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Background CCR3 is the cognate receptor for major human eosinophil chemoattractants from the eotaxin family of proteins that are elevated in asthma and correlate with disease severity. Objective This proof-of-mechanism study examined the effect of AXP1275, an oral, small-molecule inhibitor of CCR3, on airway responses to inhaled allergen challenge. Methods Twenty-one subjects with mild atopic asthma and documented early and late asthmatic responses to an inhaled aeroallergen completed a randomized double-blind cross-over study to compare early and late allergen-induced asthmatic responses, methacholine PC20 , blood and sputum eosinophils and exhaled nitric oxide after 2 weeks of treatment with once-daily doses of AXP1275 (50 mg) or placebo. Results There was a significant increase in methacholine PC20 after 12 days of AXP1275 treatment compared to placebo (increase of 0.92 doubling doses versus 0.17 doubling doses, P = .01), but this protection was lost post-allergen challenge. There was no effect of AXP1275 on allergen-induced late asthmatic responses, or eosinophils in blood and sputum. The early asthmatic response and exhaled nitric oxide levels were slightly lower with AXP1275, but this did not reach statistical significance. The number of subjects who experienced treatment-emergent adverse events while receiving AXP1275 was comparable placebo. Conclusions & clinical relevance AXP1275 50 mg administered daily was safe and well tolerated, and there was no difference in the type, severity or frequency of treatment-emergent adverse events in subjects while receiving AXP1275 compared to placebo. AXP1275 increased the methacholine PC20 ; however, the low and variable exposure to APX1275 over a short treatment period may have contributed to poor efficacy on other outcomes.

Summary Background Both prostaglandin (PG) D receptor (DP) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells)/DP2 are high-affinity receptors for PGD2. Previous studies have demonstrated that PGD2 enhances releasability and induces CRTH2/DP2-mediated migration in human basophils, but the precise effects of PGD2 on basophils as well as receptor usage have not been fully clarified. Objective We comprehensively explored the roles of DP and CRTH2/DP2 in basophil functions by using selective agonists and antagonists for each receptor. Methods DP and CRTH2/DP2 transcripts were quantified by real-time PCR. We studied the effects of selective agonists (DP: BW245C; CRTH2/DP2: 13,14-dihydro-15-keto (DK)-PGD2) and/or antagonists (DP: BWA868C; CRTH2/DP2: ramatroban) on Ca2+ mobilization, migration, degranulation, CD11b expression and survival of human basophils. Results Basophils expressed transcripts of both DP and CRTH2/DP2, but the levels of CRTH2/DP2 transcripts were ca. 100-fold higher compared with DP transcripts. Ca2+ influx was induced in basophils by either PGD2 or DK-PGD2/CRTH2 agonist but not by BW245C/DP agonist. Basophils treated with PGD2 were completely desensitized to subsequent stimulation with DK-PGD2, but not vice versa. DK-PGD2 as well as PGD2 up-regulated CD11b expression, induced migration and enhanced degranulation, and those effects were completely antagonized by ramatroban/CRTH2 antagonist. In contrast, BW245C/DP agonist exhibited an inhibitory effect on basophil migration and IgE-mediated degranulation, and the migration inhibitory effect was effectively antagonized by BWA868C/DP antagonist. On the other hand, while PGD2 significantly shortened the basophil life-span, neither DK-PGD2/CRTH2 agonist nor BW245C/DP agonist did. Conclusion CRTH2/DP2 is primarily responsible for the pro-inflammatory effects of PGD2 on human basophils, while DP introduces negative signals capable of antagonizing the effects of CRTH2/DP2 in these cells. The effects of PGD2 on longevity imply a mechanism(s) other than via DP or CRTH2/DP2. CRTH2/DP2 on basophils may afford opportunities for therapeutic targeting in allergic inflammation.

Molecular analyses of the chemokine fractalkine and its receptor CX3C-R1 in the rat brain have revealed a striking polarization: fractalkine is expressed constitutively in neurons and is up-regulated by TNF-α and IL-1β in astrocytes. Expression of its specific receptor, CX3C-R1, is restricted to astrocytes and microglia. We have analyzed the functional correlates of this expression and demonstrate that fractalkine induces microglial cell migration and activation. However, the activity of this chemokine on astrocytes may also be highly relevant in inducing astrocyte-microglia cell interactions through cytokine/mediator release leading to microglial activation.

