Differential regulation of hyaluronan-induced IL-8 and IP-10 in airway epithelial cells

Airway epithelium is emerging as a regulator of local inflammation and immune responses. However, the cellular and molecular mechanisms responsible for the immune modulation by these cells have yet to be fully elucidated. At the cellular level, the hallmarks of airway inflammation are mucus gland hypertrophy with excess mucus production, accumulation of inflammatory mediators, inflammation in the airway walls and lumen, and breakdown and turnover of the extracellular matrix. We demonstrate that fragments of the extracellular matrix component hyaluronan induce inflammatory chemokine production in primary airway epithelial cells grown at an air-liquid interface. Furthermore, hyaluronan fragments use two distinct molecular pathways to induce IL-8 and IFN-gamma-inducible protein 10 (IP-10) chemokine expression in airway epithelial cells. Hyaluronan-induced IL-8 requires the MAP kinase pathway, whereas hyaluronan-induced IP-10 utilizes the NF-kappaB pathway. The induction is specific to low-molecular-weight hyaluronan fragments as other glycosaminoglycans do not induce IL-8 and IP-10 in airway epithelial cells. We hypothesize that not only is the extracellular matrix a target of destruction in airway inflammation but it plays a critical role in perpetuating inflammation through the induction of cytokines, chemokines, and modulatory enzymes in epithelial cells. Furthermore, hyaluronan, by inducing IL-8 and IP-10 by distinct pathways, provides a unique target for differential regulation of key inflammatory chemokines.     

An IP-10 (CXCL10)-derived peptide inhibits angiogenesis

Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77-98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis.     

CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10)

The chemokine IP-10 (interferon-inducible protein of 10 kDa, CXCL10) binds the G protein-coupled receptor CXCR3, which is found mainly on activated T cells and NK cells, and plays an important role in Th1-type inflammatory diseases. IP-10 also binds to glycosaminoglycans (GAGs), an interaction thought to be important for its sequestration on endothelial and other cells. In this study, we performed an extensive mutational analysis to identify the CXCR3 and heparin binding sites of murine IP-10. The mutants were characterized for heparin binding, CXCR3 binding, and the ability to induce chemotaxis, Ca(2+) flux, and CXCR3 internalization. Double mutations neutralizing adjacent basic residues at the C terminus did not lead to a significant reduction in heparin binding, indicating that the main heparin binding site of IP-10 is not along the C-terminal alpha helix. Alanine exchange of Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, preceding the first cysteine, was critical for CXCR3 signaling. Mutations of charged and uncharged residues in the loop regions of residues 20-24 and 46-47, which caused reduced heparin binding, also resulted in reduced CXCR3 binding and signaling. CXCR3 expressing GAG-deficient Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.     

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.     

Role of NF-kappa B in cytokine production induced from human airway epithelial cells by rhinovirus infection

Infection of human epithelial cells with human rhinovirus (HRV)-16 induces rapid production of several proinflammatory cytokines, including IL-8, IL-6, and GM-CSF. We evaluated the role of NF-kappaB in HRV-16-induced IL-8 and IL-6 production by EMSA using oligonucleotides corresponding to the binding sites for NF-kappaB in the IL-6 and IL-8 gene promoters. Consistent with the rapid induction of mRNA for IL-8 and IL-6, maximal NF-kappaB binding to both oligonucleotides was detected at 30 min after infection. NF-kappaB complexes contained p65 and p50, but not c-Rel. The IL-8 oligonucleotide bound recombinant p50 with only about one-tenth the efficiency of the IL-6 oligonucleotide, even though epithelial cells produced more IL-8 protein than IL-6. Neither the potent glucocorticoid, budesonide (10-7 M), nor a NO donor inhibited NF-kappaB binding to either cytokine promoter or induction of mRNA for either IL-8 or IL-6. Sulfasalazine and calpain inhibitor I, inhibitors of NF-kappaB activation, blocked HRV-16-induced formation of NF-kappaB complexes with oligonucleotides from both cytokines, but did not inhibit mRNA induction for either cytokine. By contrast, sulfasalazine clearly inhibited HRV-16 induction of mRNA for GM-CSF in the same cells. Thus, HRV-16 induces epithelial expression of IL-8 and IL-6 by an NF-kappaB-independent pathway, whereas induction of GM-CSF is at least partially dependent upon NF-kappaB activation.     

