The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.     

IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines

IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC) belong to the non-glutamate-leucine-arginine motif CXC chemokine family and act solely through the CXCR3 receptor for potent attraction of T lymphocytes. In this study, we evaluated the capacity of the T cell-derived cytokines IL-4, IL-10, and IL-17 to modulate IP-10, Mig, and I-TAC in cultured human keratinocytes and CXCR3 expression in T cells from allergic contact dermatitis (ACD). IL-4, but not IL-10 or IL-17, significantly up-regulated IFN-gamma- or TNF-alpha-induced IP-10, Mig, and I-TAC mRNA accumulation in keratinocytes and increased the levels of IP-10 and Mig in keratinocyte supernatants. Immunohistochemistry of skin affected by ACD revealed that >70% of infiltrating cells were reactive for CXCR3 and that CXCR3 staining colocalized in CD4+ and CD8+ T cells. Nickel-specific CD4+ and CD8+ T cell lines established from ACD skin produced IFN-gamma and IL-4 and expressed moderate to high levels of CXCR3. Finally, CXCR3 agonistic chemokines released by stimulated keratinocytes triggered calcium mobilization in skin-derived nickel-specific CD4+ T cells and promoted their migration, with supernatant from keratinocyte cultures stimulated with IFN-gamma and IL-4 attracting more efficaciously than supernatant from keratinocytes activated with IFN-gamma alone. In conclusion, IL-4 exerts a proinflammatory function on keratinocytes by potentiating IFN-gamma and TNF-alpha induction of IP-10, Mig, and I-TAC, which in turn may determine a prominent recruitment of CXCR3+ T lymphocytes at inflammatory reaction sites.     

Blockade of CXCR3 receptor:ligand interactions reduces leukocyte recruitment to the lung and the severity of experimental idiopathic pneumonia syndrome

Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication after allogeneic stem cell transplantation (allo-SCT) that responds poorly to standard immunosuppressive therapy. The pathophysiology of IPS involves the secretion of inflammatory cytokines including IFN-gamma and TNF-alpha along with the recruitment of donor T cells to the lung. CXCR3 is a chemokine receptor that is expressed on activated Th1/Tc1 T cell subsets and the expression of its ligands CXCL9 (monokine induced by IFN-gamma (Mig)) and CXCL10 (IFN-gamma-inducible protein 10 (IP-10)) can be induced in a variety of cell types by IFN-gamma alone or in combination with TNF-alpha. We used a lethally irradiated murine SCT model (B6 --> bm1) to evaluate the role of CXCR3 receptor:ligand interactions in the development of IPS. We found that Mig and IP-10 protein levels were significantly elevated in the bronchoalveolar lavage fluid of allo-SCT recipients compared with syngeneic controls and correlated with the infiltration of IFN-gamma-secreting CXCR3(+) donor T cells into the lung. The in vivo neutralization of either Mig or IP-10 significantly reduced the severity of IPS compared with control-treated animals, and an additive effect was observed when both ligands were blocked simultaneously. Complementary experiments using CXCR3(-/-) mice as SCT donors also resulted in a significant decrease in IPS. These data demonstrate that interactions involving CXCR3 and its primary ligands Mig and IP-10 significantly contribute to donor T cell recruitment to the lung after allo-SCT. Therefore, approaches focusing on the abrogation of these interactions may prove successful in preventing or treating lung injury that occurs in this setting.     

Monokine induced by IFN-gamma is a dominant factor directing T cells into murine cardiac allografts during acute rejection.

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-gamma inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) in acute rejection of A/J (H-2(a)) cardiac grafts by C57BL/6 (H-2(b)) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7-9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17-19), intra-allograft expression of macrophage-inflammatory protein-1alpha, -1beta, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.     

