IP-10 Expression Research Articles

Palmitic acid induces IP-10 expression in human macrophages via NF-κB activation

It is now recognized that cross-talk between adipocytes and adipose tissue stromal cells such as macrophages contributes to local and systemic inflammation. One factor from adipocytes that may participate in this interaction and that is frequently elevated in inflammatory conditions such as obesity, insulin resistance, and type 2 diabetes is free fatty acids (FFA). To investigate the potential for FFA to enhance macrophage inflammation, we exposed U937 macrophages to physiological levels (150 μM) of FFA. Palmitic acid (PA), the predominant saturated FFA released from adipose tissue, but not unsaturated FFA, induced an ∼6-fold ( p < 0.05) increase in IP-10 gene expression (and 2- to 4-fold increases in IL-8, MCP-1, COX-2, and MIG). PA also induced an ∼2-fold increase ( p < 0.05) in active NF-κB, and two structurally distinct NF-κB inhibitors effectively blocked PA-induced IP-10 gene expression. Conditioned medium from PA-treated cells increased lymphocyte migration 41% ( p < 0.05) which was significantly reduced by IP-10-neutralizing antibody. These results suggest that elevated concentrations of PA commonly present in obese and insulin resistant individuals can increase NF-κB-mediated expression of IP-10 in macrophages. These events in turn may lead to an increasing feed-forward loop of chronic inflammation.

Targeting chemokines as a therapeutic option for hepatocellular carcinoma: A reality or just a fantasy?

Targeting chemokines as a therapeutic option for hepatocellular carcinoma: A reality or just a fantasy?

CXCR3-mediated recruitment of cytotoxic lymphocytes in lupus erythematosus profundus

Lupus erythematosus profundus (LEP) is a rare variant lupus erythematosus with unclear etiology characterized by lobular panniculitis. Recently, we observed a case of LEP involving the lower right eyelid. Our immunohistological analyses of lesional skin biopsies revealed a type I IFN signature in the context of cytotoxic lobular panniculitis. Since type I IFNs have been shown to be involved in other cutaneous LE subtypes, especially in chronic discoid LE, we hypothesized that a type I IFN driven immune response might play an important role in the pathogenesis of LEP. In addition to the above case, 9 skin biopsies taken from 5 patients with LEP were analyzed for a type I interferon signature by immunohistochemistry. Furthermore, 8 skin biopsies taken from patients with active chronic discoid LE and 5 biopsies of healthy skin were included for control purposes. The inflammatory infiltrate was characterized using monoclonal antibodies specific for CD3, CD4, CD8, CD20, CD68, and CD123. Subsequently, we analyzed the expression the type I IFN Marker MxA, the cytotoxic molecules granzyme B and Tia1, the chemokine receptor CXCR3 and its ligand, the interferon inducible protein IP10/CXCL10. LEP skin lesions were characterized by a lobular panniculitis, dominated by cytotoxic CXCR3(+) lymphocytes. Strong MxA expression indicated extensive type I IFN production within the fat lobules. Numerous plasmacytoid dendritic cells appear to be the major source of type I IFNs. Lesional expression of IP10 links the type I IFN production and recruitment of CXCR3(+) lymphocytes. The study was based on histological and immunohistological analyses in a limited number of patients, due to the rareness of the investigated disease. Our results demonstrate a type I IFN driven immune response in active LEP skin lesions. We suggest that this type I IFN driven inflammation is responsible for the recruitment of CXCR3(+) lymphocytes into fat lobules and enhance their cytotoxic capacity.

