Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints.

T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines are credited with guiding the multistep recruitment of CD8(+) T cells across tumour vessels. However, the multiplicity of chemokines within tumours has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signalling at effector sites. Here we investigate the hierarchy of chemokine receptor requirements during T-cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for Gαi-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8(+) effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8(+) effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumour vascular interface as a critical checkpoint to effective T-cell-based cancer immunotherapy.

Colonic patch and colonic SILT development are independent and differentially regulated events.

Intestinal lymphoid tissues have to simultaneously ensure protection against pathogens and tolerance toward commensals. Despite such vital functions, their development in the colon is poorly understood. Here, we show that the two distinct lymphoid tissues of the colon-colonic patches and colonic solitary intestinal lymphoid tissues (SILTs)-can easily be distinguished based on anatomical location, developmental timeframe, and cellular organization. Furthermore, whereas colonic patch development depended on CXCL13-mediated lymphoid tissue inducer (LTi) cell clustering followed by LTα-mediated consolidation, early LTi clustering at SILT anlagen did not require CXCL13, CCR6, or CXCR3. Subsequent dendritic cell recruitment to and gp38(+)VCAM-1(+) lymphoid stromal cell differentiation within SILTs required LTα; B-cell recruitment and follicular dendritic cell differentiation depended on MyD88-mediated signaling, but not the microflora. In conclusion, our data demonstrate that different mechanisms, mediated mainly by programmed stimuli, induce the formation of distinct colonic lymphoid tissues, therefore suggesting that these tissues may have different functions.

Eosinophils are necessary for pulmonary arterial remodeling in a mouse model of eosinophilic inflammation-induced pulmonary hypertension.

There is increasing evidence that inflammation plays a pivotal role in the pathogenesis of some forms of pulmonary hypertension (PH). We recently demonstrated that deficiency of adiponectin (APN) in a mouse model of PH induced by eosinophilic inflammation increases pulmonary arterial remodeling, pulmonary pressures, and the accumulation of eosinophils in the lung. Based on these data, we hypothesized that APN deficiency exacerbates PH indirectly by increasing eosinophil recruitment. Herein, we examined the role of eosinophils in the development of inflammation-induced PH. Elimination of eosinophils in APN-deficient mice by treatment with anti-interleukin-5 antibody attenuated pulmonary arterial muscularization and PH. In addition, we observed that transgenic mice that are devoid of eosinophils also do not develop pulmonary arterial muscularization in eosinophilic inflammation-induced PH. To investigate the mechanism by which APN deficiency increased eosinophil accumulation in response to an allergic inflammatory stimulus, we measured expression levels of the eosinophil-specific chemokines in alveolar macrophages isolated from the lungs of mice with eosinophilic inflammation-induced PH. In these experiments, the levels of CCL11 and CCL24 were higher in macrophages isolated from APN-deficient mice than in macrophages from wild-type mice. Finally, we demonstrate that the extracts of eosinophil granules promoted the proliferation of pulmonary arterial smooth muscle cells in vitro. These data suggest that APN deficiency may exacerbate PH, in part, by increasing eosinophil recruitment into the lung and that eosinophils could play an important role in the pathogenesis of inflammation-induced PH. These results may have implications for the pathogenesis and treatment of PH caused by vascular inflammation.

Allergic asthma: a tale of many T cells.

Asthma is a common immune-mediated disorder characterized by reversible airway inflammation, mucus production, and variable airflow obstruction with airways hyperresponsiveness (AHR). In most cases the airway inflammation characteristic of asthma is thought to result from an allergic-type reaction to an inhaled substance from the environment (so-called allergic asthma). In allergic asthma, allergen exposure stimulates eosinophilic inflammation of the airways associated with infiltration of T cells. Although the recruitment of eosinophils into the airways is an important component in the pathogenesis of asthma, the trafficking of T lymphocytes into the airways is now believed to establish and orchestrate the asthmatic inflammatory response. This review explores the roles of various T cell subsets in the pathogenesis of allergic airway inflammation and highlights the contributions of these cells in regulating asthma.