The chemokine RANTES is a chemoattractant and activating factor for T lymphocytes. Investigation of the signal transduction mechanisms induced by RANTES in T cells revealed tyrosine phosphorylation of multiple protein species with prominent bands at 70-85 and 120-130 kD. Immunoprecipitation and Western analyses revealed that a protein of 125 kD was identical to the focal adhesion kinase (FAK) pp125FAK. RANTES stimulated phosphorylation of FAK as early as 30 seconds and immunoblots using antiphosphotyrosine monoclonal antibodies revealed that there was consistent phosphorylation of a 68-70 kD species in the pp125FAK immunoprecipitates. Immunoblotting and kinase assays showed this to be two separate proteins, the tyrosine kinase zeta-associated protein (ZAP) 70, and the focal adhesion protein paxillin. These results indicate a potentially important role for RANTES in the generation of T cell focal adhesions and subsequent cell activation via a molecular complex containing FAK, ZAP-70, and paxillin.

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes. We have isolated a novel chemokine cDNA, designated CCF18, from a cDNA library of an IL-3-dependent murine pro-B cell line, Ba/F3. The cDNA encodes a protein structurally related to the C-C chemokine members. Among this family, C10 shows the highest homology to CCF18, and MIP-1 alpha also has a significant homology but to a lesser extent. CCF18 produced from COS cells induced chemotaxis and Ca2+ flux in CD4+ T cell clones. Moreover, prior administration of MIP-1 alpha desensitized the cells to CCF18. The CCF18 gene (Scya10) was mapped to a middle region of murine chromosome 11, where other genes for several C-C chemokine members are localized. These results clearly indicate that CCF18 is a new member of the C-C chemokine family. Since a high level of CCF18 mRNA is constitutively expressed in macrophage and myeloid cell lines, CCF18 may play a role in inflammatory processes.

We describe the isolation of a cDNA that encodes human lymphotactin (Ltn), a new class of lymphocyte-specific chemokine. Human Ltn shows similarity to some members of the C-C chemokine family but has lost the first and third cysteine residues that are characteristic of the C-C and C-X-C chemokines. Ltn is chemotactic for lymphocytes but not for monocytes, a characteristic that makes it unique among chemokines. In addition, calcium flux desensitization studies indicate that Ltn uses a unique receptor. The human Ltn gene maps to a different chromosome than do the C-C and C-X-C chemokine families. Taken together, these characteristics indicate that Ltn is the first example of a new class of human chemokines with preferential effects on lymphocytes.

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.

Eotaxin is a potent eosinophil chemoattractant that plays an important role in regulating eosinophil tissue levels both in healthy individuals and in diseases associated with significant eosinophil infiltrates, such as the allergic inflammation observed in asthma. Here, we demonstrate that treatment of eosinophils with eotaxin induces the phosphorylation of the mitogen-activated protein kinases (MAPKs) p42 and p44, leading to kinase activation. Blockade of MAPK activation by the MAPK kinase inhibitor PD98059 leads to a dramatic decrease in eotaxin-induced eosinophil rolling in vivo and chemotaxis in vitro. This blockade in the leukocyte migration process is consistent with the observed inhibition of actin polymerization and rearrangement within the eosinophil following treatment with MAPK inhibitor. It is suggested, therefore, that the intrinsic mechanism of eotaxin-induced eosinophil rolling and migration involves activation of the p42/p44 MAPK, possibly through regulation of the cytoskeletal rearrangements necessary for chemotaxis.

