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The chemokine monokine induced by interferon-γ (Mig) is involved in the recruitment of inflammatory cells and liver injury during hepatitis B virus (HBV) infection. HBV protein X contributes to Mig expression in vitro by activation of nuclear factor (NF)-κB; however, the molecular mechanisms by which HBV induces Mig expression in vivo are unknown. In this paper, we established a mouse model for HBV study by tail vein injection of HBV genome-containing adenovirus vectors. Host immune response to the secreted hepatitis B surface antigen and e antigen was detected and serum alanine aminotransferase (ALT) was elevated at different time points. We also demonstrated that peripheral and intrahepatic Mig expression was increased after Ad-HBV infection. This was followed by inflammatory cell migration and formation of inflammatory foci in the liver. In addition, NF-κB p65 subunit translocated from the cytoplasm to the nucleus, and phosphoinositide 3-kinase/Akt, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were to some extent phosphorylated after HBV injection. Following tail vein injection of Mig siRNA/in vivo-jetPEI-Gal complex, Mig expression was partially suppressed, inflammatory cell migration was inhibited, serum level of ALT were reduced. In conclusion, through NF-κB activation, HBV induced Mig expression in vivo, which recruited peripheral inflammatory cells to the liver and resulted in liver damage. Phosphorylation of phosphoinositide 3-kinase/Akt, ERK and JNK but not p38 might involved in the molecular mechanisms underlying HBV induced Mig expression in vivo.

Iterative affinity selection procedures were used to isolate a number of single chain Fv (scFv) antibody fragment clones from naïve Tomlinson I + J phage display libraries that specifically recognize and bind a chemokine, monokine induced by interferon-gamma (MIG/CXCL9). MIG is an important transplant rejection/biology chemokine protein. ELISA-based affinity characterization results indicate that selectants preferentially bind to MIG in the presence of key biopanning component materials and closely related chemokine proteins. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific MIG binding capability with potential applications in transplant rejection monitoring, and other biomedical applications where detection of MIG level is important.

Summary Interactions between cytokines play an important role in the development of thyroid autoimmunity. Using enzyme-linked immunosorbent assay we investigated serum concentrations of soluble interleukin-2 receptor (sIL-2R), interferon-gamma, tumour necrosis factor (TNF)-α, interleukin (IL)-10, CD30, monokine induced by interferon-gamma (MIG), cytotoxic T lymphocyte antigen-4 and markers of apoptosis decoy receptor 3 and Bcl-2 in 28 patients with hyperthyroid Graves' disease (GD), 24 patients with untreated Hashimoto's thyroiditis (HT) and 15 healthy controls. TNF-α, IL-10 and sIL-2R were higher in GD compared with HT and controls (TNF-α: 8·79 in GD versus 2·54 pg/ml in HT, P = 0·01; IL-10: 10·00 versus 3·10 versus 3·10 pg/ml, P1

The inflammatory response plays an important role during the induction of several neonatal diseases. Previous studies have shown that during newborn infections, the natural imbalance between pro- and anti-inflammatory responses shifts toward the production of pro-inflammatory cytokines. In this study, we employed an array system to detect 9 pro- and anti-inflammatory cytokines, and performed ELISA for 6 other cytokines. We then compared the immune response profiling in umbilical cord blood (UV) plasma samples with circulating levels in otherwise healthy donors (HD). Concentrations of ex vivo monokine levels, such as interleukins (IL)-18, IL-23 and IL-27, were profoundly reduced in the UV in relation to the HD group (p-values of 0.003, 0.009 and

Infection of the pancreas with lymphocytic choriomeningitis virus results in rapid and differential expression among CXCR3 chemokines. IFN-γ-inducible protein of 10 kDa (IP-10), in contrast with monokine induced by IFN-γ and IFN-inducible T cell-α chemoattractant, is strongly expressed within 24 h postinfection. Blocking of IP-10, but not monokine induced by IFN-γ, aborts severity of Ag-specific injury of pancreatic β 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.

