TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.     

Chemokine receptor expression and function in CD4+ T lymphocytes with regulatory activity

We have investigated the chemokine receptor expression and migratory behavior of a new subset of nickel-specific skin-homing regulatory CD4(+) T cells (Th(IL-10)) releasing high levels of IL-10, low IFN-gamma, and undetectable IL-4. These cells inhibit in a IL-10-dependent manner the capacity of dendritic cells to activate nickel-specific Tc1 and Th1 lymphocytes. RNase protection assay and FACS analysis revealed the expression of a vast repertoire of chemokine receptors on resting Th(IL-10), including the Th1-associated CXCR3 and CCR5, and the Th2-associated CCR3, CCR4, and CCR8, the latter at higher levels compared with Th2 cells. The most active chemokines for resting Th(IL-10), in terms of calcium mobilization and in vitro migration, were in order of potency: CCL2 (monocyte chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1beta, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1alpha, CCR1/5 ligand), CCL17 (thymus and activation-regulated chemokine, CCR4 ligand), CCL1 (I-309, CCR8 ligand), CXCL12 (stromal-derived factor-1, CXCR4), and CCL11 (eotaxin, CCR3 ligand). Consistent with receptor expression down-regulation, activated Th(IL-10) exhibited a reduced or absent response to most chemokines, but retained a significant migratory capacity to I-309, monocyte chemoattractant protein-1, and thymus and activation-regulated chemokine. I-309, which was ineffective on Th1 lymphocytes, attracted more efficiently Th(IL-10) than Th2 cells. I-309 and CCR8 mRNAs were not detected in unaffected skin and were up-regulated at the skin site of nickel-allergic reaction, with an earlier expression kinetics compared with IL-10 and IL-4. Results indicate that skin-homing regulatory Th(IL-10) lymphocytes coexpress functional Th1- and Th2-associated chemokine receptors, and that CCR8/I-309-driven recruitment of both resting and activated Th(IL-10) cells may be critically involved in the regulation of Th1-mediated skin allergic disorders.     

Differential role of CCR2 in islet and heart allograft rejection: tissue specificity of chemokine/chemokine receptor function in vivo

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-gamma-inducible protein-10), and CXCL9 (monokine induced by IFN-gamma) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2-/-). A significant prolongation of islet allograft survival was noted in CCR2-/- recipients, with median survival time of 24 and 12 days for CCR2-/- and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62L(low)CD44(high)) in CCR2-/- compared with WT recipients. In addition, CCR2-/- recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2-/- recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.     

Chemotactic responsiveness toward ligands for CXCR3 and CXCR4 is regulated on plasma blasts during the time course of a memory immune response

Plasma blasts formed during memory immune responses emigrate from the spleen to migrate into the bone marrow and into chronically inflamed tissues where they differentiate into long-lived plasma cells. In this study, we analyze the chemokine responsiveness of plasma blasts formed after secondary immunization with OVA. Starting from day 4 and within approximately 48 h, OVA-specific plasma blasts emigrate from spleen and appear in the bone marrow. Although these migratory cells have lost their responsiveness to many B cell attracting chemokines, e.g., CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant), they migrate toward CXCL12 (stromal cell-derived factor 1 alpha), and toward the inflammatory chemokines CXCL9 (monokine induced by IFN-gamma), CXCL10 (IFN-gamma-inducible protein 10), and CXCL11 (IFN-inducible T cell alpha chemoattractant). However, the responsiveness of plasma blasts to these chemokines is restricted to a few days after their emigration from the spleen, indicating a role for these molecules and their cognate receptors, i.e., CXCR3 and CXCR4, in the regulation of plasma blast migration into the bone marrow and/or inflamed tissues.     

Peroxisome proliferator-activated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily originally shown to play an important role in adipocyte differentiation and glucose homeostasis, is now known to regulate inflammatory responses. Given the importance of endothelial cell (EC)-derived chemokines in regulating leukocyte function and trafficking, we studied the effects of PPARgamma ligands on the expression of chemokines induced in ECs by the Th1 cytokine IFN-gamma. Treatment of ECs with PPARgamma activators significantly inhibited IFN-gamma-induced mRNA and protein expression of the CXC chemokines IFN-inducible protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), whereas expression of the CC chemokine monocyte chemoattractant protein-1 was not altered. PPARgamma activators decreased IFN-inducible protein of 10 kDa promoter activity and inhibited protein binding to the two NF-kappaB sites but not to the IFN-stimulated response element ISRE site. Furthermore, PPARgamma ligands inhibited the release of chemotactic activity for CXC chemokine receptor 3 (CXCR3)-transfected lymphocytes from IFN-gamma-stimulated ECs. These data suggest that anti-diabetic PPARgamma activators might attenuate the recruitment of activated T cells at sites of Th1-mediated inflammation.     