Upregulation of IP-10(CXCL10) mRNA Expression by Interleukin-18

Background : Interleukin-18 (IL-18) is one of the principal inducers of interferon- (IFN-) in lymphocytes. Materials and Methods : The effect of IL-18 on the expression of chemokine IP-10(CXCL10) mRNA in C57BL/6 mouse peritoneal macrophages was studied by using Northern blot analysis, enzyme linked immunosobent assay and electrophoretic mobility shift assay. Results : IL-18 was determined to exert no direct effect on the expression of IP-10(CXCL10) mRNA. However, IL-18 pretreatment was determined to play a cooperative role in the synergistic induction of LPS-induced IP-10(CXCL10) mRNA expression. The effect associated with IL-18 pretreatment with regard to the synergistic induction of LPS-induced IP-10 (CXCL10) mRNA expression was detected after 16 hr of IL-18 pretreatment, administered prior to LPS stimulation. The pattern of NF- binding activity during IL-18 pretreatment with LPS stimulation was found to coincide with the expression of IP-10(CXCL10) mRNA. Conclusion : Although IL-18 alone exerts no direct effect on the expression of chemokine IP-10(CXCL10), a definite period of IL-18 pretreatment induces the synergistic expression of LPS-induced IP-10(CXCL10) mRNA. NF- activation is a component of this synergistic effect of IL-18 pretreatment. These results provide useful information, which may facilitate the elucidation of the action mechanisms underlying IL-18 effect on the expression of IP-10(CXCL10) mRNA.

ROLE OF INTERFERON-γ-INDUCIBLE PROTEIN 10 IN THE PATHOGENESIS OF COXSACKIEVIRUS B3.

Background Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. It exerts its effects on its host by altering the cellular gene expression involved in viral replication, viral clearance, and maintenance of host immunity. Among the genes that are up-regulated is IP10, which is a potent chemoattractant of T cells. Our goal is to investigate the role of IP10 in the pathogenesis of CVB3-induced myocarditis using both IP-10 transgenic and knockout mouse models. Hypothesis IP10 is required for proper host immune response against CVB3 infection and knocking out IP10 delays virus clearance by causing insufficient recruitment of activated TH1 lymphocytes toward the infection. Conversely, too much IP10 causes overstimulation of the immune response; thus, overexpression of IP10 will result in a more severe myocarditis. Methods Four to 5-week-old IP10 transgenic (Tg), knockout (KO), and wild-type (WT) mice were infected with 105 pfu CVB3 by intraperitoneal injection. Their hearts were collected at 7 days postinfection (pi) and were analyzed for viral replication by plaque assay and for severity of immune response by using immunohistochemistry (IHC). Furthermore, quantitative RT-PCR (qRT-PCR) was performed to characterize the expression of chemokines and cytokines related to IP10 that might be involved in determining the myocarditis severity. Results The IHC results demonstrated a close relationship between IP-10 expression and degree of T cell infiltration, suggesting that IP10 plays a critical role in the physiologic response of activated T-cell recruitment to the heart following CVB3 infection. By qRT-PCR, mRNA of TNFα (proinflammatory cytokine), IFN-inducible T-cell alpha chemoattractant (I-TAC, CXCL11), and TH1 cytokines (IFN-γ, IL-12α), but not TH2 cytokines (IL-4, IL-5), were found to show a lower level of expression in CVB3-infected KO mice compared with the control WT mice, suggesting mainly that the TH1 immune response was impaired in IP10 KO mice. At day 7 pi, there was no difference in the virus titer of the hearts in Tg, KO, and WT mice, but we expect that a difference may be detectable at day 10 pi. Conclusion IP10 KO mice infected with CVB3 may have an impaired ability to control viral clearance in the heart, which is associated with decreased recruitment of TH1 lymphocytes into the heart and reduced levels of IFN-γ and therefore decreased I-TAC expression in the heart. IP10 Tg mice, on the other hand, may have an excessive immune response and a more severe myocarditis.