Activation and skin-selective homing of T cells and their effector functions in the skin represent sequential immunological events in the pathogenesis of atopic dermatitis (AD). Apoptosis of keratinocytes, induced mainly by T cells and mediated by IFN-γ and Fas, is the essential pathogenetic event in eczema formation. Keratinocyte apoptosis appears as activation-induced cell death in AD. By IFN-γ stimulation, chemokines such as IFN-γ-inducible protein 10, monokine induced by IFN-γ, and IFN-γ-inducible α-chemoattractant are strongly up-regulated in keratinocytes. These chemokines attract T cells bearing the specific receptor CXCR3, which is highly expressed on T cells isolated from skin biopsies of AD patients. Accordingly, an increased T cell chemotaxis was observed toward IFN-γ-treated keratinocytes. Supporting these findings, enhanced IFN-γ-inducible protein 10, monokine induced by IFN-γ, and IFN-γ-inducible α-chemoattractant expression was observed in lesional AD skin by immunohistochemical staining. These results indicate a second step of chemotaxis inside the skin after transendothelial migration of the inflammatory cells. Keratinocytes undergoing apoptosis in acute eczematous lesions release chemokines that attract more T cells toward the epidermis, which may further augment the inflammation and keratinocyte apoptosis.

CXC chemokines that lack the ELR motif, including interferon-inducible protein 10 [IP-10 (CXCL10)] and monokine induced by gamma interferon (IFN-γ) [MIG (CXCL9)], have been shown to mediate the generation of type 1 immune responses. In this study, we found that intrapulmonary administration of the gram-negative bacteriumKlebsiella pneumoniaeresulted in the local and systemic expression of IP-10, followed sequentially by MIG expression. MIG mRNA expression in the lungs ofKlebsiella-infected mice required the endogenous production of IFN-γ, whereas IP-10 was expressed in both an IFN-γ-dependent and an IFN-γ-independent fashion. Antibody-mediated neutralization of IP-10 resulted in reduced bacterial clearance and decreased survival, whereas bacterial clearance was unaltered in mice treated with anti-MIG antibody. Impaired bacterial clearance in anti-IP-10 antibody-treated mice was associated with significant reductions in the number and/or activational status of NK and NK-T cells, CD4+T cells, and γδ T cells, as well as a reduction in the expression of IFN-γ. Conversely, the transient transgenic expression of murine IP-10 using adenovirus-mediated gene transfer resulted in improved bacterial clearance when IP-10 adenovirus was given concomitant with intrapulmonary bacterial challenge. These results indicate that IP-10 is an important component of innate immunity against extracellular bacterial pathogens of the lung and may represent a candidate molecule for immunotherapy in the setting of severe respiratory tract infection.

Early diagnosis of acute allograft rejection (AR) is still decisive for long-term renal allograft survival. The aim of this study was to define the role of the chemokine monokine induced by IFN-gamma (MIG) (CXCL9) and IFN-gamma-inducible protein 10 (IP-10) (CXCL10) as early markers of AR in renal transplantation (NTX). In a prospective study, 69 de novo renal transplant recipients were monitored and urine samples were collected after NTX for a median of 29 d. In pH-adjusted urine, MIG and IP-10 were determined by modified ELISA. AR was clinically diagnosed in 15 of 69 recipients and confirmed by biopsy in 14 of 15 AR patients (Banff classification). Corresponding to CXCR3-positive infiltrates in renal tissue, urinary MIG was elevated in 14 of 15 AR patients with a median of 2809 pg/ml (quartile 25% and 75% = 870 and 13,000; n = 15), being significantly (P < 0.0001) different from both nonrejecting allograft patients (NO-AR) (median, 25%, and 75%: 96, 1.0, and 161, n = 54) and healthy controls (median, 25%, and 75%: 144, 19, and 208, n = 13). Urinary MIG predicted AR with a sensitivity of 93% and a specificity of 89%. In AR and NO-AR groups, MIG values correlated well with IP-10 (P < 0.001). MIG values indicated both imminent rejection and response to successful antirejection therapy. MIG was not related to intercurrent infections or other causes for impairment of renal function. In a multivariate analysis, MIG correlated best (P < 0.001) with AR from all AR-associated parameters. In conclusion, urinary MIG serves as a very sensitive and specific predictor for AR, mirrors response to antirejection therapy, and thus may contribute to improved long-term renal allograft survival.