Fas ligation and tumor necrosis factor α activation of murine astrocytes promote heat shock factor-1 activation and heat shock protein expression leading to chemokine induction and cell survival

Death-inducing ligands tumor necrosis factor alpha (TNFα) and Fas ligand (FasL) do not kill cultured astrocytes; instead they induce a variety of chemokines including macrophage-inflammatory protein-1α/CC chemokine ligand 3 (CCL3), monocyte chemoattractant protein-1 (CC CCL-2), macrophage-inflammatory protein-2/CXC chemokine ligand 2 (CXCL2, a murine homologue of interleukin 8), and interferon-induced protein of 10 kDa (CXCL10). Induction is enhanced by protein synthesis inhibition suggesting the existence of endogenous inhibitors. ERK, NF-κB, heat shock factor-1 (HSF-1) and heat shock proteins were examined for their possible roles in signal transduction. Inhibition of ERK activation by PD98059 partially inhibited expression of all but FasL-induced CXCL10. Although inhibition of NF-κB DNA binding inhibited chemokine induction, PD98059 did not inhibit TNFα-induced NF-κB DNA binding suggesting that ERK serves an NF-κB-independent pathway. Heat shock itself induced astrocytic chemokine expression; both TNFα and FasL induced HSF-1 DNA binding and Hsp72 production; and Hsp72-induced chemokine expression. Inhibition of either HSF-1 binding with quercetin or heat shock protein synthesis with KNK437 compromised chemokine induction without compromising cell survival. These data suggest that the induction of heat shock proteins via HSF-1 contribute to the TNFα- and FasL-induced expression of chemokines in astrocytes.     

Expression of macrophage-derived chemokine (MDC)/CCL22 in human lung cancer

Background: Ligands for CXCR3 chemokines [IFN-γ-inducible protein of 10 kD (IP-10/CXCL10), monokine induced by IFN-γ (Mig/CXCL9), IFN-inducible T cell α chemoattractant (I-TAC/CXCL11)] and those for CCR4 [macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17)] have been shown to play the central roles for T helper-cell recruitment into the tissues. To examine the role of these chemokines in tumor progression of lung cancer, we investigated their expression in human lung cancer tissues to determine the possible relationship between their expression and the prognosis of patients. Methods: Total RNA was prepared from lung cancer tissues of 40 patients (24 adenocarcinoma and 16 squamous cell carcinoma). We measured gene expression levels of chemokines (IP-10, Mig, I-TAC, MDC and TARC) by real-time quantitative RT-PCR. Results: Higher gene expression of MDC in lung cancer was significantly correlated with longer disease-free survival time and lower risk of recurrence after tumor resection. We could not find any significant relationship of IP-10, Mig, I-TAC and TARC gene expression with disease-free survival or lower risk of recurrence after surgery. Conclusions: These results suggest that increased gene expression of MDC in tumor tissues may be a predictive marker for improving the prognosis of lung cancer.Toru Nakanishi and Kazuyoshi Imaizumi equally contributed to this work.     

Chemokine receptor expressions and responsiveness of cord blood T cells

Chemokines and their receptors play a critical role in the selective attraction of various subsets of leukocytes. We examined the chemokine receptor expressions and responsiveness of cord blood (CB) T cells. Flow-cytometric analysis revealed that peripheral blood (PB) T cells expressed CCR-1, CCR-2, CCR-5, CCR-6, CXC chemokine receptor-3 (CXCR-3), and CXCR-4, while CB T cells expressed only CXCR-4 on their surface. Chemotactic migratory response of CB T cells to macrophage-inflammatory protein (MIP)-1alpha, monocyte chemoattractant protein-1, RANTES, MIP-3alpha, monokine induced by IFN-gamma, and IFN-gamma-inducible protein-10 was significantly impaired compared with those of PB T cells. In contrast, the ability of CB T cells to migrate to MIP-3beta, 6Ckine, and stromal cell-derived factor-1alpha was greater than that of PB T cells, and these events were correlated with the expression levels of CCR-7 and CXCR-4, respectively. Engagement of CD3 and CD28 specifically up-regulated CXCR-3 expression and chemotaxis to monokine induced by IFN-gamma and IFN-gamma-inducible protein-10, whereas this stimulation down-regulated CCR-7 expression and chemotaxis to MIP-3beta and 6Ckine in PB T cells, but not in CB T cells. These results suggest that PB T cells and CB T cells exhibit distinct chemokine responsiveness via different chemokine receptor repertoire.     