IP10/CXCL10 - CXCR3 Interaction: a Potential Self-recruiting Mechanism for Cytotoxic Lymphocytes in Lichen Sclerosus et Atrophicus

Lichen sclerosus et atrophicus is a chronic inflammatory skin disease of unknown aetiology. Recent studies have indicated that autoimmune mechanisms might be involved in its pathogenesis and have suggested a role for autoreactive cytotoxic T-lymphocytes. Based on recent observations we now hypothesize that a type I interferon-driven inflammation might be involved in the pathogenesis of this disease. Lesional skin biopsies were analysed by immunohistochemistry (CD3, CD4, CD8, CD68, CD123, Tia1, Granzyme B, Myxovirus resistance A, IP10/CXCL10 and CXCR3). Sequential double staining was performed to analyse co-expression of Tia1 and CXCR3. Significant expression of Myxovirus resistance A was found, indicating type I interferon production. This expression was closely associated with the expression of the interferon-inducible protein IP10 and the recruitment of CXCR3+ cytotoxic T-lymphocytes. Plasmacytoid dendritic cells appeared to be a major source of type I interferon in lichen sclerosus et atrophicus. Interestingly, several infiltrating lymphocytes contained IP10 in their granules. This is the first study providing evidence that a type I interferon-associated recruitment of CXCR3+ cytotoxic T-lymphocytes is involved in the pathogenesis of lichen sclerosus et atrophicus. Infiltrating lymphocytes, containing IP10 in their granules, could provide an important self-perpetuating mechanism.

Aspergillus fumigatus conidia induce internalization dependent IFN-ß and IP-10 expression in airway epithelial cells

Aspergillus fumigatus is a fungal pathogen of major clinical importance. Inhalation of fungal spores may cause lung diseases, ranging from local inflammation of the airways to severe and life-threatening infections of the lung. It is established that alveolar macrophages provide the first line of defence in the respiratory tract. Mice studies show that macrophage-mediated response to A. fumigatus conidia is MyD88-independent. Little is known about the role of human airway epithelial cells (HBEC) in A. fumigatus conidia induced inflammation. Here we report that airway epithelial cells activated by A. fumigatus resting conidia show a strong expression of IFN-β and IP-10 that was internalization dependent.

Selective Neutralization of the Chemokine TCA3 Reduces the Increased Injury of Partial Versus Whole Liver Transplants Induced by Cold Preservation

Given the shortage of liver donors and the development of techniques for partial liver transplantation, we compared chemokine expression and inflammatory cell infiltration of partial versus whole grafts in a mouse syngeneic liver transplant model. Orthotopic liver transplantation, using whole or partial murine liver grafts, was performed following cold preservation in ViaSpan solution for periods of one to eight hours. Partial grafts showed more severe cold ischemia/reperfusion injury and greater inflammatory cell infiltration than whole grafts, and was accompanied by the marked intrahepatic upregulation of multiple chemokines. Quantitative analysis showed that compared with expression in whole grafts harvested after the same period of cold ischemia, partial grafts had eightfold more T-cell activation gene (TCA)-3 (CCL1) chemokine messenger RNA (mRNA) expression (P<0.01) and sixfold more inducible protein (IP)-10 chemokine (CCL10) mRNA expression (P<0.01), as well as increased expression of the chemokine receptors CCR8 (receptor for TCA3) and CXCR3 (receptor for IP-10; P<0.01). Blockade of TCA3 by neutralizing monoclonal antibody significantly decreased intragraft IP-10 expression (P<0.05) but not tumor necrosis factor-alpha or interleukin-6 expression in partial grafts, and significantly decreased cold ischemia/reperfusion injury (P<0.05) and associated neutrophil and T-cell infiltration (P<0.01). These data demonstrate that the chemokine TCA3/CCL1 is important to the pathogenesis of ischemic injury of experimental partial liver grafts, and that its therapeutic targeting within such grafts can overcome the deleterious effects of prolonged cold preservation and restore liver function to the level achieved using whole liver grafts.

Effective Graft-Versus-Leukemia Responses Are Associated with the Presence of Nucleic Acid-Immunoglobulin Complexes That Stimulate TLR8 and TLR9.

