CXCR3-mediated opposite effects of CXCL10 and CXCL4 on TH1 or TH2 cytokine production

BACKGROUND: Two variants of the CXCR3 receptor exist, one (CXCR3-A) reactive with CXCL9, CXCL10, and CXCL11 and the other (CXCR3-B) also reactive with CXCL4. Both variants are contemporarily expressed by human T cells. OBJECTIVE: We sought to investigate the in vitro effects of CXCL10 and CXCL4 on the production of TH1 or TH2 cytokines. METHODS: The cytokine profile of antigen-specific human CD4+ T-cell lines obtained in the absence or presence of CXCL10 or CXCL4 was evaluated by means of quantitative RT-PCR, flow cytometry, and ELISA. RESULTS: CXCL10 upregulated IFN-gamma and downregulated IL-4, IL-5, and IL-13 production, whereas CXCL4 downregulated IFN-gamma and upregulated TH2 cytokines. Similar effects were also observed on polyclonally activated pure naive CD4+ T cells. The opposite effects of CXCL10 and CXCL4 on TH1 and TH2 cytokine production were inhibited by an anti-CXCR3 antibody able to neutralize both CXCR3-A and CXCR3-B and were apparently related to the activation of distinct signal transduction pathways. Moreover, CXCL10 upregulated mRNA levels of T-box expressed in T cells and downregulated GATA-3 expression, whereas CXCL4 downregulated T-box expressed in T cells and upregulated GATA-3. Finally, CXCL4, but not CXCL10, induced direct activation of IL-5 and IL-13 promoters. CONCLUSION: CXCL10 and CXCL4 exert opposite effects on the production of human TH1 and TH2 cytokines, likely through their respective interaction with CXCR3-A or CXCR3-B and the consequent activation of different signal transduction pathways. This might represent an internal regulatory pathway of TH cell responses and might contribute to the modulation of chronic inflammatory reactions, including allergy.     

CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11

The chemokine receptor CXCR3 is predominantly expressed on T lymphocytes, and its agonists CXCL9, CXCL10 and CXCL11 are IFN-gamma-inducible chemokines that promote Th1 responses. In contrast, the CCR3 agonists CCL11, CCL24 and CCL26 are involved in the recruitment of cells such as eosinophils and basophils during Th2 responses. Here, we report that although CCL11, CCL24 and CCL26 are neither agonists nor antagonists of CXCR3, CCL11 binds with high affinity to CXCR3. This suggests that, in vivo, CXCR3 may act as a decoy receptor, sequestering locally produced CCL11. We also demonstrate that the CXCR3 ligands inhibit CCR3-mediated functional responses of both human eosinophils and CCR3 transfectants induced by all three eotaxins, with CXCL11 being the most efficacious antagonist. The examination of CCR3-CCR1 chimeric constructs revealed that CCL11 and CXCL11 share overlapping binding sites contained within the CCR3 extracellular loops, a region that was previously shown to be essential for effective receptor-activation. Hence, eosinophil responses mediated by chemokines acting at CCR3 may be regulated by two distinct mechanisms: the antagonistic effects of CXCR3 ligands and the sequestration of CCL11 by CXCR3-expressing cells. Such interplay may serve to finely tune inflammatory responses in vivo.     

CXCL4-induced migration of activated T lymphocytes is mediated by the chemokine receptor CXCR3