Monokine induced by IFN-γ (MIG; CXC chemokine ligand (CXCL)9) is important in T lymphocyte recruitment in organ transplantation. However, it is not known whether this chemokine, in addition to its chemotactic properties, exerts any effect on T lymphocyte effector functions. For in vivo studies, we used a previously characterized murine model of chronic rejection. The recipient mice were treated with anti-MIG/CXCL9 Ab; graft-infiltrating cells were analyzed for IFN-γ production. For in vitro studies, exogenous CXCR3 ligands were added to CD4 lymphocytes in MLRs, and the proliferative responses were measured. Separate experiments quantitated the number of IFN-γ-producing cells in MLRs by ELISPOT. Neutralization of MIG/CXCL9, in the in vivo model, resulted in significant reduction in the percentage of IFN-γ-producing graft-infiltrating T lymphocytes. In vitro experiments demonstrated that 1) exogenous MIG/CXCL9 stimulated CD4 lymphocyte proliferation in a MHC class II-mismatched MLR, 2) MIG/CXCL9 also increased the number of IFN-γ-producing CD4 lymphocytes in ELISPOT, 3) neutralization of MIG/CXCL9 in MLR reduced T lymphocyte proliferation, 4) IFN-γ-inducible protein 10/CXCL10 and IFN-inducible T cell α chemoattractant/CXCL11 had similar effects on T lymphocyte proliferation, 5) MIG/CXCL9 stimulated T lymphocyte proliferation in MHC class I- and total MHC-mismatched MLRs, 6) neutralization of CXCR3 reduced MIG/CXCL9-induced T lymphocyte proliferation and the number of IFN-γ-positive spots on ELISPOT, and 7) the proliferative effects of MIG/CXCL9 were mediated via an IL-2-independent pathway and were controlled by IFN-γ. This study demonstrates that MIG/CXCL9 stimulates T lymphocyte proliferation and effector cytokine production, in addition to its chemotactic effects. This novel observation expands our current understanding of MIG/CXCL9 biology beyond that of mediating T cell trafficking.

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 α 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.

Apoptosis of histiocytes is a characteristic feature of necrotizing lymphadenitis (HNL). Recent studies have indicated that Fas and perforin-based pathways are involved in the apoptotic process of HNL. Elevated levels of serum interferon (IFN)-gamma are reported in HNL. The CXC chemokine interferon-gamma-inducible protein-10 (IP-10) and monokine induced by interferon-gamma (MIG) cause tissue necrosis, and interleukin (IL)-18 induces the expression of IFN-gamma and Fas ligand (FasL) by T and natural killer (NK) cells. This study was designed to determine the expression of IFN-gamma, IL-18, MIG and IP-10 in HNL. Ten cases of HNL were analyzed by using immunohistochemical staining and/or reverse transcriptase-polymerase chain reaction (RT-PCR). As a control, we included four cases of non-specific lymphadenitis. MIG and IP-10 proteins, which enhance the release of granzyme, showed a similar distribution pattern in viable tissues surrounding dead tissue, mostly within histiocytes, and lymphocytes in HNL. IL-18 was located within histiocytes, especially phagocytic histiocytes, but not within lymphocytes. In addition, IFN-gamma-positive lymphocytes were frequently detected in the surrounding dead tissue, and the lymphocytes in the same area were frequently positive for CXCR3, a specific receptor of MIG and IP-10. In non-specific lymphadenitis, MIG, IP-10 and IL-18 positive cells were detected, but their numbers were relatively small compared with HNL, while IFN-gamma positive cells were rarely encountered. Our findings suggest that the cytokine and chemokine pathways of IFN-gamma, IL-18, MIG and IP-10 play an important role in the pathogenesis of apoptosis associated with HNL.

