Ying Ying Xue

Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis

Previous reports have identified a circulating pool of CD45(+) collagen I(+) CXCR4(+) (CD45(+)Col I(+)CXCR4(+)) cells, termed fibrocytes, that traffic to areas of fibrosis. No studies have demonstrated that these cells actually contribute to fibrosis, however. Pulmonary fibrosis was originally thought to be mediated solely by resident lung fibroblasts. Here we show that a population of human CD45(+)Col I(+)CXCR4(+) circulating fibrocytes migrates in response to CXCL12 and traffics to the lungs in a murine model of bleomycin-induced pulmonary fibrosis. Next, we demonstrated that murine CD45(+)Col I(+)CXCR4(+) fibrocytes also traffic to the lungs in response to a bleomycin challenge. Maximal intrapulmonary recruitment of CD45(+)Col I(+)CXCR4(+) fibrocytes directly correlated with increased collagen deposition in the lungs. Treatment of bleomycin-exposed animals with specific neutralizing anti-CXCL12 Abs inhibited intrapulmonary recruitment of CD45(+)Col I(+)CXCR4(+) circulating fibrocytes and attenuated lung fibrosis. Thus, our results demonstrate, we believe for the first time, that circulating fibrocytes contribute to the pathogenesis of pulmonary fibrosis.

The CXC Chemokine Receptor 2, CXCR2, Is the Putative Receptor for ELR+ CXC Chemokine-Induced Angiogenic Activity

We have previously shown that members of the ELR+ CXC chemokine family, including IL-8; growth-related oncogenes α, β, and γ; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR+ CXC chemokines has not been determined. Because all ELR+ CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR+ CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR+ CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR+ CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR+ CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR+ CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2−/− mice. We thus conclude that CXCR2 is the receptor responsible for ELR+ CXC chemokine-mediated angiogenesis.

Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury

Mortality related to adult respiratory distress syndrome (ARDS) ranges from 35% to 65%. Lung-protective ventilator strategies can reduce mortality during ARDS. The protective strategies limit tidal volumes and peak pressures while maximizing positive end-expiratory pressure. The efficacy of this approach is due to a reduction of shear-stress of the lung and release of inflammatory mediators. Ventilator-induced lung injury (VILI) is characterized by inflammation. The specific mechanism(s) that recruit leukocytes during VILI have not been elucidated. Because the murine CXC chemokines KC/CXCL1 and MIP-2/CXCL2/3, via CXCR2, are potent neutrophil chemoattractants, we investigated their role in a murine model of VILI. We compared two ventilator strategies in C57BL/6 mice: high peak pressure and high stretch (high peak pressure/stretch) versus low peak pressure/stretch for 6 hours. Lung injury and neutrophil sequestration from the high-peak pressure/stretch group were greater than those from the low-peak pressure/stretch group. In addition, lung expression of KC/CXCL1 and MIP-2/CXCL2/3 paralleled lung injury and neutrophil sequestration. Moreover, in vivo inhibition of CXCR2/CXC chemokine ligand interactions led to a marked reduction in neutrophil sequestration and lung injury. These findings were confirmed using CXCR2(-/-) mice. Together these experiments support the notion that increased expression of KC/CXCL1 and MIP-2/CXCL2/3 and their interaction with CXCR2 are important in the pathogeneses of VILI.

Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival after lung transplantation. Acute rejection, its main risk factor, is characterized by perivascular/bronchiolar leukocyte infiltration. BOS is characterized by persistent peribronchiolar leukocyte recruitment leading to airway fibrosis and obliteration. The specific mechanism(s) by which these leukocytes are recruited are unknown. Because MCP-1, acting through its receptor CCR2, is a potent mononuclear cell chemoattractant, we hypothesized that expression of this chemokine during an allogeneic-response promotes persistent recruitment of leukocytes and, ultimately, rejection. We found that elevated levels of biologically active MCP-1 in human bronchial lavage fluid (BALF) were associated with the continuum from acute to chronic allograft rejection. Translational studies in a murine model of BOS demonstrated increased MCP-1 expression paralleling mononuclear cell recruitment and CCR2 expression. Loss of MCP-1/CCR2 signaling, as seen in CCR2(-/-) mice or in WT mice treated with neutralizing antibodies to MCP-1, significantly reduced recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS. Lymphocyte infiltration was not reduced under these conditions. We suggest that MCP-1/CCR2 signaling plays an important role in recruitment of mononuclear phagocytes, a pivotal event in the pathogenesis of BOS.

Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor in non-small cell lung cancer

We report here the role of the CXC chemokine, epithelial neutrophil activating peptide (ENA-78), as an angiogenic factor in human non-small cell lung cancer (NSCLC). In freshly isolated human specimens of NSCLC, elevated levels of ENA-78 were found that strongly correlated with the vascularity of the tumors. In a SCID mouse model of human NSCLC tumorigenesis, expression of ENA-78 in developing tumors correlated with tumor growth in two different NSCLC cell lines. Furthermore, passive immunization of NSCLC tumor-bearing mice with neutralizing anti-ENA-78 antibodies reduced tumor growth, tumor vascularity, and spontaneous metastases, while having no effect on the proliferation of NSCLC cells either in vitro or in vivo. These findings suggest that ENA-78 is an important angiogenic factor in human NSCLC.