To elucidate the basis of the potent graft versus leukemia (GvL) immune response that is initiated with donor lymphocyte infusion (DLI), we hypothesized the existence of an endogenous adjuvant that enhances antigen-specific immunity. In patients with chronic myeloid leukemia (CML) who achieved molecular remission after DLI, we previously identified a panel of CML-associated antigens that are targets of antibodies present in post-DLI sera and are predicted to bind nucleic acids. Recent studies in SLE have identified immunostimulatory nucleic-acid antibody complexes in patient sera, which can stimulate Toll-like receptors (TLRs) in plasmacytoid dendritic cells (pDCs). To identify immunostimulatory factors associated with GvL, we studied 6 patients that responded to DLI without clinically significant GvHD. Consistent with our prediction that sera from DLI responders contain an immunostimulatory activity, we measured a 3 to 50-fold increase in the expression of MIP-1α, TNF-α, IP-10 and IFN-α transcripts in normal peripheral blood mononuclear cells (PBMC) induced by exposure to 5 of 6 post-DLI sera. No increase in cytokine/chemokine expression was observed upon exposure to sera from pre DLI sera from the same patients, from 3 of 3 DLI non-responders, nor from 3 of 3 CML patients who achieved molecular remission after imatinib treatment. To identify the cell type that responds to the immunostimulatory activity in post-DLI sera, we isolated B, T, NK cells, monocytes, myeloid DCs and pDCs, and cultured them with pre- or post-DLI responder sera. In contrast to published findings that SLE sera stimulate pDCs, we found that post DLI sera induced normal monocytes to express high levels of MIP-1α, IP-10, MCP, TNF-α, IFN-α, IL-6, IL-8 and IL-12. Pretreatment of post-DLI sera with DNase, RNase, papain or pepsin resulted in marked decrease in IL-8 induction, demonstrating that this endogenous immunostimulatory factor requires both nucleic acid and protein for its adjuvant activity. Four active post-DLI sera were then used to stimulate stable HEK transfectants expressing TLR2, 3, 4, 8 or 9. IL-8 expression increased only in the TLR8 and TLR9-expressing cells lines that are known to be responsive to RNA and DNA respectively. IL-8 expression was further induced by post-DLI sera in TLR9-expressing cell lines co-transfected with CD32, suggesting that internalization by FcR may enhance delivery of nucleic acids to endosomal TLR8/9. None of the TLR transfectants could be activated by pre-DLI sera from the same responder patients, nor by post-DLI sera from non-responders. Finally, sera from responders collected between 2 weeks to several months after DLI consistently activated TLR8 and TLR9 suggesting that endogenous TLR8/9 activation may contribute to the early immunologic events that lead to an effective DLI response. Ongoing studies are focused on precisely identifying the TLR8/TLR9 agonists in DLI responders, studying the immunologic effects of this endogenous adjuvant and determining the association of our previously identified CML antigens with nucleic acids that activate TLR8 and TLR9. In summary, we demonstrate for the first time that effective tumor immunity is correlated with the presence of endogenous adjuvant-immunoglobulin complexes in patient sera, and propose that these observations form the basis for novel tumor vaccine strategies.

Targeting the NF-κB pathway through pharmacological inhibition of IKK2 prevents human cytomegalovirus replication and virus-induced inflammatory response in infected endothelial cells

Endothelial cells are important reservoirs for human cytomegalovirus (HCMV) replication, dissemination and persistence. HCMV infection of endothelial cells has been associated with a proinflammatory response characterized by an increased expression of chemokines and adhesion molecules and modulation of angiogenesis. Many of the host proinflammatory genes augmented in HCMV-infected endothelial cells are regulated, at least in part, by the NF-κB pathway. HCMV is a potent activator of NF-κB through the IKK-IκB signaling axis. To explore whether inhibition of HCMV-induced NF-κB activation may interfere with the onset of virus-associated inflammatory response, we measured the effects of the specific IKK2 inhibitor AS602868 on the expression of a panel of proinflammatory genes in HUVEC cells infected with a clinical isolate. Treatment of infected HUVEC with AS602868 was shown to impair HCMV-induced NF-κB activity, IE gene expression, viral replication and to prevent HCMV-induced upregulation of ICAM-1, IL-8, RANTES, IP-10, I-TAC and COX-2 gene expression. Consistent with these results, HCMV-mediated upregulation of another NF-κB-dependent gene, the plasminogen inhibitor type-1, a regulatory factor of endothelial proliferation and angiogenesis, was abrogated by AS602868. These results suggest that inhibition of HCMV-induced IKK-NF-κB activation may be of interest to limit the virus-induced inflammatory response of infected endothelial cells.