Variant eotaxin: its effects on the asthma phenotype.

BACKGROUND: Eotaxin, a CC chemokine expressed in the asthmatic lung, has been associated with impaired lung function. The role of its variant form is unknown. OBJECTIVE: The purpose of this study was to detect the population frequency and effects of a known single-nucleotide polymorphism in the eotaxin gene in which a threonine residue (THR(23)) is substituted for the wild-type alanine (ALA(23)) at the 23rd amino acid at the terminus of the peptide leader sequence. METHODS: We measured eotaxin protein secretion in 293 cells transfected with expression vectors and in PBMCs obtained from individuals bearing the alternative forms of the gene. A case-control study of plasma eotaxin levels and eosinophil counts, a comparison of baseline lung function by genotype in a population of 806 subjects with asthma, and a comparison of the allele frequency with a nonasthmatic population were performed. RESULTS: Human 293 cells and PBMCs with THR(23) variant eotaxin secreted significantly less eotaxin protein than did ALA(23)-bearing cells. In the case-control study, THR(23)-THR(23) individuals had lower plasma levels of eotaxin (310 [240-350] vs 420 [270-700] pg/mL; P < .05) and eosinophil counts (120 [5-220] vs 190 [110-470] cells/microL; P < .05) than ALA(23)-ALA(23) subjects; heterozygous subjects had intermediate levels. Higher levels of lung function were associated with THR(23) eotaxin (percent of predicted FEV(1), 65% +/- 3.5% [THR(23)-THR(23)] vs 58% +/- 0.9% [THR(23)-ALA(23)] and 56% +/- 0.5% [ALA(23)-ALA(23)]; P < .05). CONCLUSION: The THR(23) variant is associated with both decreased eosinophil counts and higher levels of lung function in subjects with asthma.

CXCR3 internalization following T cell-endothelial cell contact: preferential role of IFN-inducible T cell alpha chemoattractant (CXCL11).

Chemokine receptors are rapidly desensitized and internalized following ligand binding, a process that attenuates receptor-mediated responses. However, the physiological settings in which this process occurs are not clear. Therefore, we examined the fate of CXCR3, a chemokine receptor preferentially expressed on activated T cells following contact with endothelial cells. By immunofluorescence microscopy and flow cytometry, we found that CXCR3 was rapidly internalized when T cells were incubated with IFN-gamma-activated human saphenous vein endothelial cells (HSVEC), but not with resting HSVEC. Similar results were obtained using human CXCR3-transfected murine 300-19 B cells. CXCR3 down-regulation was significantly more pronounced when T cells were in contact with HSVEC than with their supernatants, suggesting that CXCR3 ligands were efficiently displayed on the surface of HSVEC. Using neutralizing mAbs to IFN-induced protein-10 (CXCL10), monokine induced by IFN-gamma (CXCL9), and IFN-inducible T cell alpha chemoattractant (I-TAC; CXCL11), we found that even though I-TAC was secreted from IFN-gamma-activated HSVEC to lower levels than IFN-induced protein-10 or the monokine induced by IFN-gamma, it was the principal chemokine responsible for CXCR3 internalization. This correlated with studies using recombinant chemokines, which revealed that I-TAC was the most potent inducer of CXCR3 down-regulation and of transendothelial migration. Known inhibitors of chemokine-induced chemotaxis, such as pertussis toxin or wortmannin, did not reduce ligand-induced internalization, suggesting that a distinct signal transduction pathway mediates internalization. Our data demonstrate that I-TAC is the physiological inducer of CXCR3 internalization and suggest that chemokine receptor internalization occurs in physiological settings, such as leukocyte contact with an activated endothelium.

IL-4 differentially regulates eotaxin and MCP-4 in lung epithelium and circulating mononuclear cells.