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

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

Distinct CXC chemokines mediate tumorigenicity of prostate cancer cells.

Prostate cancer is the second leading cause of malignancy-related mortality in males in the United States. As a solid tumor, clinically significant tumor growth and metastasis are dependent on nutrients and oxygen supplied by tumor-associated neovasculature. As such, there is a selective tumorigenic advantage for those neoplasms that can produce angiogenic mediators. We show here that human prostate cancer cell lines can constitutively produce angiogenic CXC chemokines. Tumorigenesis of PC-3 prostate cancer cells was shown to be attributable, in part, to the production of the angiogenic CXC chemokine, interleukin (IL)-8. Neutralizing antisera to IL-8 inhibits PC-3 tumor growth in a human prostate cancer/SCID mouse model. Furthermore, angiogenic activity in PC-3 tumor homogenates was attributable to IL-8. In contrast, the Du145 prostate cancer cell line uses a different angiogenic CXC chemokine, GRO-alpha, to mediate tumorigenicity. Neutralizing antisera to GRO-alpha but not IL-8 reduced tumor growth in vivo and reduced the angiogenic activity in tumor homogenates. Thus, prostate cancer cell lines can use distinct CXC chemokines to mediate their tumorigenicity.

The Role of the CC Chemokine, RANTES, in Acute Lung Allograft Rejection

Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.

The CXC chemokine, monokine induced by interferon-gamma, inhibits non-small cell lung carcinoma tumor growth and metastasis.

Angiogenesis is an absolute requirement for tumor growth beyond 2 mm3 in size. The balance in expression between opposing angiogenic and angiostatic factors controls the angiogenic process. The CXC chemokines are a group of chemotactic cytokines that possess disparate activity in the regulation of angiogenesis. Non-small cell lung carcinoma (NSCLC) has an imbalance in expression of ELR+ (angiogenic) compared with ELR- (angiostatic) CXC chemokines that favors angiogenesis and progressive tumor growth. We found that the level of the ELR- CXC chemokine MIG (monokine induced by interferon gamma) in human specimens of NSCLC was not significantly different from that found in normal lung tissue. These results suggested that the increased expression of ELR+ CXC chemokines found in these tumor samples is not counterregulated by a concomitant increase in the expression of the angiostatic ELR-CXC chemokine MIG. This would result in an even more profound imbalance in the expression of regulatory factors of angiogenesis that would favor neovascularization. We hypothesized that MIG might be an endogenous inhibitor of NSCLC tumor growth in vivo and that reconstituion of MIG in the tumor microenvironment would result in the inhibition of tumor growth and metastasis. In support of this hypothesis, we demonstrate here that overexpression of the ELR-CXC chemokine MIG, by three different strategies including gene transfer, results in the inhibition of NSCLC tumor growth and metastasis via a decrease in tumor-derived vessel density. These findings support the importance of the ELR- CXC chemokine MIG in inhibiting NSCLC tumor growth by attenuation of tumor-derived angiogenesis. Furthermore, these findings demonstrate the potential of gene therapy as an alternative means to deliver and overexpress a potent angiostatic CXC chemokine.

Role of CXCR2/CXCR2 ligands in vascular remodeling during bronchiolitis obliterans syndrome

Angiogenesis and vascular remodeling support fibroproliferative processes; however, no study has addressed the importance of angiogenesis during fibro-obliteration of the allograft airway during bronchiolitis obliterans syndrome (BOS) that occurs after lung transplantation. The ELR(+) CXC chemokines both mediate neutrophil recruitment and promote angiogenesis. Their shared endothelial cell receptor is the G-coupled protein receptor CXC chemokine receptor 2 (CXCR2). We found that elevated levels of multiple ELR(+) CXC chemokines correlated with the presence of BOS. Proof-of-concept studies using a murine model of BOS not only demonstrated an early neutrophil infiltration but also marked vascular remodeling in the tracheal allografts. In addition, tracheal allograft ELR(+) CXC chemokines were persistently expressed even in the absence of significant neutrophil infiltration and were temporally associated with vascular remodeling during fibro-obliteration of the tracheal allograft. Furthermore, in neutralizing studies, treatment with anti-CXCR2 Abs inhibited early neutrophil infiltration and later vascular remodeling, which resulted in the attenuation of murine BOS. A more profound attenuation of fibro-obliteration was seen when CXCR2(-/-) mice received cyclosporin A. This supports the notion that the CXCR2/CXCR2 ligand biological axis has a bimodal function during the course of BOS: early, it is important for neutrophil recruitment and later, during fibro-obliteration, it is important for vascular remodeling independent of neutrophil recruitment.