Induction of the chemokine interferon-γ-inducible protein-10 in human pancreatic islets during enterovirus infection

Enterovirus infections have long been suspected to be environmental factors that may cause type 1 diabetes, but the pathways leading from infection to beta cell destruction are still unknown. We therefore examined whether enterovirus infection of human islets leads to upregulation of interferon-gamma-inducible protein (IP-10, now known as chemokine [C-X-C motif] ligand 10 [CXCL10]), a chemokine important for the induction of insulitis. Isolated human islets were infected with three different strains of Coxsackie B4 virus. IP-10 expression and secretion from the infected human islets were then measured using RT-PCR and ELISA at several time points. IP-10 was clearly upregulated in and secreted from human islets during enterovirus infection. This was demonstrated with three different strains of Coxsackie B4 virus, two of which are lytic to islets and one which is non-lytic and can establish a persistent infection in human islets. We propose that enterovirus-induced upregulation of IP-10 during infection of the islets in vivo is the first step towards destructive insulitis. Our findings support the idea that enterovirus infection triggers immune-mediated beta cell destruction, and for the first time suggest a possible mechanism behind enterovirus-induced diabetes.

Interferon-γ-inducible Protein (IP)-10 mRNA Stabilized by RNA-binding Proteins in Monocytes Treated with S100b

Chemokines mediate the recruitment and activation of blood monocyte/macrophages and lymphocytes to sites of inflammation. Expression of the chemokine IP-10 (interferon-gamma-inducible protein) has been documented in several inflammatory and autoimmune disorders including type 1 diabetes. However, the mechanism of its expression in monocytes or its functional role in diabetes is not known. Advanced glycation end products acting via their receptor, RAGE, play major roles in diabetic complications. In this study, we observed for the first time that S100b, an inflammatory protein as well as a specific RAGE ligand, significantly increased IP-10 mRNA and protein levels in THP-1 monocytes as well as peripheral blood monocytes. Promoter luciferase assays showed that IP-10 mRNA accumulation by S100b was not via increased transcription. On the other hand, S100b significantly increased IP-10 mRNA half-life and stability. This appeared to be mediated by S100b-induced binding of specific RNA-binding protein(s) to a 3'-untranslated region-responsive region of the IP-10 mRNA. Our results demonstrate for the first time that diabetic stimuli such as RAGE ligands can induce inflammatory gene expression in monocytes via increased message stability.

Constitutive and regulated expression and secretion of interferon-γ-inducible protein 10 (IP-10/CXCL10) in human adipocytes

Chemokine secretion by adipocytes has been postulated to initiate leukocyte infiltration in adipose tissue and might mediate an important step in the establishment of obesity-related chronic immune activation. The chemokine interferon (IFN)gamma-inducible protein-10 (IP-10/CXCL10) is a chemoattractant for various leukocyte subsets and has been implicated in the pathogenesis of atherosclerosis. This study investigates whether IP-10 is expressed in human adipocytes and whether its release is regulated by body mass index (BMI) or immunological stimuli. In cultures of human mature adipocytes and in vitro differentiated adipocytes, IP-10 expression under basal conditions and in the presence of IFNgamma, lipopolysaccharide (LPS) or interleukin (IL)-4 was characterized by reverse transcriptase-polymerase chain reaction, Luminex technology and immunofluorescence. IP-10 was expressed and secreted constitutively in most cultures of mature adipocytes from omental and subcutaneous (s.c.) depots. The association between IP-10 release and donor BMI was not significant. In in vitro differentiated adipocytes from s.c. and mammary depots and in mature s.c. adipocytes, IP-10 secretion was strongly upregulated by IFNgamma, whereas LPS or IL-4 did not affect IP-10 expression in s.c. mature adipocytes. Immunofluorescence confirmed IP-10 expression in adipocytes with abundant lipid droplets. Mature human adipocytes express and secrete the chemokine IP-10 and are thus identified as a novel cellular source of this disease-related immune mediator. IP-10 expression could be significantly induced by IFNgamma, but not by LPS, which points to both similar reactivities and functional differences between adipocytes and innate immune cells.