To investigate the mechanisms of eosinophil recruitment in allergic airway inflammation, we examined the effects of interleukin (IL)-4, a Th2-type cytokine, on eotaxin and monocyte chemoattractant protein-4 (MCP-4) expression in human peripheral blood mononuclear cells (PBMCs; n = 10), in human lower airway mononuclear cells (n = 5), in the human lung epithelial cell lines A549 and BEAS-2B, and in human cultured airway epithelial cells. IL-4 inhibited eotaxin and MCP-4 mRNA expression induced by IL-1 beta and tumor necrosis factor-alpha in PBMCs but did not significantly inhibit expression in epithelial cells. Eotaxin and MCP-4 mRNA expression was not significantly induced by proinflammatory cytokines in lower airway mononuclear cells. IL-1 beta-induced eotaxin and MCP-4 protein production was also inhibited by IL-4 in PBMCs, whereas IL-4 enhanced eotaxin protein production in A549 cells. In contrast, dexamethasone inhibited eotaxin and MCP-4 expression in both PBMCs and epithelial cells. The divergent effects of IL-4 on eotaxin and MCP-4 expression between PBMCs and epithelial cells may create chemokine concentration gradients between the subepithelial layer and the capillary spaces that may promote the recruitment of eosinophils to the airway in Th2-type responses.

Eotaxin and monocyte chemoattractant protein-1 in chronic eosinophilic pneumonia.

Chronic eosinophilic pneumonia (CEP) is characterized by chronic or recurrent pulmonary infiltrates with eosinophils, but the precise mechanism of eosinophil accumulation has not been fully elucidated. Eotaxin is one of the CC chemokines that selectively recruits eosinophils and contributes to the pathogenesis of allergic airway diseases including asthma, but its roles in pathogenesis of CEP have not been fully elucidated. The authors measured concentrations of eotaxin and other CC chemokines, monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T-cell expressed and secreted, macrophage inflammatory protein-1alpha, and the eosinophil activating Th2 cytokine interleukin (IL)-5 in bronchoalveolar lavage (BAL) fluid from CEP patients (n=11), and compared these concentrations with those from control subjects (n = 6). The eotaxin (904 +/- 203 versus 29 +/- 7 pg x mL(-1), p = 0.0001), MCP-1 (194 +/- 57 versus 15 +/- 2 pg x mL(-1), p < 0.05), and IL-5 (7.8 +/- 2.0 versus 2.7 +/- 0.6 pg x mL(-1), p < 0.05) levels were significantly higher for cases with CEP in comparison to those serving as controls. Proportions of eosinophil and lymphocyte counts were greater in BAL fluid from CEP patients. Eotaxin and IL-5 levels correlated with the proportion of eosinophils in BAL fluid from CEP patients. MCP-1 correlated with the relative lymphocyte numbers. In short, eotaxin, interleukin-5, and monocyte chemoattractant protein-1 levels were higher in the BAL fluid of CEP patients and these levels may contribute to eosinophil and lymphocyte recruitment and activation in the airways as found with this disorder.

Noncytolytic inhibition of X4 virus by bulk CD8(+) cells from human immunodeficiency virus type 1 (HIV-1)-infected persons and HIV-1-specific cytotoxic T lymphocytes is not mediated by beta-chemokines.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) mediate immunologic selection pressure by both cytolytic and noncytolytic mechanisms. Non cytolytic mechanisms include the release of beta-chemokines blocking entry of R5 HIV-1 strains. In addition, CD8(+) cells inhibit X4 virus isolates via release of as yet poorly characterized soluble factors. To further characterize these factors, we performed detailed analysis of CTL as well as bulk CD8(+) T lymphocytes from six HIV-1-infected individuals and from six HIV-1-seronegative individuals. Kinetic studies revealed that secreted suppressive activities of HIV-1-specific CTL and bulk CD8(+) T lymphocytes from all HIV-1-infected persons are significantly higher than that of supernatants from seronegative controls. The suppressive activity could be blocked by monensin and brefeldin A, was heat labile, and appeared in a pattern different from that of secretion of chemokines (MDC, I-309, MIP-1alpha, MIP-1beta, and RANTES), cytokines (gamma interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor), and interleukins (interleukin-13 and interleukin-16). This suppression activity was characterized by molecular size exclusion centrifugation and involves a suppressive activity of >50 kDa which could be bound to heparin and a nonbinding inhibitory activity of <50 kDa. Our data provide a functional link between CD8(+) cells and CTL in the noncytolytic inhibition of HIV-1 and suggest that suppression of X4 virus is mediated through proteins. The sizes of the proteins, their affinity for heparin, and the pattern of release indicate that these molecules are not chemokines.