CCL7 and CXCL10 orchestrate oxidative stress-induced neutrophilic lung inflammation.

Oxidative stress from ozone (O(3)) exposure augments airway neutrophil recruitment and chemokine production. We and others have shown that severe and sudden asthma is associated with airway neutrophilia, and that O(3) oxidative stress is likely to augment neutrophilic airway inflammation in severe asthma. However, very little is known about chemokines that orchestrate oxidative stress-induced neutrophilic airway inflammation in vivo. To identify these chemokines, three groups of BALB/c mice were exposed to sham air, 0.2 ppm O(3), or 0.8 ppm O(3) for 6 h. Compared with sham air, 0.8 ppm O(3), but not 0.2 ppm O(3), induced pronounced neutrophilic airway inflammation that peaked at 18 h postexposure. The 0.8 ppm O(3) up-regulated lung mRNA of CXCL1,2,3 (mouse growth-related oncogene-alpha and macrophage-inflammatory protein-2), CXCL10 (IFN-gamma-inducible protein-10), CCL3 (macrophage-inflammatory protein-1alpha), CCL7 (monocyte chemoattractant protein-3), and CCL11 (eotaxin) at 0 h postexposure, and expression of CXCL10, CCL3, and CCL7 mRNA was sustained 18 h postexposure. O(3) increased lung protein levels of CXCL10, CCL7, and CCR3 (CCL7R). The airway epithelium was identified as a source of CCL7. The role of up-regulated chemokines was determined by administering control IgG or IgG Abs against six murine chemokines before O(3) exposure. As expected, anti-mouse growth-related oncogene-alpha inhibited neutrophil recruitment. Surprisingly, Abs to CCL7 and CXCL10 also decreased neutrophil recruitment by 63 and 72%, respectively. These findings indicate that CCL7 and CXCL10, two chemokines not previously reported to orchestrate neutrophilic inflammation, play a critical role in mediating oxidative stress-induced neutrophilic airway inflammation. These observations may have relevance in induction of neutrophilia in severe asthma.     

IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking

IFN-gamma-inducible protein 10 (IP-10, CXCL10), a chemokine secreted from cells stimulated with type I and II IFNs and LPS, is a chemoattractant for activated T cells. Expression of IP-10 is seen in many Th1-type inflammatory diseases, where it is thought to play an important role in recruiting activated T cells into sites of tissue inflammation. To determine the in vivo function of IP-10, we constructed an IP-10-deficient mouse (IP-10(-/-)) by targeted gene disruption. Immunological analysis revealed that IP-10(-/-) mice had impaired T cell responses. T cell proliferation to allogeneic and antigenic stimulation and IFN-gamma secretion in response to antigenic challenge were impaired in IP-10(-/-) mice. In addition, IP-10(-/-) mice exhibited an impaired contact hypersensitivity response, characterized by decreased ear swelling and reduced inflammatory cell infiltrates. T cells recovered from draining lymph nodes also had a decreased proliferative response to Ag restimulation. Furthermore, IP-10(-/-) mice infected with a neurotropic mouse hepatitis virus had an impaired ability to control viral replication in the brain. This was associated with decreased recruitment of CD4(+) and CD8(+) lymphocytes into the brain, reduced levels of IFN-gamma and the IFN-gamma-induced chemokines monokine induced by IFN-gamma (Mig, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11) in the brain, decreased numbers of virus-specific IFN-gamma-secreting CD8(+) cells in the spleen, and reduced levels of demyelination in the CNS. Taken together, our data suggest a role for IP-10 in both effector T cell generation and trafficking in vivo.     