Eotaxin has been characterized as a chemokine involved in eosinophil activation; however, mRNA for this C-C chemokine has been shown to be constitutively expressed in thymus. Immunohistochemical analysis showed a punctate distribution pattern, with eotaxin expression localized mainly in the medulla and in Hassle's corpuscles. Moreover, the receptor for eotaxin, CCR-3, was detected on thymocytes, with the highest level of expression being on the CD8 single-positive population. Equilibrium binding analyses on unfractionated thymocytes demonstrated specific 125I-eotaxin binding profiles comparable with CCR-3 transfectants. Eotaxin induced cell migration and mobilization of intracellular calcium in all thymocytes except the immature CD4(-)/CD8(-) population. Eotaxin also induced the secretion of the chemokines interleukin-8, RANTES, and macrophage inflammatory protein-1beta from thymocyte cultures in vitro. These results suggest that eotaxin-induced thymocyte activation may have important physiological implications for lymphocyte mobilization within and from this lymphoid organ.

We have isolated a novel human C-C chemokine, MIP-1 delta from a human fetal spleen cDNA library. The human MIP-1 delta cDNA has an unusually long 400-bp 5-prime untranslated region and a predicted 113-amino acid protein of 10 kDa. The coding sequence contains a signal peptide of 21 amino acids, indicating that the mature protein has 92 amino acids (8 kDa). Recombinant human MIP-1 delta produced by transfected human embryonic kidney 293 cells produced an 8-kDa protein, which confirmed the presence of a signal peptide. Compared with other human C-C chemokines, human MIP-1 delta shows the highest homology with human HCC-1, CK beta-8, murine C10, and CCF18 (MIP-1 gamma). The human MIP-1 delta gene is localized on chromosome 17 where most of the C-C chemokine superfamily is located. Human MIP-1 delta is expressed in T and B lymphocytes, NK cells, monocytes, and monocyte-derived dendritic cells, but not in bone marrow-derived dendritic cells. Its expression can be induced by other proinflammatory cytokines in monocytes and dendritic cells. Human MIP-1 delta is chemotactic for T cells and monocytes, but not for neutrophils, eosinophils, or B cells. Human MIP-1 delta induced calcium flux in human CCR1-transfected cells.

The CC chemokine macrophage inflammatory protein 1beta (MIP-1beta), has been shown to be a chemoattractant preferentially activating CD4(+) CD45RA+ T lymphocytes. Further analysis of chemokine action on lymphocytic cells has shown the potent migration-promoting capacity of MIP-1beta on human thymocytes. The responding cells were the CD4(+) and CD8(+) single-positive (SP), as well as the CD4(+) CD8(+) double-positive (DP) populations, with little if any migratory activity on the double-negative (DN) population. The activation of thymocytes by MIP-1beta appeared to be a direct, receptor-mediated event as evidenced by the rapid mobilization of intracellular calcium, increase in proteins phosphorylated on tyrosine, and activation of the mitogen-activated protein kinase (MAPK) pathway. Radioligand binding analyses showed specific and displaceable binding of MIP-1beta to thymocytes with a Kd of approximately 1 nmol/L, a profile that was comparable with MIP-1beta binding to CCR-5-transfected NIH 3T3 cells. In addition, CCR-5 mRNA was detected in total thymocyte populations indicating that activation of thymocytes by MIP-1beta may occur through binding to CCR-5. Further dissection of the subpopulations showed that only the DP and CD8(+) SP populations expressed CCR-5 and expression data on these two populations was confirmed using anti-CCR-5 monoclonal antibody. These data may be suggestive of a role for MIP-1beta in human thymocyte activation, and show a potential route for HIV infectivity in the developing immune system.

The chemokine RANTES induces a unique biphasic cytoplasmic Ca2+ signal in T cells. The first phase of this signal, similar to that of other chemokines, is G-protein mediated and chemotaxis associated. The second phase of this signal, unique to RANTES and evident at concentrations greater than 100 nM, is tyrosine kinase linked and results in a spectrum of responses similar to those seen with antigenic stimulation of T cells. We show here that certain jurkat T cells responded to RANTES solely through this latter pathway. A direct correlation between the RANTES-induced second phase response and CD3 expression was demonstrated in these cells. Sorting the Jurkat cells into CD3(high) and CD3(low) populations revealed that only the CD3(high) cells were responsive to RANTES. Furthermore, stimulation of these Jurkat cells with anti-CD3 mAb significantly depresses their subsequent response to RANTES. While a RANTES-specific chemokine receptor is expressed at a low level on these Jurkat cells, the RANTES-induced activation is dependent on the presence of the TCR. Thus, stimulation through TCR may partially account for RANTES' unique pattern of signaling in T cells.