The chemokine CXCL4/platelet factor-4 is released by activated platelets in micromolar concentrations and is a chemoattractant for leukocytes via an unidentified receptor. Recently, a variant of the human chemokine receptor CXCR3 (CXCR3-B) was described, which transduced apoptotic but not chemotactic signals in microvascular endothelial cells following exposure to high concentrations of CXCL4. Here, we show that CXCL4 can induce intracellular calcium release and the migration of activated human T lymphocytes. CXCL4-induced chemotaxis of T lymphocytes was inhibited by a CXCR3 antagonist and pretreatment of cells with pertussis toxin (PTX), suggestive of CXCR3-mediated G-protein signaling via Galphai-sensitive subunits. Specific binding by T lymphocytes of the CXCR3 ligand CXCL10 was not effectively competed by CXCL4, suggesting that the two are allotopic ligands. We subsequently used expression systems to dissect the potential roles of each CXCR3 isoform in mediating CXCL4 function. Transient expression of the CXCR3-A and CXCR3-B isoforms in the murine pre-B cell L1.2 produced cells that migrated in response to CXCL4 in a manner sensitive to PTX and a CXCR3 antagonist. Binding of radiolabeled CXCL4 to L1.2 CXCR3 transfectants was of low affinity and appeared to be mediated chiefly by glycosaminoglycans (GAGs), as no specific CXCL4 binding was observed in GAG-deficient 745-Chinese hamster ovary cells stably expressing CXCR3. We suggest that following platelet activation, the CXCR3/CXCL4 axis may play a role in T lymphocyte recruitment and the subsequent amplification of inflammation observed in diseases such as atherosclerosis. In such a setting, antagonism of the CXCR3/CXCL4 axis may represent a useful, therapeutic intervention.     

Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation

CXCR3 is a chemokine receptor predominantly expressed on T lymphocytes, and binds the chemokines CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC). Here, we have investigated the role of the extracellular domains of CXCR3 in ligand selectivity and receptor activation by assessing the ligand binding and chemotactic responses of chimeric CXCR3/CXCR1 constructs. Our data reveal that the secondextracellular loop of CXCR3 is essential for receptor activation in response to all CXCR3 ligands. In contrast, the N terminus and first extracellular loop of CXCR3 play some role in CXCL10- and CXCL11-mediated activation but are dispensable for CXCL9-induced signaling. The third extracellular loop of CXCR3 is important only for CXCL9- and CXCL10-induced chemotaxis. Binding studies suggest that the CXCR3 ligands bind to distinct sites composed of multiple domains of CXCR3 and that high-affinity binding and receptor activation are disparate functions. Collectively, our data support a multi-site model for CXCR3 interactions with its agonists, in which several extracellular domains of CXCR3 contribute to ligand binding and the induction of receptor activation. The development of antagonists targeting the second extracellular loop of CXCR3 should impede receptor activation and aid the treatment of several human inflammatory disorders.     

A crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12-triggered integrin activation but not in chemokine-triggered motility of human T lymphocytes and CD34+ cells

The chemokine CXCL12 promotes migration of human leukocytes, hematopoietic progenitors, and tumor cells. The binding of CXCL12 to its receptor CXCR4 triggers Gi protein signals for motility and integrin activation in many cell types. CXCR7 is a second, recently identified receptor for CXCL12, but its role as an intrinsic G-protein-coupled receptor (GPCR) has been debated. We report that CXCR7 fails to support on its own any CXCL12-triggered integrin activation or motility in human T lymphocytes or CD34(+) progenitors. CXCR7 is also scarcely expressed on the surface of both cell types and concentrates right underneath the plasma membrane with partial colocalization in early endosomes. Nevertheless, various specific CXCR7 blockers get access to this pool and attenuate the ability of CXCR4 to properly rearrange by surface-bound CXCL12, a critical step in the ability of the GPCR to trigger optimal CXCL12-mediated stimulation of integrin activation in T lymphocytes as well as in CD34(+) cells. In contrast, CXCL12-triggered CXCR4 signaling to early targets, such as Akt as well as CXCR4-mediated chemotaxis, is insensitive to identical CXCR7 blocking. Our findings suggest that although CXCR7 is not an intrinsic signaling receptor for CXCL12 on lymphocytes or CD34(+) cells, its blocking can be useful for therapeutic interference with CXCR4-mediated activation of integrins.     