Nerve growth factor modulates human rhinovirus infection in airway epithelial cells by controlling ICAM-1 expression

Human rhinoviruses (HRV) are the most common agent of upper respiratory infections and an important cause of lower respiratory tract symptoms. Our previous research with other viral pathogens has shown that virus-induced airway inflammation and hyperreactivity involve neurotrophic pathways that also affect tropism and severity of the infection. The goals of this study were to analyze systematically the expression of key neurotrophic factors and receptors during HRV-16 infection of human airway epithelial cells and to test the hypothesis that neurotrophins modulate HRV infection by controlling the expression of a major cellular receptor for this virus, the intercellular adhesion molecule 1 (ICAM-1). Neurotrophins and ICAM-1 expression were analyzed at the mRNA level by real-time PCR and at the protein level by flow cytometry and immunocytochemistry. A small inhibitory RNA (siRNA) or a specific blocking antibody was utilized to suppress nerve growth factor (NGF) expression and measure its effects on viral replication and virus-induced cell death. Nasal and bronchial epithelial cells were most susceptible to HRV-16 infection at 33°C and 37°C, respectively, and a significant positive relationship was noted between expression of NGF and tropomyosin-related kinase A (TrkA) and virus copy number. ICAM-1 expression was dose dependently upregulated by exogenous NGF and significantly downregulated by NGF inhibition with corresponding decrease in HRV-16 replication. NGF inhibition also increased apoptotic death of infected cells. Our results suggest that HRV upregulates the NGF-TrkA pathway in airway epithelial cells, which in turn amplifies viral replication by increasing HRV entry via ICAM-1 receptors and by limiting apoptosis.     

CXCL10/IP-10 in infectious diseases pathogenesis and potential therapeutic implications

C–X–C motif chemokine 10 (CXCL10) also known as interferon γ-induced protein 10 kDa (IP-10) or small-inducible cytokine B10 is a cytokine belonging to the CXC chemokine family. CXCL10 binds CXCR3 receptor to induce chemotaxis, apoptosis, cell growth and angiostasis. Alterations in CXCL10 expression levels have been associated with inflammatory diseases including infectious diseases, immune dysfunction and tumor development. CXCL10 is also recognized as a biomarker that predicts severity of various diseases. A review of the emerging role of CXCL10 in pathogenesis of infectious diseases revealed diverse roles of CXCL10 in disease initiation and progression. The potential utilization of CXCL10 as a therapeutic target for infectious diseases is discussed.     

Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis

Introduction  

Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum.     

Role of IP-10/CXCL10 in the progression of pancreatitis-like injury in mice after murine retroviral infection

Exocrinopathy and pancreatitis-like injury were developed in C57BL/6 (B6) mice infected with LP-BM5 murine leukemia virus, which is known to induce murine acquired immunodeficiency syndrome (MAIDS). The role of chemokines, especially CXCL10/interferon (IFN)-gamma-inducible protein 10 (IP-10), a chemokine to attract CXCR3+ T helper 1-type CD4+ T cells, has not been investigated thoroughly in the pathogenesis of pancreatitis. B6 mice were inoculated intraperitoneally with LP-BM5 and then injected every week with either an antibody against IP-10 or a control antibody. Eight weeks after infection, we analyzed the effect of IP-10 neutralization. Anti-IP-10 antibody treatment did not change the generalized lymphadenopathy and hepatosplenomegaly of mice with MAIDS. The treatment significantly reduced the number of IP-10- and CXCR3-positive cells in the mesenteric lymph nodes (mLNs) but not the phenotypes and gross numbers of cells. In contrast, IP-10 neutralization reduced the number of mononuclear cells infiltrating into the pancreas. Anti-IP-10 antibody treatment did not change the numbers of IFN-gamma+ and IL10+ cells in the mLN but significantly reduced their numbers, especially IFN-gamma+ and IL-10+ CD4+ T cells and IFN-gamma+ Mac-1+ cells, in the pancreas. IP-10 neutralization ameliorated the pancreatic lesions of mice with MAIDS probably by blocking the cellular infiltration of CD4+ T cells and IFN-gamma+ Mac-1+ cells into the pancreas at least at 8 wk after infection, suggesting that IP-10 and these cells might play a key role in the development of chronic autoimmune pancreatitis.     