The chemokine RANTES is a potent agonist of T cell activation. In an investigation of signal-transduction events activated by this chemokine, we have shown that RANTES stimulates dose-dependent phospholipase D (PLD) activity in Jurkat cells. Equilibrium-binding analyses using 125I-labeled RANTES indicated the presence of a receptor for RANTES on these cells, which has a Kd of 0.1 nM, is expressed at approximately 600 sites per cell, and a binding specificity that was not comparable with that of any of the known chemokine receptors, since 125I-labeled RANTES was displaced by macrophage-inflammatory protein-1 beta (but not macrophage-inflammatory protein-1 alpha), monocyte-chemotactic protein-1 (MCP-1), MCP-3, MCP-4, and eotaxin. RANTES-induced PLD activation was augmented by GTP gamma S, but not GDP beta S, and inhibited by the protein kinase C inhibitor bisindolylmaleimide, as well as the fungal metabolite brefeldin A, and C3 exoenzyme (Clostridium botulinum), implicating the activation of RhoA. RANTES also induced GTP-GDP exchange of immunoprecipitated RhoA. RANTES-stimulated PLD activity was dependent on an ADP-ribosylation factor(s), as assessed by inhibition studies using a synthetic inhibitory peptide of the N-terminal 16 amino acids of ADP-ribosylation factor 1. These studies indicate the potential existence of a novel receptor-mediated mechanism for activation of T cells by the chemokine RANTES.

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes. We have isolated a novel chemokine cDNA, designated CCF18, from a cDNA library of an IL-3-dependent murine pro-B cell line, Ba/F3. The cDNA encodes a protein structurally related to the C-C chemokine members. Among this family, C10 shows the highest homology to CCF18, and MIP-1 alpha also has a significant homology but to a lesser extent. CCF18 produced from COS cells induced chemotaxis and Ca2+ flux in CD4+ T cell clones. Moreover, prior administration of MIP-1 alpha desensitized the cells to CCF18. The CCF18 gene (Scya10) was mapped to a middle region of murine chromosome 11, where other genes for several C-C chemokine members are localized. These results clearly indicate that CCF18 is a new member of the C-C chemokine family. Since a high level of CCF18 mRNA is constitutively expressed in macrophage and myeloid cell lines, CCF18 may play a role in inflammatory processes.

We describe the isolation of a cDNA that encodes human lymphotactin (Ltn), a new class of lymphocyte-specific chemokine. Human Ltn shows similarity to some members of the C-C chemokine family but has lost the first and third cysteine residues that are characteristic of the C-C and C-X-C chemokines. Ltn is chemotactic for lymphocytes but not for monocytes, a characteristic that makes it unique among chemokines. In addition, calcium flux desensitization studies indicate that Ltn uses a unique receptor. The human Ltn gene maps to a different chromosome than do the C-C and C-X-C chemokine families. Taken together, these characteristics indicate that Ltn is the first example of a new class of human chemokines with preferential effects on lymphocytes.

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.

In order to study the initial events during infection of target cells by the enteric pathogen Entamoeba histolytica, we developed a quantitative adhesion assay based on the use of a human colonic cell line (CaCo-2) and biotinylated amoebae tagged with fluorescein. To prevent the strong and rapid lytic activity of Entamoeba histolytica on colonic cells, which would otherwise impede the study of the primary adhesion steps, parasites were mildly fixed, biotinylated and labelled with streptavidin-FITC. After labelled parasites have bound to enterocytes, nonadhered amoebae are removed by washing and attached parasites quantified by means of an automated fluorescence plate reader. The bioassay is simple, nonhazardous and can be completed in 1.5 h. We were able to detect ranges from 200 to 20,000 fluorescent parasites per microwell in a 96-well plate, containing approximately 10(5) colonic cells. Fluorescence intensity (arbitrary units) increased in direct relationship to the number of parasites added per well, and was not limited by the size of the culture plate (96, 24 or six wells). As an example of the value of this assay, two proinflammatory cytokines (interleukin-1, (IL-1 beta) and interferon-gamma (IFN-gamma) known to influence the adhesion properties of endothelial and epithelial cells, were used to assess their effects upon enterocyte-entamoeba binding. The increase in amoebae binding revealed by cytokine treatment to enterocytes suggests that the parasite may take advantage of inflammatory stimuli in order to increase its binding to colonic epithelium. We believe this rapid, sensitive and simple method offers the potential for large scale screening assays to study the immunobiology of this protozoal infection by analysing the mechanisms involved in the primary interactions between Entamoeba histolytica and enterocytes.