The IFN-γ-inducible proteins monokine induced by IFN-γ (Mig) and chemokine responsive to γ-2 (Crg-2) can contribute to IL-12-induced antiangiogenic and leukocyte-recruiting activities, but the extent to which leukocytes vs parenchymal cells in different organs contribute to the production of these molecules remains unclear. The results presented herein show that IFN-γ-dependent induction of Mig and Crg-2 gene expression can occur in many nonlymphoid organs, and these genes are rapidly induced in purified hepatocytes isolated from mice treated with IL-2 plus IL-12, or from Hepa 1-6 hepatoma cells treated in vitro with IFN-γ. In addition to depending on IFN-γ, the ability of IL-12 or IL-2/IL-12 to induce Mig and Crg-2 gene expression in purified hepatocytes also is accompanied by the coordinate up-regulation of the IFN-γ R α and β-chains, in the absence of IL-12R components. Supernatants of primary hepatocytes obtained from mice treated in vivo with IL-2/IL-12 or from hepatocytes treated in vitro with IFN-γ contain increased chemotactic activity for enriched human and mouse CD3+ T cells, as well as mouse DX5+ NK cells. The hepatocyte-derived chemotactic activity for mouse T cells but not NK cells was ablated by Abs specific for Mig and Crg-2. These results suggest that parenchymal cells in some organs may contribute substantially to initiation and/or amplification of inflammatory or antitumor responses.

We have previously shown that interferon-inducible protein-10 (IP-10), a chemokine for activated lymphocytes, was specifically induced in the liver of Concanavalin A (Con A)-treated mice. The aim of this study was to investigate the time course profile and cell-type-specific hepatic production of monokine induced by interferon-gamma (MIG), a chemokine which shares its receptor and most of its activity with IP-10, in Con A-treated mice and to compare them with those of IP-10.Hepatic mRNA expression of MIG and IP-10 was studied by means of Northern blot analysis and in situ hybridization in Con A-treated mice. The levels of MIG and IP-10 in the serum and culture supernatants of murine hepatoma-, hepatic sinusoidal endothelial cell-, hepatic stellate cell- and macrophage-derived cell lines were determined by means of specific enzyme-linked immunosorbent assays.The serum level of MIG slowly reached a maximum at 12 h after Con A injection and remained elevated for a long time, whereas that of IP-10 reached a maximum at 3 h and declined quickly, a finding supported by Northern blot analysis. Using in situ hybridization, the mRNA of MIG as well as IP-10 was found to be expressed in hepatocytes and hepatic non-parenchymal cells. Similar to IP-10, MIG was produced by hepatoma-, hepatic sinusoidal endothelial cell-, hepatic stellate cell- and macrophage-derived cell lines in vitro.Although both MIG and IP-10 were produced by hepatocytes and hepatic non-parenchymal cells in Con A-treated mice, the time course profile of MIG was distinguishable from that of IP-10. The fact that hepatic MIG and IP-10 were produced sequentially in this hepatitis model may suggest that a non-redundant role is played by these two chemokines in the process of hepatic necro-inflammation.

BACKGROUND Because recombinant human granulocyte colony-stimulating factor (rhG-CSF) is known to modulate function of antigen-presenting cells, we examined effects of pretransplant host treatment with rhG-CSF on allograft survival. METHODS In DA-to-Lewis rat heart transplantation, hosts were given pretransplant injections of rhG-CSF (250 microg/kg/day subcutaneously from day -5-0) and/or posttransplant injections of tacrolimus (2 mg/kg/day intramuscularly from day 0-3). Cytokine mRNA levels in grafts were measured by real-time reverse-transcription polymerase chain reaction. RESULTS rhG-CSF pretreatment was effective in prolonging allograft survival only in tacrolimus-treated hosts (P

CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon γ (IFN-γ)–inducible protein 10 (γIP-10) and monokine induced by IFN-γ (Mig). We report the novel finding that CXCR3 is also expressed on CD34+ hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34+ progenitors. Freshly isolated CD34+progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase–polymerase chain reaction technique. γIP-10 and Mig induced chemotaxis of GM-CSF–stimulated CD34+ progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block γIP-10–induced and Mig-induced CD34+ progenitor chemotaxis. These chemotactic attracted CD34+ progenitors are colony-forming units—granulocyte-macrophage. γIP-10 and Mig also induced GM-CSF–stimulated CD34+ progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of γIP-10 and Mig but not of chemokine stromal cell–derived factor 1α. γIP-10–induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF–stimulated CD34+progenitors. Moreover, γIP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF–stimulated CD34+progenitors. These results indicate that CXCR3–γIP-10 and CXCR3–Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34+ hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34+ hematopoietic progenitors.