Early up‐regulation of CXC‐chemokine expression is associated with strong cellular immune responses to murine skin xenografts

The dynamic pattern of the cellular infiltration and mRNA expression of chemokines in rat-to-mouse skin xenografts were examined to gain an understanding of the possible role of chemokines in the stronger cellular immune responses to xenografts compared with the allo-response. The mean survival time of the xenografts was approximately 2 days less than that of allografts (10.7 +/- 0.9 days vs. 8.9 +/- 0.7 days, P < 0.05). In comparison with the allografts, the xenografts were characterized by a very early infiltration of monocytes/macrophages (day 3) and a larger number of CD4+ and CD8+ cells, as well as neutrophil infiltration in the early phase (day 5), and larger number of CD8+ and CD11b+ cells, as well as macrophage infiltration, in the later phase (day 7). Xenografts showed stronger interferon (IFN)-inducible 10-kDa protein (IP-10) and monokine induced by IFN (MIG) mRNA expression levels, which appeared earlier than in the allografts. In the later phase, strong expression of regulated on activation, normal T cell expressed and secreted was observed. Cytokine mRNA expression in the xenografts could be summarized by higher expression of IFN-gamma, interleukin (IL)1beta, IL6, and transforming growth factor -beta1 mRNA than in the allografts. These results suggest that the early increased CXC-chemokine expression, such as IP-10 and MIG, which has been known to be mainly produced by macrophages, may play a critical role in the stronger cellular xenograft rejection compared with allograft rejection. Therefore, MIG antiserum or CXCR3 antiserum was administered to the rat skin-engrafted mice every other day until rejection. Compared with the control normal rabbit serum-treated mice, either the MIG antiserum- or CXCR3 antiserum-treated mice showed a delayed rejection of approximately 2 days (8.3 +/- 0.5 days vs. 10.6 +/- 0.5 days or 10.8 +/- 1.9 days, respectively, P < 0.05). Overall, these results suggest that the more aggressive rejection of xenografts compared with allografts is due to the earlier expression of CXC-chemokines, IP-10 and MIG, and subsequent adjuvant effects of proinflammatory cytokines.

Antiangiogenic and Antitumor Activities of IL-27

IL-27 is a novel IL-6/IL-12 family cytokine playing an important role in the early regulation of Th1 responses. We have recently demonstrated that IL-27 has potent antitumor activity, which is mainly mediated through CD8(+) T cells, against highly immunogenic murine colon carcinoma. In this study, we further evaluated the antitumor and antiangiogenic activities of IL-27, using poorly immunogenic murine melanoma B16F10 tumors, which were engineered to overexpress single-chain IL-27 (B16F10 + IL-27). B16F10 + IL-27 cells exerted antitumor activity against not only s.c. tumor but also experimental pulmonary metastasis. Similar antitumor and antimetastatic activities of IL-27 were also observed in IFN-gamma knockout mice. In NOD-SCID mice, these activities were decreased, but were still fairly well-retained, suggesting that different mechanisms other than the immune response are also involved in the exertion of these activities. Immunohistochemical analyses with Abs against vascular endothelial growth factor and CD31 revealed that B16F10 + IL-27 cells markedly suppressed tumor-induced neovascularization in lung metastases. Moreover, B16F10 + IL-27 cells clearly inhibited angiogenesis by dorsal air sac method, and IL-27 exhibited dose-dependent inhibition of angiogenesis on chick embryo chorioallantoic membrane. IL-27 was revealed to directly act on HUVECs and induce production of the antiangiogenic chemokines, IFN-gamma-inducible protein (IP-10) and monokine induced by IFN-gamma. Finally, augmented mRNA expression of IP-10 and monokine induced by IFN-gamma was detected at the s.c. B16F10 + IL-27 tumor site, and antitumor activity of IL-27 was partially inhibited by the administration of anti-IP-10. These results suggest that IL-27 possesses potent antiangiogenic activity, which plays an important role in its antitumor and antimetastatic activities.