Antagonism of the chemokine receptors CXCR3 and CXCR4 reduces the pathology of experimental autoimmune encephalomyelitis

Abstract Chemokines regulate lymphocyte trafficking under physiologic and pathologic conditions. In this study, we have investigated the role of CXCR3 and CXCR4 in the activation of T lymphocytes and their migration to the central nervous system (CNS) using novel mutant chemokines to antagonize CXCR3 and CXCR4 specifically. A series of truncation mutants of CXCL11, which has the highest affinity for CXCR3, were synthesized, and an antagonist, CXCL11((4-79)), was obtained. CXCL11((4-79)) strongly inhibited the migration of activated mouse T cells in response to all three high-affinity CXCR3 ligands, CXCL9, 10 and 11. CXCL12((P2G2)), while exhibiting minimal agonistic activity, potently inhibited the migration of activated mouse T cells in response to CXCL12. Interfering with the action of CXCR3 and CXCR4 with these synthetic receptor antagonists inhibited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis and reduced the accumulation of CD4(+) T cells in the CNS. Further investigation demonstrated that CXCL12((P2G2)) inhibited the sensitization phase, whereas CXCL11((4-79)) inhibited the effector phase of the immune response. Our data suggest that simultaneous targeting of CXCR4 and CXCR3 may be of benefit in the treatment of the CNS autoimmune disease.     

Chemokines and other GPCR ligands synergize in receptor-mediated migration of monocyte-derived immature and mature dendritic cells

Dendritic cells (DCs) are potent antigen presenting cells, described as the initiators of adaptive immune responses. Immature monocyte-derived DCs (MDDC) showed decreased CD14 expression, increased cell surface markers DC-SIGN and CD1a and enhanced levels of receptors for the chemokines CCL3 (CCR1/CCR5) and CXCL8 (CXCR1/CXCR2) compared with human CD14⁺ monocytes. After further MDDC maturation by LPS, the markers CD80 and CD83 and the chemokine receptors CXCR4 and CCR7 were upregulated, whereas CCR1, CCR2 and CCR5 expression was reduced. CCL3 dose-dependently synergized with CXCL8 or CXCL12 in chemotaxis of immature MDDC. CXCL12 augmented the CCL3-induced ERK1/2 and Akt phosphorylation in immature MDDC, although the synergy between CCL3 and CXCL12 in chemotaxis of immature MDDC was dependent on the Akt signaling pathway but not on ERK1/2 phosphorylation. CCL2 also synergized with CXCL12 in immature MDDC migration. Moreover, two CXC chemokines not sharing receptors (CXCL12 and CXCL8) cooperated in immature MDDC chemotaxis, whereas two CC chemokines (CCL3 and CCL7) sharing CCR1 did not. Further, the non-chemokine G protein-coupled receptor ligands chemerin and fMLP synergized with respectively CCL7 and CCL3 in immature MDDC signaling and migration. Finally, CXCL12 and CCL3 did not cooperate, but CXCL12 synergized with CCL21 in mature MDDC chemotaxis. Thus, chemokine synergy in immature and mature MDDC migration is dose-dependently regulated by chemokines via alterations in their chemokine receptor expression pattern according to their role in immune responses.     

Slit-2/Robo-1 modulates the CXCL12/CXCR4-induced chemotaxis of T cells

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal, dendritic, and leukocyte migration. However, the molecular mechanism by which the Slit/Robo complex inhibits the migration of cells is not well defined. Here, we showed that Slit-2 can inhibit the CXCL12-induced chemotaxis and transendothelial migration of T cells and monocytes. We observed that CXCR4 associates with Robo-1 and that Slit-2 treatment enhances this association with the Robo-1 receptor. Robo-1 is a single-pass transmembrane receptor whose intracellular region contains four conserved motifs designated as CC0, CC1, CC2, and CC3. Structural and functional analyses of Robo receptors revealed that interaction of the CC3 motif with the CXCR4 receptor may regulate the CXCL12-induced chemotaxis of T cells. We further characterized Slit-2-mediated inhibition of the CXCL12/CXCR4 chemotactic pathway and found that Slit-2 can block the CXCL12-induced activation of the Src and Lck kinases but not Lyn kinase. Although Slit-2 did not inhibit the CXCL12-induced activation of MAPKs, it did inhibit the Akt phosphorylation and Rac activation induced by this chemokine. Altogether, our studies indicate a novel mechanism by which the Slit/Robo complex may inhibit the CXCR4/CXCL12-mediated chemotaxis of T cells.     