Cross reactivity of three T cell attracting murine chemokines stimulating the CXC chemokine receptor CXCR3 and their induction in cultured cells and during allograft rejection.

Recent work identified the murine gene homologous to the human T cell attracting chemokine CXC receptor ligand 11 (CXCL11, also termed I-TAC, SCYB11, ss-R1, H174, IP-9). Here, the biological activity and expression patterns of murine CXCL11 relative to CXCL9 (MIG) and CXCL10 (IP-10/crg-2), the other two CXCR3 ligands, were assessed. Calcium mobilization and chemotaxis experiments demonstrated that murine CXCL11 stimulated murine CXCR3 at much lower doses than murine CXCL9 or murine CXCL10. Murine CXCL11 also evoked calcium mobilization in CHO cells transfected with human CXCR3 and was chemotactic for CXCR3-expressing human T lymphocytes as well as for 300--19 pre-B cells transfected with human or murine CXCR3. Moreover, murine CXCL11 blocked the chemotactic effect of human CXCL11 on human CXCR3 transfectants. Depending on cell type (macrophage-like cells RAW264.7, J774A.1, fetal F20 and adult dermal fibroblasts, immature and mature bone marrow-derived dendritic cells) and stimulus (interferons, LPS, IL-1 beta and TNF-alpha), an up to 10,000-fold increase of CXCL9, CXCL10 and CXCL11 mRNA levels, quantified by real-time PCR, was observed. In vivo, the three chemokines are constitutively expressed in various tissues from healthy BALB/c mice and were strongly up-regulated during rejection of allogeneic heart transplants. Chemokine mRNA levels exceeded those of CXCR3 and IFN-gamma which were induced with similar kinetics by several orders of magnitude.

SDF-1 alpha induces chemotaxis and enhances Sonic hedgehog-induced proliferation of cerebellar granule cells.

The chemokine SDF-1 alpha (CXC12) and its receptor CXCR4 have been shown to play a role in the development of normal cerebellar cytoarchitecture. We report here that SDF-1 alpha both induces chemotactic responses in granule precursor cells and enhances granule cell proliferative responses to Sonic hedgehog. Chemotactic and proliferative responses to SDF-1 alpha are greater in granule cells obtained from cerebella of animals in the first postnatal week, coinciding with the observed in vivo peak in cerebellar CXCR4 expression. SDF-1 alpha activation of neuronal CXCR4 differs from activation of CXCR4 in leukocytes in that SDF-1 alpha-induced calcium flux is activity dependent, requiring predepolarization with KCl or pretreatment with glutamate. However, as is the case in leukocytes, neuronal responses to SDF-1 alpha are all abolished by pretreatment of granule cells with pertussis toxin, suggesting they occur through G(alpha i) activation. In conclusion, SDF-1 alpha plays a role in two important processes of granule cell maturation - proliferation and migration - assisting in the achievement of appropriate cell number and position in the cerebellar cortex.

CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1-mediated demyelinating disease of the CNS that serves as a model for multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific and Ag-nonspecific T lymphocytes into the CNS. In the present report, we investigated the role of the CXC chemokine CXCL10 (IFN-gamma-inducible protein-10) in the pathogenesis of EAE. Production of CXCL10 in the CNS correlated with the development of clinical disease. Administration of anti-CXCL10 decreased clinical and histological disease incidence, severity, as well as infiltration of mononuclear cells into the CNS. Anti-CXCL10 specifically decreased the accumulation of encephalitogenic PLP(139-151) Ag-specific CD4+ T cells in the CNS compared with control-treated animals. Anti-CXCL10 administration did not affect the activation of encephalitogenic T cells as measured by Ag-specific proliferation and the ability to adoptively transfer EAE. These results demonstrate an important role for the CXC chemokine CXCL10 in the recruitment and accumulation of inflammatory mononuclear cells during the pathogenesis of EAE.

Eotaxin expression after segmental allergen challenge in subjects with atopic asthma.

Expression of pulmonary eotaxin protein and mRNA was determined in six subjects with atopic asthma and five nonatopic normal subjects. Levels of eotaxin expression and eosinophil mobilization were compared before and after segmental allergen challenge in subjects with atopic asthma. In the absence of allergen challenge, we found significantly higher levels of eotaxin in the bronchoalveolar lavage (BAL) fluid of subjects with asthma than in that of normal subjects (25 +/- 3 versus 15 +/- 2 pg/ml, p < 0.05). BAL eotaxin levels increased after segmental allergen challenge in all six subjects with atopic asthma tested, with a mean increase from 22 +/- 4 to 53 +/- 10 pg/ml (p = 0.013). Segmental allergen challenge was associated with a significant increase in the percentage of BAL macrophages and eosinophils that were immunopositive for eotaxin. Eotaxin mRNA was detectable by northern analysis in BAL cells exclusively from allergen-challenged segments. Allergen- induced increases in eotaxin levels were strongly associated with increases in BAL eosinophil recovery (r(2) = 0.88, p = 0.0036). Segmental allergen challenge also increased eotaxin expression in airway epithelial and endothelial cells obtained by endobronchial biopsy. These findings demonstrate, for the first time, that the airways of subjects with allergic asthma respond to allergen by increasing eotaxin expression. The tissue loci of eotaxin expression, the levels of eotaxin recovered in BAL fluid, and the association of eotaxin levels with eosinophil mobilization suggest either that eotaxin plays a mechanistic role in allergen-induced airway eosinophilia or that it serves as a biomarker for the causal mechanisms.

Signal transducer and activator of transcription 6 controls chemokine production and T helper cell type 2 cell trafficking in allergic pulmonary inflammation.

Antigen-specific CD4 T helper type 2 (Th2) cells play a pivotal role in the induction of allergic asthma, but the mechanisms regulating their recruitment into the airways are unknown. Signal transducer and activator of transcription factor (Stat)6 is a transcription factor essential for Th2 cell differentiation. Here we show that Stat6 also controls Th2 cell recruitment and effector function in allergic inflammation in vivo. To isolate the role of Stat6 in regulating Th2 cell trafficking and effector function from its role in Th2 cell differentiation, we used a murine model of asthma in which in vitro-differentiated Stat6(+/+) antigen-specific Th2 cells were adoptively transferred into naive Stat6(-/-) and Stat6(+/+) mice followed by aerosol antigen challenge. We found that all of the features of asthma, including Th2 cell accumulation, Th2 and eosinophil-active chemokine production, and airway eosinophilia, mucus production, and hyperresponsiveness seen in Stat6(+/+) mice, were dramatically absent in Stat6(-/)- mice that received Stat6(+/)+ antigen-specific Th2 cells. Our findings establish Stat6 as essential for Th2 cell trafficking and effector function and suggest that interruption of Stat6 signaling in resident cells of the lung is a novel approach to asthma therapy.

IFN-gamma-inducible protein-10 is essential for the generation of a protective tumor-specific CD8 T cell response induced by single-chain IL-12 gene therapy.