Type I interferon-associated skin recruitment of CXCR3+ lymphocytes in dermatomyositis

Dermatomyositis (DM) is an autoimmune disease of unknown origin affecting skin and muscles. Infiltrating autoreactive T lymphocytes are thought to play an important pathogenetic role, but it is unclear which mechanisms are involved in the recruitment of these cells. Recent studies provided evidence that a type I interferon (IFN)-driven immune response, including the recruitment of T cells via IP10/CXCR3 interactions, might be important for the generation of skin lesions of cutaneous lupus erythematosus (CLE), an autoimmune disease that shares some clinical and histopathological features with DM. We hypothesized that a similar mechanism might also be involved in the pathogenesis of DM skin lesions. Skin biopsies of 23 donors (11 DM, 5 healthy controls, 7 CLE controls) were analysed by immunohistochemistry using monoclonal antibodies against CD3, CD4, CD8, CD20, CD68, CD123, the chemokine receptor CXCR3 and its ligand IP10/CXCL10, and the myxovirus-resistance protein A (MxA)-protein, which is a specific marker for type I IFNs. We detected strong expression of the MxA protein in all DM skin biopsies, indicating involvement of type I IFNs. Expression of MxA was closely associated with expression of the interferon-inducible protein IP10/CXCL10 and the recruitment of CXCR3+ lymphocytes. Plasmacytoid dendritic cells appear to be an important source of type I IFNs in DM. Our results support the hypothesis that lesional type I IFN signalling, induction of IP10 expression, and recruitment of potentially autoreactive T cells via IP10/CXCR3 interaction are involved in the pathogenesis of DM skin lesions.

Differential expression of retinal pigment epithelium (RPE) IP-10 and interleukin-8

Interferon-γ induced protein of 10 kDa (IP-10) is a C–X–C chemokine that attracts T lymphocytes and inhibits angiogenesis. In this study, we investigated the expression of IP-10 by human retinal pigment epithelial cells (HRPE) and compared IP-10 expression to that of interleukin-8 (IL-8), which is a leukocytic chemoattractant and pro-angiogenic factor. Cultured HRPE cells were incubated with either IL-1β (0.2–20 ng/ml) or tumor necrosis factor (TNF)-α (0.2–20 ng/ml) alone or in combination with interferon-γ (IFN-γ) (1000 U/ml). HRPE cells were also incubated with: (1) media conditioned by activated human T lymphocytes (CM), or (2) the same CM treated with neutralizing antibodies to IL-1, TNF, and/or IFN-γ. IL-8 and IP-10 protein levels were measured by ELISA and mRNA levels by Northern blot analysis of HRPE cells. HRPE cells produced very high levels of IP-10 in response to either IL-1β/IFN-γ, TNF-α/IFN-γ or CD3-activated T-lymphocyte CM. The levels of IP-10 were at least tenfold higher ( p < .001) than IL-8 measured in the same samples. Neutralizing antibodies to TNF and IFN-γ, but not to IL-1, abrogated the ability of the CD3-activated T lymphocytes CM to induce HRPE IP-10 ( p < .001). HRPE cells produce differential levels of IP-10 and IL-8 in response to various combinations of recombinant and T-lymphocyte-secreted pro-inflammatory cytokines. This may be important in evolving inflammatory and angiogenic ocular responses.