Tumor-associated macrophages promote the metastasis of ovarian carcinoma cells by enhancing CXCL16/CXCR6 expression

This study investigated the underlying mechanism by which C-X-C motif chemokine ligand 16 (CXCL16)/C-X-C motif chemokine receptor 6 (CXCR6) signaling is activated by tumor-associated macrophages and assists in regulating the metastasis of ovarian carcinoma. Specimens of ovarian carcinoma tissue and adjacent tissue were collected from 20 ovarian carcinoma patients. Human THP-1 cells were induced to differentiate into macrophages, which were then co-cultured with SKOV3 cells and low concentrations of tumor necrosis factor-α (TNF-α) to simulate the inflammatory microenvironment of ovarian carcinoma. Additionally, small interfering RNA (siRNA) targeting CXCR6 was transfected into SKOV3 cells; after which, the levels of nuclear factor kappa B p65 (NF-κB p65) protein and phosphorylated PI3K and Akt were measured. The migration and invasion abilities of the SKOV3 cells were also tested. The levels of TNF-α, interluekin-6 (IL-6), NF-κB p65, CXCL16, and CXCR6 expression in the ovarian carcinoma tissues were higher than those in the precancerous tissues. CXCR6 expression was positively correlated with TNF-α, IL-6, and CXCL16 expression. Co-culture of SKOV3 cells with macrophages significantly promoted CXCL16, CXCR6, NF-κB, and p65 expression by the SKOV3 cells, increased their levels of phosphorylated PI3K and Akt, and increased the migration and invasion abilities of SKOV3 cells. Silencing of CXCR6 or blocking the PI3K/Akt signal pathway markedly attenuated the expression of NF-κB p65 and phosphorylation of PI3K and Akt, as well as the migration and invasion abilities of SKOV3 cells. These findings demonstrate that macrophages can promote the migration and invasion of ovarian carcinoma cells by affecting the CXCL16/CXCR6 pathway.     

Cbl and Akt regulate CXCL8-induced and CXCR1- and CXCR2-mediated chemotaxis

CXCL8 (IL-8) plays an important role in the pathogenesis of a variety of inflammatory diseases. However, little is known about the signaling pathways that regulate CXCL8-induced chemotaxis. Here, we found that CXCL8 treatment of CXCR1- and CXCR2-over-expressing L1.2 cells (CXCR1-L1.2 and CXCR2-L1.2, respectively) induced the phosphorylation of Cbl and Akt. The tyrosine kinase inhibitor Tyrphostin A9, phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 as well as proteasome inhibitors significantly blocked the CXCL8-induced chemotaxis of L1.2 cells and human neutrophils. We further found that stimulation with CXCL8 enhanced the association of the PI3K subunit p85 with Cbl. Additionally, over-expression of wild-type Cbl and G306E-Cbl (mutation in the tyrosine kinase-binding domain) inhibited chemotaxis by approximately 50% as compared with the vector control, whereas the 70Z mutant (deletion in the RING finger domain) did not reduce migration. However, wild-type Cbl or its mutants had no effect on the CXCL8-induced activation of MAPK, indicating that Cbl specifically modulated CXCL8-induced chemotaxis. Furthermore, over-expression of the kinase-dead Akt mutant decreased CXCL8-induced chemotaxis by 60% and diminished Cbl phosphorylation as compared with the vector control. The CXCL8-induced phosphorylation of Cbl was also reduced when cells were pre-treated with the PI3K inhibitor LY294002. Lastly, we have shown that pre-treatment of L1.2 cells with the proteasome inhibitor Lactacystin blocks CXCL8-induced internalization of the CXCR1 and CXCR2 receptors. These studies provide new information regarding CXCL8-induced signaling pathways that may regulate chemotaxis and receptor internalization.     