The successful induction of T cell-mediated protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. Here, we demonstrate that the induction of tumor-protective immunity by IL-12 in a murine neuroblastoma model depends entirely on the CXC chemokine IFN-gamma-inducible protein 10 (IP-10). This was established by in vivo depletion of IP-10 with mAbs in mice vaccinated against NXS2 neuroblastoma by gene therapy with a linearized, single-chain (sc) version of the heterodimeric cytokine IL-12 (scIL-12). The efficacy of IP-10 depletion was indicated by the effective abrogation of scIL-12-mediated antiangiogenesis and T cell chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2 cells. These findings were extended by data demonstrating that IP-10 is directly involved in the generation of a tumor-protective CD8+ T cell-mediated immune response during the early immunization phase. Four lines of evidence support this contention: First, A/J mice vaccinated with NXS2 scIL-12 and depleted of IP-10 by two different anti-IP-10 mAbs revealed an abrogation of systemic-protective immunity against disseminated metastases. Second, CD8+ T cell-mediated MHC class I Ag-restricted tumor cell lysis was inhibited in such mice. Third, intracellular IFN-gamma expressed by proliferating CD8+ T cells was substantially inhibited in IP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth, systemic tumor protective immunity was completely abrogated in mice depleted of IP-10 in the early immunization phase, but not if IP-10 was depleted only in the effector phase. These findings suggest that IP-10 plays a crucial role during the early immunization phase in the induction of immunity against neuroblastoma by scIL-12 gene therapy.

CXCR-4 desensitization is associated with tissue localization of hemopoietic progenitor cells.

The chemokine stroma-derived factor (SDF)-1, and its receptor, CXCR-4, have been shown to be essential for the translocation of hemopoietic stem cells from the fetal liver to the bone marrow (BM). We hypothesized that if CXCR-4 plays a crucial role in the localization of human hemopoiesis, stem cells from distinct tissue sources should demonstrate distinct CXCR-4 expression or signaling profiles. CD34(+) cells from BM were compared with blood: either mobilized peripheral blood or umbilical cord blood. Unexpectedly, significantly higher levels of CXCR-4 surface expression on CD34(+) cells from blood sources, mobilized peripheral blood, or cord blood were observed compared with BM (p = 0.0005 and p = 0.002, respectively). However, despite lower levels of CXCR-4, responsiveness of the cells to SDF-1 as measured by either calcium flux or transmigration was proportionally greatest in cells derived from BM. Further, internalization of CXCR-4 in response to ligand, associated with receptor desensitization, was significantly lower on BM-derived cells. Therefore, preserved chemokine receptor signaling was highly associated with marrow rather than blood localization. To test the functional effects of perturbing CXCR-4 signaling, adult mice were exposed to the methionine-SDF-1beta analog that induces prolonged down-regulation/desensitization of CXCR-4 and observed mobilization of Lin(-), Sca-1(+), Thy-1(low), and c-kit(+) hemopoietic progenitor cells to the peripheral blood with a >30-fold increase compared with PBS control (p = 0.0007 day 1 and p = 0.004 day 2). These data demonstrate that CXCR-4 expression and function can be dissociated in progenitor cells and that desensitization of CXCR-4 induces stem cell entry into the circulation.

Donor-derived IP-10 initiates development of acute allograft rejection.