Early post-transplant urinary IP-10 expression after kidney transplantation is predictive of short- and long-term graft function

The early identification of renal transplant recipients at enhanced risk of developing acute and subclinical rejection would allow individualized adjustment of immunosuppression before functional graft injury occurs and would exclude these patients from drug-weaning studies. Protein and reverse transcriptase-polymerase chain reaction-based analyses of candidate markers in urine open the opportunity to closely monitor kidney-transplanted patients non-invasively. The chemokine interferon-inducible protein 10 (IP-10; CXCL10) might be an interesting candidate to uncover ongoing immune processes within the graft. Urine samples from kidney-transplanted recipients were retrospectively analyzed for IP-10 mRNA and protein expression. IP-10 levels were correlated with the incidence of acute rejection episodes proven by histology and long-term graft function assessed by the glomerular filtration rate 6 months post transplantation. IP-10 expression in urine identified patients with ongoing acute rejection episodes several days before a biopsy was indicated by rising serum creatinine levels. Most importantly, elevated levels of urinary IP-10 protein within the first four postoperative weeks were predictive of graft function at 6 months even in the absence of acute rejection. These data reveal a correlation between elevated IP-10 expression in urine at early time points post-transplantation and intragraft immune activation that leads to acute rejection and compromised long-term graft function.

Expression of IP-10/CXCL10 Is Upregulated by Double-Stranded RNA in BEAS-2B Bronchial Epithelial Cells

Background: Interferon (IFN)-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a potent chemoattractant for activated T and NK cells, and elevated levels of IP-10 are identified in bronchoalveolar lavage fluids from patients with pulmonary disorders related to Th-1-type immunity, which is a prerequisite for elimination of viral pathogens. Bronchial epithelial cells play an important role in respiratory infections as the initiator of airway inflammation by releasing chemokines and expressing cell surface membrane molecules involved in leukocyte adhesion. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA) and induces antiviral reactions in cells. Objectives: We investigated the regulation of IP-10 in BEAS-2B bronchial epithelial cells in response to poly IC, and also addressed the possible role of retinoic-acid-inducible gene-I (RIG-I) and IFN-regulatory factor 3 (IRF-3), two genes involved in the signaling induced by viral infection. Methods: The expressions of IP-10 mRNA and protein were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. The overexpression of RIG-I or IRF-3 was performed by transfection of BEAS-2B cells with each cDNA. Results: Poly IC enhanced the expression of IP-10 mRNA and protein in concentration- and time-dependent manners. Overexpression of RIG-I or IRF-3 potentiated the poly-IC-induced upregulation of IP-10. Conclusions: IP-10 may contribute to antiviral activity through the activation of Th-1-type immunity, and RIG-I and IRF-3 may be involved in this reaction.

Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis

IL-23 is a member of the IL-12 cytokine family that drives a highly pathogenic T cell population involved in the initiation of autoimmune diseases. We have shown that IL-23-dependent, pathogenic T cells produced IL-17 A, IL-17 F, IL-6, and TNF but not IFN-gamma or IL-4. We now show that T-bet and STAT1 transcription factors are not required for the initial production of IL-17. However, optimal IL-17 production in response to IL-23 stimulation appears to require the presence of T-bet. To explore the clinical efficacy of targeting the IL-23 immune pathway, we generated anti-IL-23p19-specific antibodies and tested to determine whether blocking IL-23 function can inhibit EAE, a preclinical animal model of human multiple sclerosis. Anti-IL-23p19 treatment reduced the serum level of IL-17 as well as CNS expression of IFN-gamma, IP-10, IL-17, IL-6, and TNF mRNA. In addition, therapeutic treatment with anti-IL-23p19 during active disease inhibited proteolipid protein (PLP) epitope spreading and prevented subsequent disease relapse. Thus, therapeutic targeting of IL-23 effectively inhibited multiple inflammatory pathways that are critical for driving CNS autoimmune inflammation.