Interaction of the chemokines I-TAC (CXCL11) and SDF-1 (CXCL12) in the regulation of tumor angiogenesis of colorectal cancer

The chemokine CXCL12 has a decisive role in tumor progression by mediating pro-angiogenic and pro-metastatic effects through its receptor CXCR4. The CXCL12 pathway is connected with another chemokine, CXCL11, through its second receptor CXCR7. CXCL11 also binds to the CXCR3 receptor. CXCL11 function in tumor angiogenesis is likely receptor dependent because CXCR3 predominantly mediates angiostatic signals whereas CXCR7 mediated signaling is rather angiogenic. We therefore studied the interaction of CXCL12 and CXCL11 in an in vivo model of colorectal cancer metastasis. GFP-transfected CT26.WT colorectal cancer cells were implanted into the dorsal skinfold chamber of syngeneic BALB/c mice. The animals received either peritumoral application of CXCL11 or intraperitoneal injections with neutralizing antibodies against CXCL11, CXCL12 or both. Tumor growth characteristics, angiogenesis, cell migration, invasive tumor growth, tumor cell proliferation and apoptosis were studied by intravital fluorescence microscopy and immunohistochemistry during an observation period of 14 days. Local exposure to CXCL11 significantly stimulated tumor growth compared to controls and enhanced invasive growth characteristics without affecting tumor angiogenesis and tumor cell migration. Neither CXCL11 nor CXCL12-blockade had a significant impact on tumor growth and angiogenesis, whereas the combined neutralization of CXCL11 and CXCL12 almost completely abrogated tumor vessel formation. As a consequence, tumor growth and invasive growth characteristics were reduced compared to the other groups. The results of the present study underline the interaction of CXCL12 and CXCL11 during tumor angiogenesis. The combined blockade of both signaling pathways may provide an interesting anti-angiogenic approach for anti-tumor therapy.     

I-TAC/CXCL11 is a natural antagonist for CCR5

The selective CXC chemokine receptor 3 (CXCR3) agonists, monokine induced by interferon-gamma (IFN- gamma)/CXC chemokine ligand 9 (CXCL9), IFN-inducible protein 10/CXCL10, and IFN-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, attract CXCR3+ cells such as CD45RO+ T lymphocytes, B cells, and natural killer cells. Further, all three chemokines are potent, natural antagonists for chemokine receptor 3 (CCR3) and feature defensin-like, antimicrobial activities. In this study, we show that I-TAC, in addition to these effects, acts as an antagonist for CCR5. I-TAC inhibited the binding of macrophage-inflammatory protein-1alpha (MIP-1alpha)/CC chemokine ligand 3 (CCL3) to cells transfected with CCR5 and to monocytes. Furthermore, cell migration evoked by regulated on activation, normal T expressed and secreted (RANTES)/CCL5 and MIP-1beta/CCL4, the selective agonist of CCR5, was inhibited in transfected cells and monocytes, respectively. In two other functional assays, namely the release of free intracellular calcium and actin polymerization, I-TAC reduced CCR5 activities to minimal levels. Sequence and structure analyses indicate a potential role for K17, K49, and Q51 of I-TAC in CCR5 binding. Our results expand on the potential role of I-TAC as a negative modulator in leukocyte migration and activation, as I-TAC would specifically counteract the responses mediated by many "classical," inflammatory chemokines that act not only via CCR3 but via CCR5 as well.     

Expression of rat I-TAC/CXCL11/SCYA11 during central nervous system inflammation: comparison with other CXCR3 ligands

The chemokines are a large gene superfamily with critical roles in development and immunity. The chemokine receptor CXCR3 appears to play a major role in the trafficking of activated Th1 lymphocytes. There are at least three major ligands for CXCR3: mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, and of these three ligands, CXCL11 is the least well-characterized. In this study, we have cloned a rat ortholog of CXCL11, evaluated its function, and examined its expression in the Th-1-mediated disease, experimental autoimmune encephalomyelitis (EAE) in the rat. Based on its predicted primary amino-acid sequence, rat I-TAC/CXCL11 was synthesized and shown to induce chemotaxis of activated rat T lymphocytes in vitro and the in vivo migration of T lymphocytes when injected into the skin. I-TAC/CXCL11 expression, as determined by RT-PCR, increased in lymph node and spinal cord tissue collected from rats in which EAE had been actively induced, and in spinal cord tissue from rats in which EAE had been passively induced. The kinetics of expression were similar to that of CXCR3 and IP-10/CXCL10, although expression of both CXCR3 and IP-10/CXCL10 was more intense than that of I-TAC/CXCL11 and increased more rapidly in both lymph nodes and the spinal cord. Only minor levels of expression of the related chemokine mig/CXCL9 were observed. Immunohistochemistry revealed that the major cellular source of I-TAC/CXCL11 in the central nervous system (CNS) during EAE is likely to be the astrocyte. Together, these data indicate that I-TAC/CXCL11 is expressed in the CNS during the clinical phase of EAE. However, the observation that I-TAC/CXCL11 is expressed after receptor expression is detected suggests that it is not essential for the initial migration of CXCR3-bearing cells into the CNS.     