An allograft is often considered an immunologically inert playing field on which host leukocytes assemble and wreak havoc. However, we demonstrate that graft-specific physiologic responses to early injury initiate and promulgate destruction of vascularized grafts. Serial analysis of allografts showed that intragraft expression of the three chemokine ligands for the CXC chemo-kine receptor CXCR3 was induced in the order of interferon (IFN)-gamma-inducible protein of 10 kD (IP-10, or CXCL10), IFN-inducible T cell alpha-chemoattractant (I-TAC; CXCL11), and then monokine induced by IFN-gamma (Mig, CXCL9). Initial IP-10 production was localized to endothelial cells, and only IP-10 was induced by isografting. Anti-IP-10 monoclonal antibodies prolonged allograft survival, but surprisingly, IP-10-deficient (IP-10(-/-)) mice acutely rejected allografts. However, though allografts from IP-10(+/+) mice were rejected by day 7, hearts from IP-10(-/-) mice survived long term. Compared with IP-10(+/+) donors, use of IP-10(-/-) donors reduced intragraft expression of cytokines, chemokines and their receptors, and associated leukocyte infiltration and graft injury. Hence, tissue-specific generation of a single chemokine in response to initial ischemia/reperfusion can initiate progressive graft infiltration and amplification of multiple effector pathways, and targeting of this proximal chemokine can prevent acute rejection. These data emphasize the pivotal role of donor-derived IP-10 in initiating alloresponses, with implications for tissue engineering to decrease immunogenicity, and demonstrate that chemokine redundancy may not be operative in vivo.

Expression of IFN-gamma-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in TH1- and TH2-mediated lung diseases.

BACKGROUND: Chemokines are involved in the influx of leukocytes into the airways in inflammatory lung diseases. The differential cell recruitment characteristic of T(H)1 versus T(H)2 immune responses may be associated with differential chemokine expression. OBJECTIVE: We investigated the expression of chemokines; monocyte chemotactic proteins (MCPs) 1, 3, and 4; eotaxin; and IFN-gamma-inducible protein 10 (IP-10) in both T(H)1- and T(H)2-mediated lung diseases. METHODS: By using immunocytochemistry and in situ hybridization, we examined the protein and mRNA expression, respectively, in bronchoalveolar lavage and biopsy samples in subjects with asthma, tuberculosis, sarcoidosis, and chronic bronchitis. RESULTS: Increased immunoreactivity and mRNA expression of IP-10 and of the MCPs was found in the bronchoalveolar lavage fluid and biopsy specimens of subjects with asthma and tuberculosis compared with that of control subjects (P <.005). IP-10, however, was particularly increased in subjects with sarcoidosis (P <.001). Eotaxin, on the other hand, was increased only in patients with asthma when compared with control subjects (P <.005). CONCLUSION: This study demonstrates that MCP-1, MCP-3, and MCP-4 expression is not specifically associated with lung diseases characterized by a particular cytokine profile. In contrast, IP-10 is mostly expressed in T(H)1-mediated diseases, and eotaxin expression seems to be specifically associated with lung diseases of a T(H)2 cytokine profile.

The CC chemokines MDC and TARC induce platelet activation via CCR4.

While chemokines have received considerable attention for their role in leukocyte chemotaxis, their effects on platelets have not been well described. We found that two CC chemokine receptor 4 (CCR4) ligands, macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC) induce concentration-dependent platelet aggregation and calcium flux. Flow cytometric analysis revealed the expression of CCR4 on platelets and a monoclonal antibody (mAb) to CCR4 inhibited MDC- and TARC-induced platelet aggregation, confirming that this effect is mediated through their common receptor CCR4. MDC fully desensitized TARC-induced calcium mobilization in platelets, while TARC was unable to completely desensitize a subsequent MDC response, which is similar to observations made in Th2 CD4(+) lymphocytes and CCR4-transfected cells. Aspirin (ASA) treatment of platelets allowed reversible primary aggregation but inhibited irreversible complete aggregation, suggesting that MDC- and TARC-induced full platelet aggregation is dependent on cyclooxygenase metabolites of arachidonic acid. MDC and TARC were unable to induce platelet aggregation and platelet secretion in washed human platelets, even though they induced a calcium flux, suggesting that plasma components are required for MDC- and TARC-induced platelet aggregation. Since Th2-type cytokines induce the release of MDC and TARC from cells and the expression of these chemokines is increased in Th2-type inflammation, we hypothesize that MDC and TARC may play a role in platelet activation seen in Th2 diseases, such as asthma and atopic dermatitis.