CXCR3 expression on CD34(+) hemopoietic progenitors induced by granulocyte-macrophage colony-stimulating factor: II. Signaling pathways involved

CXCR3, known to have four ligands (IFN-gamma inducible protein 10 (gamma IP-10), monokine induced by IFN-gamma (Mig), I-TAC, and 6Ckine), is predominantly expressed on memory/activated T lymphocytes. We recently reported that GM-CSF induces CXCR3 expression on CD34(+) hemopoietic progenitors, in which gamma IP-10 and Mig induce chemotaxis and adhesion. Here we further report that stimulation with GM-CSF causes phosphorylation of Syk protein kinase, but neither Casitas B-lineage lymphoma (Cbl) nor Cbl-b in CD34(+) hemopoietic progenitors can be blocked by anti-CD116 mAb. Specific Syk blocking generated by PNA antisense completely inhibits GM-CSF-induced CXCR3 expression in CD34(+) progenitors at both mRNA and protein as well as at functional levels (chemotaxis and adhesion). Cbl and Cbl-b blocking have no such effects. Thus, GM-CSF binds to its receptor CD116, and consequently activates Syk phosphorylation, which leads to induce CXCR3 expression. gamma IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34(+) progenitors by means of CXCR3. gamma IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34(+) hemopoietic progenitors, whereas SDF-1alpha induces both chemotaxis and adhesion in these cells. Interestingly, gamma IP-10 and Mig can induce chemotaxis and adhesion in GM-CSF-stimulated Syk- or Cbl-blocked CD34(+) hemopoietic progenitors. Thus, Cbl-b, but not Syk and Cbl phosphorylation, is essential for gamma IP-10- and Mig-induced chemotaxis and adhesion in CD34(+) hemopoietic progenitors. This study provides a useful insight into novel signaling transduction pathways of the functions of CXCR3/gamma IP-10 and Mig, which may be especially important in the cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells.     

Cytokine and chemokine mRNA expression in neutrophils from CBA/NSlc mice infected with Plasmodium berghei ANKA that induces experimental cerebral malaria.

To investigate the role of neutrophils in experimental cerebral malaria (ECM), in a previous study we found that early neutrophil depletion prevented the development of ECM and down regulated the expression of Th1 cytokines in the brain. To further clarify the mechanisms responsible for these findings, in the present study, using RT-PCR, we examined the expression of cytokine and chemokine mRNAs in neutrophils and macrophages after PbA infection. We found that, after infection, neutrophils not only expressed cytokines IL-2, IL-12p40, IL-18, IFN-gamma and TNF-alpha mRNAs, but also mRNAs for Th1 chemoattractive chemokines, monokine-induced by IFN-gamma (MIG), macrophage-inflammatory protein-1alpha (MIP-1alpha) and IFN-gamma inducible protein-10 (IP-10). Neutrophil depletion down regulated the expression of IL-18 and MIG mRNAs in macrophages, but did not affect the expression of IFN-gamma, TNF-alpha, MIP-1alpha and IP-10 mRNAs. Therefore, this study confirms our hypothesis that neutrophils may play a role in the pathogenesis of ECM via their expression of cytokines or chemokines.     

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.     

CpG oligodeoxynucleotides directly induce CXCR3 chemokines in human B cells

CpG oligodeoxynucleotides (CpG ODN) are known to elicit Th1 immune responses via TLR9. However, the precise mechanisms through which B cells are involved in this phenomenon are not fully understood. We investigated the effect of CpG ODN on the induction of Th1-chemoattractant CXCR3 chemokines, IP-10, Mig, and I-TAC, in B cells. Cells from the RPMI 8226 human B cell line and human peripheral B cells were stimulated with three distinct classes of CpG ODN. As a result, CXCR3 chemokines were strongly up-regulated by CpG-B and CpG-C, but only weakly by CpG-A. Though CXCR3 chemokines are known to be induced by IFNs, blocking mAbs against IFN receptors did not inhibit their induction by CpG-B. Induction of CXCR3 chemokines was blocked by two NF-kappaB inhibitors and a p38 inhibitor. These results strongly suggest that CXCR3 chemokines are directly induced by CpG ODN via NF-kappaB- and p38-dependent pathways in human B cells.     