Cannabinoid receptor CB2 modulates the CXCL12/CXCR4-mediated chemotaxis of T lymphocytes

Cannabinoids have been shown to influence the immune system. However, their immunomodulatory effects have not been extensively studied. In this investigation, we have observed that both primary and Jurkat T cells express a functional cannabinoid receptor 2 (CB₂). Furthermore, both the synthetic cannabinoids CP55,940 and WIN55,212-2, as well as the CB₂-selective agonist JWH-015, caused a significant inhibition of the chemokine CXCL12-induced and CXCR4-mediated chemotaxis of Jurkat T cells, as well as their transendothelial migration. Involvement of the CB₂ receptor was further confirmed by partial reversal of the inhibition using the CB₂-specific antagonist, AM630. Similarly, CP55,940 and JWH-015 inhibited the CXCL12-induced chemotaxis of primary CD4⁺ and CD8⁺ T lymphocytes. Further investigation of signaling studies to delineate the mechanism of inhibition revealed that cannabinoids enhance CXCL12-induced p44/42 MAP kinase activity. However, enhanced MAP kinase activity was not responsible for the inhibition of chemotaxis. This suggests that cannabinoids differentially regulate CXCR4-mediated migration and MAP kinase activation in T cells. Cannabinoids were also found to downregulate the PMA-enhanced enzyme activity of matrix metalloproteinase-9, which is known to play an important role in transendothelial migration. This study provides novel information regarding cannabinoid modulation of functional effects in T cells.     

CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages

Interferon-inducible protein-10 (IP-10)/CXCL10, which is a ligand for CXC chemokine receptor 3 (CXCR3), is known to be involved in the pathogenesis of pulmonary sarcoidosis. However, the roles of monokine induced by interferon gamma (Mig)/CXCL9 and interferon-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, which are also CXCR3 ligands, remain unclear. Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 in both bronchoalveolar lavage fluid (BALF) and serum in patients with pulmonary sarcoidosis were measured by enzyme-linked immunosorbent assay (ELISA). The expression of these chemokines in alveolar macrophages was examined using ELISA, quantitative real-time polymerase chain reaction and immunostaining. In BALF, Mig/CXCL9 and IP-10/CXCL10 were significantly elevated in stage II sarcoidosis as compared with the levels in healthy volunteers. In serum, Mig/CXCL9 and I-TAC/CXCL11 were increased in stage II of the disease. The levels of all CXCR3 ligands in BALF were correlated with the numbers of both total and CD4(+) lymphocytes. Alveolar macrophages were stained positive for all CXCR3 ligands and produced increased amounts of these chemokines. Positive staining of the three chemokines was also observed in the epithelioid and giant cells in the sarcoid lungs. These findings suggest that Mig/CXCL9 and I-TAC/CXCL11 as well as IP-10/CXCL10 play important roles in the accumulation of Th1 lymphocytes in sarcoid lungs.     

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.     

Distribution and kinetics of SR-PSOX/CXCL16 and CXCR6 expression on human dendritic cell subsets and CD4+ T cells