CC-chemokine ligand 16 induces a novel maturation program in human immature monocyte-derived dendritic cells

Dendritic cells (DCs) are indispensable for initiation of primary T cell responses and a host's defense against infection. Many proinflammatory stimuli induce DCs to mature (mDCs), but little is known about the ability of chemokines to modulate their maturation. In the present study, we report that CCL16 is a potent maturation factor for monocyte-derived DCs (MoDCs) through differential use of its four receptors and an indirect regulator of Th cell differentiation. MoDCs induced to mature by CCL16 are characterized by increased expression of CD80 and CD86, MHC class II molecules, and ex novo expression of CD83 and CCR7. They produce many chemokines to attract monocytes and T cells and are also strong stimulators in activating allogeneic T cells to skew toward Th1 differentiation. Interestingly, they are still able to take up Ag and express chemokine receptors usually bound by inflammatory ligands and can be induced to migrate to different sites where they capture Ags. Our findings indicate that induction of MoDC maturation is an important property of CCL16 and suggest that chemokines may not only organize the migration of MoDCs, but also directly regulate their ability to prime T cell responses.     

Regulation of conceptus adhesion by endometrial CXC chemokines during the implantation period in sheep

To gain a better understanding of biochemical mechanisms of conceptus adhesion to the maternal endometrium in ruminant ungulates, the present study was performed to clarify roles of chemokines and extracellular matrix (ECM) components in the regulation of ovine blastocyst attachment to the endometrium. In addition to the chemokine, interferon-gamma inducible protein 10 kDa (IP-10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN-gamma (MIG, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11). Similar to CXCL10, CXCL9, and CXCL11 were expressed in the uterus during the peri-implantation period, and CXCL9 mRNA expression was stimulated in endometrial explants from day 14 cyclic ewes by the addition of IFN-tau or IFN-gamma. Without ECM components, conceptus cell adhesion was low on day 14 of gestation and exhibited a 2.5-fold increase on day 17; adhesiveness on day 20 was 1/10 of that on day 14. Among various ECM components examined, trophoblast adhesion was greatest when fibronectin was used. Although day 14 conceptuses did not show much adhesive activity to fibronectin, day 17 trophoblast, and day 20 chorionic membrane exhibited 2.3-fold and 50-fold increase, respectively, which was enhanced by treatment with CXCL9 or CXCL10. These results indicate that through endometrial fibronectin and chemokines, ovine conceptus cells gain the ability to attach to the endometrium during pre-implantation period; however, elucidation of molecular mechanisms by which the conceptus acquires the adhesive ability during this time period awaits further investigation.     

Critical role for CXCR3 chemokine biology in the pathogenesis of bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival post-lung transplantation and is characterized by a persistent peribronchiolar inflammation that eventually gives way to airway fibrosis/obliteration. Acute rejection is the main risk factor for the development of BOS and is characterized by a perivascular/bronchiolar leukocyte infiltration. The specific mechanism(s) by which these leukocytes are recruited have not been elucidated. The CXC chemokines (monokine induced by IFN-gamma (MIG)/CXC chemokine ligand (CXCL)9, IP-10/CXCL10, and IFN-inducible T cell alpha chemoattractant (ITAC)/CXCL11) act through their shared receptor, CXCR3. Because they are potent leukocyte chemoattractants and are involved in other inflammation/fibroproliferative diseases, we hypothesized that the expression of these chemokines during an allogeneic response promotes the persistent recruitment of mononuclear cells, leading to chronic lung rejection. We found that elevated levels of MIG/CXCL9, IFN-inducible protein 10 (IP-10)/CXCL10, and ITAC/CXCL11 in human bronchoalveolar lavage fluid were associated with the continuum from acute to chronic rejection. Translational studies in a murine model demonstrated increased expression of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 paralleling the recruitment of CXCR3-expressing mononuclear cells. In vivo neutralization of CXCR3 or its ligands MIG/CXCL9 and IP-10/CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated BOS. This supports the notion that ligand/CXCR3 biology plays an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of BOS.     

Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant gram-positive bacteria to CD4+ T lymphocytes.

Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.     

Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients

Rheumatoid arthritis (RA) is a chronic symmetric polyarticular joint disease that primarily affects the small joints of the hands and feet. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to proliferation of synoviocytes and destruction of cartilage and bone. In RA synovial tissue, the infiltrating cells such as macrophages, T cells, B cells and dendritic cells play important role in the pathogenesis of RA. Migration of leukocytes into the synovium is a regulated multi-step process, involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. Chemokines are small, chemoattractant cytokines which play key roles in the accumulation of inflammatory cells at the site of inflammation. It is known that synovial tissue and synovial fluid from RA patients contain increased concentrations of several chemokines, such as monocyte chemoattractant protein-4 (MCP-4)/CCL13, pulmonary and activation-regulated chemokine (PARC)/CCL18, monokine induced by interferon-gamma (Mig)/CXCL9, stromal cell-derived factor 1 (SDF-1)/CXCL12, monocyte chemotactic protein 1 (MCP-1)/CCL2, macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3, and Fractalkine/CXC3CL1. Therefore, chemokines and chemokine-receptors are considered to be important molecules in RA pathology.     

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.     

IFN-gamma-induced chemokines synergize with pertussis toxin to promote T cell entry to the central nervous system

Inflammation of the CNS, which occurs during multiple sclerosis and experimental autoimmune encephalomyelitis, is characterized by increased levels of IFN-gamma, a cytokine not normally expressed in the CNS. To investigate the role of IFN-gamma in CNS, we used intrathecal injection of a replication-defective adenovirus encoding murine IFN-gamma (AdIFNgamma) to IFN-gamma-deficient (GKO) mice. This method resulted in stable, long-lived expression of IFN-gamma that could be detected in cerebrospinal fluid using ELISA and Luminex bead immunoassay. IFN-gamma induced expression in the CNS of message and protein for the chemokines CXCL10 and CCL5, to levels comparable to those seen during experimental autoimmune encephalomyelitis. Other chemokines (CXCL2, CCL2, CCL3) were not induced. Mice lacking the IFN-gammaR showed no response, and a control viral vector did not induce chemokine expression. Chemokine expression was predominantly localized to meningeal and ependymal cells, and was also seen in astrocytes and microglia. IFN-gamma-induced chemokine expression did not lead to inflammation. However, when pertussis toxin was given i.p. to mice infected with the IFN-gamma vector, there was a dramatic increase in the number of T lymphocytes detected in the CNS by flow cytometry. This increase in blood-derived immune cells in the CNS did not occur with pertussis toxin alone, and did not manifest as histologically detectable inflammatory pathology. These results show that IFN-gamma induces a characteristic glial chemokine response that by itself is insufficient to promote inflammation, and that IFN-gamma-induced CNS chemoattractant signals can synergize with a peripheral infectious stimulus to drive T cell entry into the CNS.     

IFN-gamma/STAT1 acts as a proinflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines and adhesion molecules: a critical role of IRF-1

We have previously shown that IFN-gamma/STAT1 plays an essential role in concanavalin A (ConA)-induced T cell hepatitis via activation of apoptotic signaling pathways. Here we demonstrate that IFN-gamma/STAT1 also plays a crucial role in leukocyte infiltration into the liver in T cell hepatitis. After injection of ConA, leukocytes were significantly infiltrated into the liver, which was suppressed in IFN-gamma(-/-) and STAT1(-/-) mice. Disruption of the IFN regulatory factor-1 (IRF-1) gene, a downstream target of IFN-gamma/STAT1, abolished ConA-induced liver injury and suppressed leukocyte infiltration into the liver. Additionally, ConA injection induced expression of a wide variety of chemokines and adhesion molecules in the liver. Among them, expression of ICAM-1, VCAM-1, monokine induced by IFN-gamma (Mig), CC chemokine ligand-20, epithelial cell-derived neutrophil-activating peptide (ENA)-78, IFN-inducible T cell-alpha chemoattractant (I-TAC), and IFN-inducible protein-10 (IP-10) was markedly attenuated in IFN-gamma(-/-), STAT1(-/-), and IRF-1(-/-) mice. In primary mouse hepatocytes, Kupffer cells, and endothelial cells, in vitro treatment with IFN-gamma activated STAT1, STAT3, and IRF-1, and induced expression of VCAM-1, ICAM-1, Mig, ENA-78, I-TAC, and IP-10 mRNA. Induction of these chemokines and adhesion molecules was markedly diminished in STAT1(-/-) and IRF-1(-/-) hepatic cells compared with wild-type hepatic cells. These findings suggest that in addition to induction of apoptosis, previously well documented, IFN-gamma also stimulated hepatocytes, sinusoidal endothelial cells, and Kupffer cells partly via an STAT1/IRF-1-dependent mechanism to produce multiple chemokines and adhesive molecules responsible for promoting infiltration of leukocytes and, ultimately, resulting in hepatitis.