretogranin II that is released from sensory afferent C-fibers by capsaicin. Described functions of secretoneurin include chemotaxis of monocytes and endothelial cells, and inhibition of endothelial cell proliferation. Inhibition of monocyte chemotaxis by staurosporine indicated involvement of specific signaling pathways. We have tested effects of SN, substance P (SP), and interleukin-8 (IL-8) on eosinophil migration in modified Boyden chambers including signaling mechanisms of neuropeptide and cytokine stimulation of human eosinophils. Experiments showed SN as eosinophil chemoattractant comparable in its potency to IL-8. Checkerboard analysis, usage of a specific anti-SN-antibody, and receptor desensitization experiments confirmed the chemotactic activity. Preincubation of the cells with effective concentrations of staurosporine or tyrphostin-23 showed no effect, whereas treatment with wortmannin (WTN) or 3-isobutyl-1-methylxantin (IBMX) completely blocked SN-induced migration. Additionally, experiments ruled out tyrphostin-23- and WTN-sensitive signaling pathways for SP-induced chemotaxis of eosinophils. We conclude that SN-stimulated human eosinophil chemotaxis is mediated via a unique and specific signal transduction pathway that involves activation of phosphodiesterases and WTN-sensitive enzymes, ie, phospholipase D and phosphatidylinositol-3-kinase. In contrast, we report that activation of the latter and tyrosine kinases is required for SP-induced chemotaxis of eosinophils.     

Inhibitory effects of formoterol on platelet-activating factor induced eosinophil chemotaxis and degranulation

A new long-acting beta 2-agonist, formoterol, has been reported to have a greater efficacy and duration of action in asthmatic patients as compared to conventional beta 2-agonists. We recently demonstrated that formoterol inhibited antigen-induced late asthmatic response (LAR) and accompanying airway eosinophilia in guinea pigs. In this study, we investigated the direct effect of formoterol in vitro on human eosinophil function, focusing on platelet-activating factor (PAF)-induced eosinophil chemotaxis and eosinophil cationic protein (ECP) release. Purified normodense eosinophils were separated by discontinuous gradient from 12 mild asthmatic patients. Formoterol in concentrations of 1-100 microM significantly inhibited PAF-induced eosinophil chemotaxis in a dose-dependent manner with a concentration of drug required to produce 50% inhibition (IC50) of 10.16 microM; % inhibition: 22.9 +/- 13.0% (1 microM), 51.6 +/- 12.7% (10 microM), 75.0 +/- 11.3% (100 microM). When formyl-methionyl-leucyl-phenylamine (FMLP) was used as a chemoattractant, a similar inhibition of eosinophil chemotaxis by formoterol was observed; % inhibition: 13.1 +/- 5.0% (1 microM). 47.7 +/- 7.6% (10 microM), 65.5 +/- 16.5% (100 microM). A conventional beta 2-agonist, salbutamol, at doses to 100 microM did not show any inhibitory effects on PAF-induced eosinophil chemotaxis. Formoterol in concentrations of 1-100 microM also significantly inhibited PAF-induced ECP release from eosinophils; % inhibition: 21.7 +/- 9.0% (1 microM), 39.3 +/- 7.4% (10 microM), 39.6 +/- 8.4% (100 microM). In the presence of phosphodiesterase inhibitors, theophylline or isobutylmethyl xanthine (IBMX), the inhibition by formoterol on PAF-induced ECP release was enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transmembrane Ca2+ gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum C(a2+)-ATPase

The sarcoplasmic reticulum (SR) C(a2+)-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role of phospholipids, especially phosphatidylcholine (PC), in the modulation of C(a2+)-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows. (i) Incubated with phospholipids, the enzyme activity of the delipidated C(a2+)-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1,000 mumol/L:50 mumol/L, similar to the physiological condition), the inhibition of C(a2+)-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50:50 molar ratio of PC:PE (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of C(a2+)-ATPase is observed in vesicles containing DPPC:PE and DOPC:PE, while no inhibition in DMPC:PE vesicles (iv) If the transmembrane Ca2+ gradient is in the inverse direction, the enzyme activity of C(a2+)-ATPase is inhibited whenever reconstituted with acidic or neutral phospholipids.     

Changes with ageing in the modulation of murine lymphocyte chemotaxis by CCK-8S, GRP and NPY

The general immunodepression found in ageing organisms may be related to changes in the neuroimmune network. In the present study, the migration capacity of lymphocytes from BALB/c mice of three different ages: young (12 +/- 2 weeks), adult (24 +/- 2 weeks) and old (72 +/- 2 weeks), has been assayed in vitro in response to three neuropeptides: sulfated cholecystokinin octapeptide (CCK-8s), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY) in a physiological range of concentrations (10(-8)-10(-12) M). The capacity of migration to a chemical gradient or chemotaxis was studied by the Boyden's technique using f-met-leu-phe at 10(-8) M as chemoattractant. The results show a different response of lymphocytes to the different neuropeptides, as wells as to age, concentrations and locations studied. However, some similarities were found, for instance the three neuropeptides inhibited chemotaxis in thymus. The stimulatory effects that GRP and NPY exerted in young and adult mice were not observed in old animals. CCK-8s inhibited the chemotaxis in every organ studied, with the effect being more striking in old mice. Our conclusion is that stimulatory effects of the neuropeptides disappear or become inhibitory with ageing.     

TCR activation inhibits chemotaxis toward stromal cell-derived factor-1: evidence for reciprocal regulation between CXCR4 and the TCR

Stromal cell-derived factor-1 (SDF-1), a C-X-C family chemokine, is a potent T lymphocyte chemoattractant. We investigated the effects of T cell activation on the chemotactic response to SDF-1. Anti-CD3 Ab stimulation of either Jurkat T cells or murine peripheral CD4+ T lymphocytes produced a dramatic inhibition of SDF-1-induced chemotaxis. In contrast, the SDF-1 responses of Jurkat clones with deficiencies in key TCR signaling components (Lck, CD45, and TCR-beta), were only marginally reduced by anti-CD3 stimulation. Similar to PMA treatment, which abolished both CXCR4 receptor expression and the chemotactic response of Jurkat cells to SDF-1, anti-CD3 Ab treatment reduced cell surface expression of CXCR4 to 65% of the control value, an effect that was blocked by protein kinase C inhibitors. Our data suggest that initial T cell activation events inhibit the response of Jurkat T cells to CXCR4 stimulation. In contrast, SDF-1 treatment resulted in a reduction of tyrosine phosphorylation of the TCR downstream effectors, ZAP-70, SLP-76, and LAT (linker for activation of T cells), suggesting that this chemokine potentially regulates the threshold for T cell activation.