Dendritic cells (DCs) coordinate T cell responses by producing T cell-attracting chemokines and by inducing the expression of chemokine receptors on T cells. Scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXC chemokine ligand 16 (CXCL16) is a unique chemokine that also functions as an endocytic receptor and an adhesion molecule in its membrane-bound form. SR-PSOX/CXCL16 is the only known ligand of CXC chemokine receptor 6 (CXCR6) that is expressed on activated T cells and thus, may play an important role in enhancing effector functions of T cells. Here, we investigated the expression of SR-PSOX/CXCL16 on human DC subsets and that of CXCR6 on T cell subpopulations to elucidate the dynamics of CXCL16/CXCR6 interaction in DC/T cell responses. Membrane-bound SR-PSOX/CXCL16 was expressed on macrophages, monocyte-derived DCs, and blood myeloid DCs, and the expression increased after DC maturation. Myeloid antigen-presenting cells constitutively secreted SR-PSOX/CXCL16 for an extended period, suggesting the involvement of CXCL16 in peripheral and lymphoid tissues. Plasmacytoid DCs hardly expressed SR-PSOX/CXCL16 on their surfaces but secreted significant amounts of SR-PSOX/CXCL16. A subset of CD4+ effector memory T (T(EM)) cells constitutively expressed CXCR6, whereas central memory T cells (T(CM)) and naive T cells did not. Upon stimulation with mature DCs, however, the expression of CXCR6 on T(CM) cells was markedly up-regulated, whereas the expression on naive T cells was induced only weakly. These results suggest that the interaction between SR-PSOX/CXCL16 and CXCR6 plays an important role in enhancing T(CM) cell responses by mature DCs in lymphoid tissues and in augmenting T(EM) cell responses by macrophages in peripheral inflamed tissues.     

Identification of p53 peptides recognized by CD8(+) T lymphocytes from patients with bladder cancer

In many types of cancer, p53 frequently accumulates in tumor cells and anti-p53 antibodies can be detected. However, only four CD8(+) T-cell epitopes from p53 have been identified in humans so far. To further analyze the development of a T-cell response against p53, peptides having binding motifs specific for HLA-A1, -A2, -A3, -A24, -B7, -B35, -B44, and -B51 molecules have been defined. The HLA-binding capacity of those peptides was tested, and the stability of formed complexes was defined. Thirteen peptides that bound to HLA-A24 and -B44 molecules are presented. The positive peptides were then used to detect the anti-p53 response of CD8(+) T lymphocytes from patients with bladder cancer. Six peptides, presented by HLA-A2, -B51, or -A24, were able to stimulate T cells from two patients (among 16) with tumor cells that strongly accumulated p53. On the contrary, p53 peptides systematically failed to stimulate T cells from healthy donors or patients with low or undetectable levels of p53 in their tumor cells. These results have led to the identification of four new potential T CD8(+) epitopes from p53: 194-203 associating with HLA-B51 and 204-212, 211-218, and 235-243 associating with HLA-A24.     

A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection

ABSTRACT: BACKGROUND: The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738. RESULTS: In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment. CONCLUSIONS: SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.     

IL-18 enhances IFN-gamma-induced production of CXCL9, CXCL10, and CXCL11 in human keratinocytes

IL-18 is involved in the pathogenesis of atopic dermatitis, psoriasis, and allergic contact dermatitis. CXCL9, CXCL10, and CXCL11 recruit type 1 T cells, and the production of these chemokines by keratinocytes is enhanced in these dermatoses. We examined the in vitro effects of IL-18 on IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production in human keratinocytes. IL-18 enhanced the IFN-gamma-induced secretion and mRNA expression of CXCL9, CXCL10, and CXCL11 in parallel to the activation of NF-kappaB, STAT1, and IFN-regulatory factor (IRF)-1. Antisense oligonucleotides against NF-kappaB p50, p65, or STAT1 suppressed CXCL9, CXCL10, and CXCL11 production, and antisense IRF-1 suppressed CXCL11 production. Inhibitors of PI3 K, p38 MAPK, and MEK suppressed IL-18 plus IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production and NF-kappaB, STAT1, and IRF-1 activities. IL-18 induced phosphorylation of ERK and Akt, while IFN-gamma induced phosphorylation of p38 MAPK. These results suggest that IL-18 may potentiate IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production in keratinocytes by activating NF-kappaB, STAT1, or IRF-1 through PI3 K/Akt and MEK/ERK pathways. These effects of IL-18 may promote the infiltration of type 1 T cells into lesions with inflammatory dermatoses and amplify the skin inflammation. IL-18 may act as a pro-inflammatory cytokine in these dermatoses and thus is a candidate therapeutic target.