IFN-gamma-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inflammation in a mouse model of asthma

Allergic asthma is an inflammatory disease of the airways characterized by eosinophilic inflammation and airway hyper-reactivity. Cytokines and chemokines specific for Th2-type inflammation predominate in asthma and in animal models of this disease. The role of Th1-type inflammatory mediators in asthma remains controversial. IFN-gamma-inducible protein 10 (IP-10; CXCL10) is an IFN-gamma-inducible chemokine that preferentially attracts activated Th1 lymphocytes. IP-10 is up-regulated in the airways of asthmatics, but its function in asthma is unclear. To investigate the role of IP-10 in allergic airway disease, we examined the expression of IP-10 in a murine model of asthma and the effects of overexpression and deletion of IP-10 in this model using IP-10-transgenic and IP-10-deficient mice. Our experiments demonstrate that IP-10 is up-regulated in the lung after allergen challenge. Mice that overexpress IP-10 in the lung exhibited significantly increased airway hyperreactivity, eosinophilia, IL-4 levels, and CD8(+) lymphocyte recruitment compared with wild-type controls. In addition, there was an increase in the percentage of IL-4-secreting T lymphocytes in the lungs of IP-10-transgenic mice. In contrast, mice deficient in IP-10 demonstrated the opposite results compared with wild-type controls, with a significant reduction in these measures of Th2-type allergic airway inflammation. Our results demonstrate that IP-10, a Th1-type chemokine, is up-regulated in allergic pulmonary inflammation and that this contributes to the airway hyperreactivity and Th2-type inflammation seen in this model of asthma.     

Interferon-independent, human immunodeficiency virus type 1 gp120-mediated induction of CXCL10/IP-10 gene expression by astrocytes in vivo and in vitro.

The CXC chemokine gamma interferon (IFN-gamma)-inducible protein CXCL10/IP-10 is markedly elevated in cerebrospinal fluid and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1) and is implicated in the pathogenesis of HIV-associated dementia (HAD). To explore the possible role of CXCL10/IP-10 in HAD, we examined the expression of this and other chemokines in the central nervous system (CNS) of transgenic mice with astrocyte-targeted expression of HIV gp120 under the control of the glial fibrillary acidic protein (GFAP) promoter, a murine model for HIV-1 encephalopathy. Compared with wild-type controls, CNS expression of the CC chemokine gene CCL2/MCP-1 and the CXC chemokine genes CXCL10/IP-10 and CXCL9/Mig was induced in the GFAP-HIV gp120 mice. CXCL10/IP-10 RNA expression was increased most and overlapped the expression of the transgene-encoded HIV gp120 gene. Astrocytes and to a lesser extent microglia were identified as the major cellular sites for CXCL10/IP-10 gene expression. There was no detectable expression of any class of IFN or their responsive genes. In astrocyte cultures, soluble recombinant HIV gp120 protein was capable of directly inducing CXCL10/IP-10 gene expression a process that was independent of STAT1. These findings highlight a novel IFN- and STAT1-independent mechanism for the regulation of CXCL10/IP-10 expression and directly link expression of HIV gp120 to the induction of CXCL10/IP-10 that is found in HIV infection of the CNS. Finally, one function of IP-10 expression may be the recruitment of leukocytes to the CNS, since the brain of GFAP-HIV gp120 mice had increased numbers of CD3(+) T cells that were found in close proximity to sites of CXCL10/IP-10 RNA expression.     

Bronchial epithelial cells release IL-6, CXCL1 and CXCL8 upon mast cell interaction

Although mast cells have been found in increased numbers in bronchial epithelium in asthma patients, the pathogenic role of the interaction of mast cells with bronchial epithelial cells in the development of local inflammation in asthma is not well understood. In this study, primary human bronchial epithelial cells and a human mast cell line (HMC-1) were cultured either together or separately in the presence or absence of various signaling molecule inhibitors or dexamethasone. Cytokine IL-6, and chemokines including CXCL1 and CXCL8 in cell culture supernatant were assayed by enzyme-linked immunosorbent assay (ELISA), and the activity of mitogen-activated protein kinases (MAPKs), or nuclear factor-κB (NF-κB) in co-culture system was analyzed by ELISA. Co-culture of bronchial epithelial cells and mast cells induced a significant elevation of IL-6, CXCL1 and CXCL8 in bronchial epithelial cells, and both IL-17A and IL-17F could further enhance the release of these inflammatory mediators from co-culture. The induction of IL-6, CXCL1 and CXCL8 upon the interaction of bronchial epithelial cells with mast cells was mediated by MAPKs and NF-κB signaling pathways. These data indicate that the interaction of mast cells with bronchial epithelial cells may represent a crucial mechanism of regulating local inflammatory response in allergic asthma.     