BACKGROUND Our goal was to test the intragraft mRNA expression and production of two chemokines that are potent chemoattractants for antigen-primed T cells, interferon-gamma inducible protein 10 (IP-10) and monokine-induced by IFN-gamma, (Mig), in allogeneic heart grafts. METHODS Syngeneic or allogeneic A/J (H-2a) hearts were heterotopically transplanted to wild-type, CD4-/-, CD8alpha-/-, or IFN-gamma-/- C57BL/6 (H-2b) recipients. To test expression of IP-10 and Mig, grafts were removed 1-8 days posttransplant for RNA isolation and Northern blot analysis. To test the potential recipient leukocyte populations mediating intraallograft expression of IP-10 and Mig, recipients were treated with anti-NK 1.1, anti-CD4, and/or anti-CD8 monoclonal antibodies before transplantation. RESULTS Allogeneic heart grafts transplanted to wild-type, but not IFN-gamma-/-, recipients expressed IP-10 and Mig at day +2 posttransplant that increased thereafter until rejection was completed. Expression of IP-10 and Mig in isografts was low or undetectable. Cardiac allografts from CD8+ T cell depleted, but not NK cell or CD4+ T cell depleted, recipients had low to undetectable expression of IP-10 and Mig on day +2 posttransplant. Similarly, cardiac allografts from CD8-/-, but not CD4-/-, recipients had low to undetectable expression of IP-10 and Mig on day +2 posttransplant. CONCLUSIONS Early intraallograft expression of Mig and IP-10 during primary rejection of cardiac allografts is dependent on the activities of recipient CD8+ T cells.

In intact tissue, the extracellular matrix (ECM) provides support and helps maintain homeostasis but is considered biologically inert. In the setting of inflammation, not only is the ECM the target of inflammation, but its breakdown products modulate the magnitude and quality of an immune response. Fragments of the ECM component hyaluronan (HA) induce macrophage expression of chemokines, cytokines, and growth factors as well greatly enhance IFN-gamma-induced MIG expression. In this report, we demonstrate that the synergistic induction of MIG by HA and IFN-gamma occurs at the level of transcription via NF-kappaB. Using electrophoretic mobility shift assays and reporter assays, we have identified two NF-kappaB sites proximal to the IFN-gamma-responsive element-1 (gammaRE-1) that mediate this effect. Interestingly, our experiments also revealed a critical role for NF-kappaB in mediating IFN-gamma-induced MIG expression independent of HA. These data emphasize the ability of "degraded self" to activate/modify immune responses through the NF-kappaB pathway.

Previous reports have indicated that the early induction of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), IL-1beta, and IL-10 is crucial for the establishment and regulation of host cell-mediated immunity to the intracellular protozoan parasite Toxoplasma gondii. In this study, we demonstrate that a soluble tachyzoite extract (soluble tachyzoite antigen) can trigger the expression of these four monokines by murine inflammatory macrophages. Further characterization revealed that the parasite molecules in soluble tachyzoite antigen responsible for monokine induction are heat stable at 100 degree C but differ in sensitivity to protease digestion. Thus, the tachyzoite factors that stimulate TNF-alpha and IL-to expression were found to be more resistant to treatment with proteinase K than those responsible for IL-12 and IL-10 induction. Similarly, while the factors responsible for the induction of all four monokines were found to be sensitive to periodate oxidation, the TNF-alpha-stimulating activity was partially resistant to treatment with the compound at a low concentration (1 mM). A further dichotomy in monokine induction signals was inferred from experiments with isoquinoline sulfonamide protein kinase inhibitors. The latter work suggested that the pathways for TNF-alpha and IL-1beta are protein kinase C dependent, while expression of IL-12 and expression of IL-10 share distinct signal transduction mechanisms involving other kinases. Together, these data argue that monokine induction by T. gondii is mediated by glycoproteins that may belong to distinct groups in terms of their biochemical properties and intracellular signaling pathways.