Expression of IFN-gamma induced CXCR3 agonist chemokines and compartmentalization of CXCR3+ cells in the periphery and lymph nodes of rhesus macaques during simian immunodeficiency virus infection and acquired immunodeficiency syndrome

Dysregulation of cytokines and chemokines during human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV) infection is thought to be critical in the progression of acquired immunodeficiency syndrome (AIDS). To evaluate the potential role of Th1-agonist chemokines in disease progression during AIDS, we assessed CXCL9/MIG and CXCL10/IP-10 expression simultaneously in the periphery and lymphoid tissues of SIV-infected animals at a single-cell level by flow cytometry. We optimized intracellular staining and analysis of CXCL9/MIG and CXCL10/IP-10 production in human leukocyte antigen (HLA)-DR+ macaque cells by flow cytometry using cross-reactive antibodies against human chemokines. We observed an upregulation of CXCL9/MIG and CXCL10/IP-10 production in both the periphery and lymph nodes of infected animals compared with na?ve controls. Animals with higher viral loads had higher levels of CXCL9/MIG and CXCL10/IP-10 producing cells compared with animals with low viral loads. Analysis of cells bearing the receptor (CXCR3) for CXCL9/MIG and CXCL10/IP-10 revealed increased number of CXCR3+ cells in the lymph nodes of infected animals. Importantly, an inverse correlation (P < 0.05) between CXCL9/MIG and CXCL10/IP-10 production, both in the periphery and lymph nodes, and peripheral CD4+ T-cell numbers was observed. These findings provide further evidence that dysregulation of Th1 agonist chemokines might contribute to the ultimate immunopathology during AIDS.     

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.     

Differential expression of the IFN-gamma-inducible CXCR3-binding chemokines,IFN-inducible protein 10, monokine induced by IFN,and IFN-inducible T cell alpha chemoattractant in human cardiac allografts: association with cardiac allograft vasculopathy and acute rejection

CXCR3 chemokines exert potent biological effects on both immune and vascular cells. The dual targets suggest their important roles in cardiac allograft vasculopathy (CAV) and rejection. Therefore, we investigated expression of IFN-inducible protein 10 (IP-10), IFN-inducible T cell alpha chemoattractant (I-TAC), monokine induced by IFN (Mig), and their receptor CXCR3 in consecutive endomyocardial biopsies (n = 133) from human cardiac allografts and corresponding normal donor hearts (n = 11) before transplantation. Allografts, but not normal hearts, contained IP-10, Mig, and I-TAC mRNA. Persistent elevation of IP-10 and I-TAC was associated with CAV. Allografts with CAV had an IP-10-GAPDH ratio 3.7 +/- 0.8 compared with 0.8 +/- 0.2 in those without CAV (p = 0.004). Similarly, I-TAC mRNA levels were persistently elevated in allografts with CAV (6.7 +/- 1.9 in allografts with vs 1.5 +/- 0.3 in those without CAV, p = 0.01). In contrast, Mig mRNA was induced only during rejection (2.4 +/- 0.9 with vs 0.6 +/- 0.2 without rejection, p = 0.015). In addition, IP-10 mRNA increased above baseline during rejection (4.1 +/- 2.3 in rejecting vs 1.8 +/- 1.2 in nonrejecting biopsies, p = 0.038). I-TAC did not defer significantly with rejection. CXCR3 mRNA persistently elevated after cardiac transplantation. Double immunohistochemistry revealed differential cellular distribution of CXCR3 chemokines. Intragraft vascular cells expressed high levels of IP-10 and I-TAC, while Mig localized predominantly in infiltrating macrophages. CXCR3 was localized in vascular and infiltrating cells. CXCR3 chemokines are induced in cardiac allografts and differentially associated with CAV and rejection. Differential cellular distribution of these chemokines in allografts indicates their central roles in multiple pathways involving CAV and rejection. This chemokine pathway may serve as a monitor and target for novel therapies to prevent CAV and rejection.     