CXCL10/XCL1 fusokine elicits in vitro and in vivo chemotaxis

Fusokines are proteins formed by the fusion of two cytokines. They have greater bioavailability and therapeutic potential than individual cytokines or a combination of different cytokines. Interferon-gamma-inducible protein 10 (CXCL10) and lymphotactin (XCL1) are members of the chemotactic family of cytokines, which induce tumor regression by eliciting immune-system cell chemotaxis. We engineered a replication-deficient adenoviral system expressing CXCL10/XCL1 fusokine (Ad FIL) and assessed its chemotactic response in vitro and in vivo. The CXCL10/XCL1 fusokine elicited a greater chemotactic effect in IL-2 stimulated lymphocytes than individual or combined cytokines in vitro. CXCL10/XCL1 fusokine biological activity was demonstrated in vivo by intratumoral chemoattraction of CXCR3+ cells. Thus, this novel CXCL10/XCL1 fusokine may represent a potential tool for gene therapy treatment of cancer and other illnesses that require triggering immune-system cell recruitment.     

Effects of dexamethasone on rhinovirus infection in cultured human tracheal epithelial cells

To examine the effects of glucocorticoid on rhinovirus (RV) infection, primary cultures of human tracheal epithelial cells were infected with either RV2 or RV14. Viral infection was confirmed by demonstrating that viral RNA in infected cells and viral titers of supernatants and lysates from infected cells increased with time. RV14 infection upregulated the expression of mRNA and protein of intercellular adhesion molecule-1 (ICAM-1), the major RV receptor, on epithelial cells, and it increased the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor-alpha in supernatants. Dexamethasone reduced the viral titers of supernatants and cell lysates, viral RNA of infected cells, and susceptibility of RV14 infection in association with inhibition of cytokine production and ICAM-1 induction. In contrast to RV14 infection, dexamethasone did not alter RV2 infection, a minor group of RVs. These results suggest that dexamethasone may inhibit RV14 infection by reducing the surface expression of ICAM-1 in cultured human tracheal epithelial cells. Glucocorticoid may modulate airway inflammation via reducing the production of proinflammatory cytokines and ICAM-1 induced by rhinovirus infection.     

Human thymic epithelial cells produce granulocyte and macrophage colony-stimulating factors

The development of culture conditions for growing normal human thymic epithelial (TE) cells free from contamination with other stromal cells has allowed us to identify and characterize TE cell-derived cytokines. In this study, we report that cultured human TE cells produced CSF that supported the growth of clonal hematopoietic progenitor cells in the light density fraction of human bone marrow cells. Thymic epithelial supernatants (TES) induced growth of granulocyte/macrophage colonies (CFU-GM), mixed granulocyte/erythrocyte/monocyte/megakaryocyte colonies (CFU-GEMM), and early burst-forming unit erythroid colonies (BFU-E). In addition, TES induced differentiation of the promyelocyte leukemic cell line HL-60 and stimulated growth of both granulocyte (CFU-G) and monocyte (CFU-M) colonies from murine bone marrow cells. Using anion exchange column chromatography, pluripotent CSF activities in TES were separated and shown to be distinct from an IL-1-like cytokine that has been shown as a TE cell-derived cytokine (TE-IL-1). Colony-stimulating activity supporting the growth of bone marrow CFU-GEMM, BFU-E, and CFU-GM co-eluted at 150 to 180 mM NaCl. A separate peak of CFU-GM-stimulating activity eluted early in the gradient at 20 mM NaCl. In Northern blot analysis of enriched RNA, synthetic oligonucleotide probes complementary to human G-CSF and M-CSF coding sequence each hybridized with a single RNA species of 1.7 and 4.4 kb, respectively. These data suggest that normal human TE cells synthesize G-CSF and M-CSF that promote differentiation of non-lymphoid hematopoietic cell precursors.     

In vivo glycosylation of MUC1 in airway epithelial cells

The O-glycans that decorate mucin glycoproteins contribute to the biophysical and biochemical properties of these molecules and hence their function as a barrier and lubricant on epithelial surfaces. Alterations in mucin O-glycosylation in certain diseases may contribute to pathology. It is known that both the host cell type and the amino acid sequence of the mucin tandem repeat contribute to the O-glycosylation of a mucin molecule. We expressed an epitope-tagged MUC1 mucin cDNA construct in the airway cell line 16HBE14o- and the colon carcinoma cell line Caco2 and used Fast Atom Bombardment Mass Spectrometry to evaluate the contribution of the host cell to differences in O-glycosylation of a single mucin. Many of the glycans detected on the MUC1 mucin were common to both cell types, as would be predicted from biosynthetic constraints. However, MUC1 synthesized in the airway cell line showed comparatively low levels of sialylation but carried a range of oligo-N-acetyllactosamine structures that were not seen in the colon carcinoma cell line.