The role of locally released cytokines in inducing lymphocyte activation and infiltration in the skin lesions of mycosis fungoides has been investigated. The levels of selected cytokines were measured in chamber fluid samples from lesional and control skin. Biologically active interleukin 6 was significantly elevated in lesional samples and a recombinant form of this cytokine was shown to induce lymphocyte migration in an in vitro assay. Biologically active interleukin 1 was detected in all control chamber fluid samples. Significantly reduced levels of this cytokine were present in lesional samples, which may be the result of the release of preformed material. Interleukin 2 and tumour necrosis factor activity, and gamma interferon and granulocyte macrophage colony-stimulating factor immunoreactivity, were not detectable in any of the samples. Interleukins 1 and 6 may play a role in the pathogenesis of the lesional lymphocyte infiltrates in mycosis fungoides.

Rationale Both PGD receptor (DP) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells)/DP2 are high-affinity receptors for PGD 2 . Previous studies have demonstrated that PGD 2 enhances releasability and induces CRTH2/DP2-mediated migration in human basophils. We comprehensively explored the precise effects of PGD 2 as well as receptor usage on human basophils. Methods DP and CRTH2/DP2 transcripts were quantified by real-time PCR. We studied the effects of selective agonists (DP: BW245C; CRTH2/DP2: DK-PGD 2 ) and/or antagonists (DP: BWA868C; CRTH2/DP2: ramatroban) on Ca 2+ mobilization, migration, degranulation, CD11b expression and survival of human basophils. Results Basophils expressed ∼100-fold higher levels of CRTH2/DP2 transcripts compared with DP transcripts. Ca 2+ influx was induced by either PGD 2 or DK-PGD 2 but not by BW245C. Basophils treated with PGD 2 were completely desensitized to subsequent stimulation with DK-PGD 2 , but not vice versa. DK-PGD 2 as well as PGD 2 induced migration and enhanced degranulation, and those effects were completely antagonized by ramatroban. In contrast, BW245C exhibited inhibitory effects on basophil migration. CD11b expression was also up-regulated by PGD 2 or DK-PGD 2 but not by BW245C. Although PGD 2 significantly shortened the basophil life-span, neither DK-PGD 2 nor BW245C did. Conclusions CRTH2/DP2 activation is necessary and sufficient for the pro-inflammatory effects of PGD 2 (migration, degranulation and CD11b expression), while DP activation may mediate anti-inflammatory effects by negatively regulating basophil functions. The effects of PGD 2 on longevity imply a mechanism(s) other than via DP or CRTH2/DP2. CRTH2/DP2 on basophils may afford opportunities for therapeutic targeting in allergic inflammation.

1. The chemotactic activity of 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE), 12(S)-HETE and leukotriene B4 (LTB4) for human mixed peripheral blood lymphocytes has been assessed in a 48-well microchemotaxis assay. Responses to the standard lymphocyte chemoattractants, zymosan-activated plasma, casein and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were also measured. 2. 12(R)-HETE was shown to be chemotactic for lymphocytes over the range 5 x 10(-7) to 5 x 10(-5) M. In contrast, negligible chemotactic responses to 12(S)-HETE were obtained. 3. LTB4 was 200 times more potent than 12(R)-HETE as a lymphocyte chemoattractant, although maximal responses to the two agonists were similar. 4. 12(R)-HETE and LTB4, which are present in extracts of samples from the skin lesions of psoriasis, may be, at least in part, responsible for the lymphocyte infiltrate which is a characteristic feature of this disease.