Among CXCR3 chemokines, IFN-gamma-inducible protein of 10 kDa (CXC chemokine ligand (CXCL) 10) but not monokine induced by IFN-gamma (CXCL9) imprints a pattern for the subsequent development of autoimmune disease

Infection of the pancreas with lymphocytic choriomeningitis virus results in rapid and differential expression among CXCR3 chemokines. IFN-gamma-inducible protein of 10 kDa (IP-10), in contrast with monokine induced by IFN-gamma and IFN-inducible T cell-alpha chemoattractant, is strongly expressed within 24 h postinfection. Blocking of IP-10, but not monokine induced by IFN-gamma, aborts severity of Ag-specific injury of pancreatic beta cells and abrogates type 1 diabetes. Mechanistically, IP-10 blockade impedes the expansion of peripheral Ag-specific T cells and hinders their migration into the pancreas. IP-10 expression was restricted to viruses infecting the pancreas and that are capable of causing diabetes. Hence, virus-induced organ-specific autoimmune diseases may be dependent on virus tropism and its ability to alter the local milieu by selectively inducing chemokines that prepare the infected tissue for the subsequent destruction by the adaptive immune response.     

Serum levels of ifn-inducible PROTEIN-10 relating to the activity of systemic lupus erythematosus

IFN-inducible protein-10 (IP-10) is supposed to act as a specific chemoattractant for Th(1)cells. Since Th(1)cells and IFN-gamma are shown to be important for developing systemic lupus erythematosus (SLE), we examined the relationship between serum IP-10 levels and the disease activity. Serum IP-10 levels were markedly increased in the SLE patients depending on the level of disease activity, whereas not in the patients with rheumatoid arthritis (RA). On the other hand, serum MCP-1 levels were increased to a similar extent both in RA and inactive SLE patients, and a little more elevated in active SLE patients. Serum IP-10 levels in SLE patients correlated positively and negatively with levels of anti-DNA antibody and complements, respectively, whereas MCP-1 levels correlated less or not at all. These results suggest that serum IP-10 levels could be a good indicator for the activity of SLE and that IP-10 could play an important immunological role in SLE.     

CXCR3 expression and activation of eosinophils: role of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for both IFN-gamma-inducible protein-10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report a novel finding that CXCR3 is also expressed on eosinophils. gamma IP-10 and Mig induce eosinophil chemotaxis via CXCR3, as documented by the fact that anti-CXCR3 mAb blocks gamma IP-10- and Mig-induced eosinophil chemotaxis. gamma IP-10- and Mig-induced eosinophil chemotaxis are up- and down-regulated by IL-2 and IL-10, respectively. Correspondingly, CXCR3 protein and mRNA expressions in eosinophils are up- and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-time quantitative RT-PCR technique. gamma IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinase A signaling pathways. The fact that gamma IP-10 and Mig induce an increase in intracellular calcium in eosinophils confirms that CXCR3 exists on eosinophils. Besides induction to chemotaxis, gamma IP-10 and Mig also activate eosinophils to eosinophil cationic protein release. These results indicate that CXCR3-gamma IP-10 and -Mig receptor-ligand pairs as well as the effects of IL-2 and IL-10 on them may be especially important in the cytokine/chemokine environment for the pathophysiologic events of allergic inflammation, including initiation, progression, and termination in the processes.     

Gene expression and production of the monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein-10 (IP-10) chemokines by human neutrophils

Monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein of 10 kDa (IP-10) are related members of the CXC chemokine subfamily that bind to a common receptor, CXCR3, and that are produced by different cell types in response to IFN-gamma. We have recently reported that human polymorphonuclear neutrophils (PMN) have the capacity to release IP-10. Herein, we show that PMN also have the ability to produce MIG and to express I-TAC mRNA in response to IFN-gamma in combination with either TNF-alpha or LPS. While IFN-gamma, alone or in association with agonists such as fMLP, IL-8, granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF, failed to influence MIG, IP-10, and I-TAC gene expression, IFN-alpha, in combination with TNF-alpha, LPS, or IL-1beta, resulted in a considerable induction of IP-10 release by neutrophils. Furthermore, IL-10 and IL-4 significantly suppressed the expression of MIG, IP-10, and I-TAC mRNA and the extracellular production of MIG and IP-10 in neutrophils stimulated with IFN-gamma plus either LPS or TNF-alpha. Finally, supernatants harvested from stimulated PMN induced migration and rapid integrin-dependent adhesion of CXCR3-expressing lymphocytes; these activities were significantly reduced by neutralizing anti-MIG and anti-IP-10 Abs, suggesting that they were mediated by MIG and IP-10 present in the supernatants. Since MIG, IP-10, and I-TAC are potent chemoattractants for NK cells and Th1 lymphocytes, the ability of neutrophils to produce these chemokines might contribute not only to the progression and evolution of the inflammatory response, but also to the regulation of the immune response.     

Overexpression of IFN-induced protein 10 and its receptor CXCR3 in myasthenia gravis

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. Microarray technology was used to identify new potential drug targets for treatment of myasthenia that would reduce the need for the currently used nonspecific immunosuppression. The chemokine IFN-gamma-inducible protein 10 (IP-10; CXCL10), a CXC chemokine, and its receptor, CXCR3, were found to be overexpressed in lymph node cells of EAMG rats. Quantitative real-time PCR confirmed these findings and revealed up-regulated mRNA levels of another chemoattractant that activates CXCR3, monokine induced by IFN-gamma (Mig; CXCL9). TNF-alpha and IL-1beta, which act synergistically with IFN-gamma to induce IP-10, were also up-regulated. These up-regulations were observed in immune response effector cells, namely, lymph node cells, and in the target organ of the autoimmune attack, the muscle of myasthenic rats, and were significantly reduced after suppression of EAMG by mucosal tolerance induction with an AChR fragment. The relevance of IP-10/CXCR3 signaling in myasthenia was validated by similar observations in MG patients. A significant increase in IP-10 and CXCR3 mRNA levels in both thymus and muscle was observed in myasthenic patients compared with age-matched controls. CXCR3 expression in PBMC of MG patients was markedly increased in CD4(+), but not in CD8(+), T cells or in CD19(+) B cells. Our results demonstrate a positive association of IP-10/CXCR3 signaling with the pathogenesis of EAMG in rats as well as in human MG patients.     

Intraallograft chemokine RNA and protein during rejection of MHC-matched/multiple minor histocompatibility-disparate skin grafts

Chemokines direct leukocyte recruitment into sites of tissue inflammation and may facilitate recruitment of leukocytes into allografts following transplantation. Although the expression of chemokines during rejection of MHC-disparate allografts has been examined, chemokine expression in MHC-matched/multiple minor histocompatibility Ag-disparate allografts has not been tested. The intraallograft RNA expression of several C-X-C and C-C chemokines was tested during rejection of full thickness skin grafts from B10. D2 donors on control Ig-, anti-CD4 mAb-, and anti-CD8 mAb-treated BALB/c recipients. In all recipients, two patterns of intragraft chemokine expression were observed during rejection of these grafts: 1) macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, GRO-alpha (KC), JE, and IFN-gamma-inducible protein (IP-10) were expressed at equivalent levels in allo- and isografts for 2-4 days posttransplant and then returned to low or undetectable levels; and 2) IP-10 and monokine induced by IFN-gamma (Mig) were expressed in the allografts 3 days before rejection was completed, suggesting a possible role in recruiting primed T cells into the allograft. Three days before completion of rejection, intraallograft IP-10 protein was restricted to the epidermis, whereas Mig was located in the lower dermis and associated with the intense infiltration of mononuclear cells. Treatment of B10.D2 recipients with rabbit antiserum to Mig, but not to IP-10, delayed rejection of the allografts 3-4 days. The results suggest that Mig mediates optimal recruitment of T cells into MHC-matched/multiple minor histocompatibility Ag-disparate allografts during rejection.     

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.