Michael P. Keane

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.

CXC chemokines in angiogenesis

A variety of factors have been identified that regulate angiogenesis, including the CXC chemokine family. The CXC chemokines are a unique family of cytokines for their ability to behave in a disparate manner in the regulation of angiogenesis. CXC chemokines have four highly conserved cysteine amino acid residues, with the first two cysteine amino acid residues separated by one non‐conserved amino acid residue (i.e., CXC). A second structural domain within this family determines their angiogenic potential. The NH2 terminus of the majority of the CXC chemokines contains three amino acid residues (Glu‐Leu‐Arg: the ELR motif), which precedes the first cysteine amino acid residue of the primary structure of these cytokines. Members that contain the ELR motif (ELR+) are potent promoters of angiogenesis. In contrast, members that are inducible by interferons and lack the ELR motif (ELR−) are potent inhibitors of angiogenesis. This difference in angiogenic activity may impact on the pathogenesis of a variety of disorders.

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.

Depletion of CXCR2 Inhibits Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer

The Glu-Leu-Arg+ (ELR+) CXC chemokines are potent promoters of angiogenesis and have been demonstrated to induce a significant portion of nonsmall cell lung cancer-derived angiogenic activity and support tumorigenesis. ELR+ CXC chemokines share a common chemokine receptor, CXCR2. We hypothesized that CXCR2 mediates the proangiogenic effects of ELR+ CXC chemokines during tumorigenesis. To test this postulate, we used syngeneic murine Lewis lung cancer (LLC; 3LL, H-2b) heterotopic and orthotopic tumor model systems in C57BL/6 mice replete (CXCR2+/+) and deficient in CXCR2 (CXCR2−/−). We first demonstrated a correlation of the expression of endogenous ELR+ CXC chemokines with tumor growth and metastatic potential of LLC tumors. Next, we found that LLC primary tumors were significantly reduced in growth in CXCR2−/− mice. Moreover, we found a marked reduction in the spontaneous metastases of heterotopic tumors to the lungs of CXCR2−/− mice. Morphometric analysis of the primary tumors in CXCR2−/− mice demonstrated increased necrosis and reduced vascular density. These findings were further confirmed in CXCR2+/+ mice using specific neutralizing Abs to CXCR2. The results of these studies support the notion that CXCR2 mediates the angiogenic activity of ELR+ CXC chemokines in a preclinical model of lung cancer.

The role of CXC chemokines in pulmonary fibrosis

The CXC chemokine family is a pleiotropic family of cytokines that are involved in promoting the trafficking of various leukocytes, in regulating angiogenesis and vascular remodeling, and in promoting the mobilization and trafficking of mesenchymal progenitor cells such as fibrocytes. These functions of CXC chemokines are important in the pathogenesis of pulmonary fibrosis and other fibroproliferative disorders. In this Review, we discuss the biology of CXC chemokine family members, specifically as it relates to their role in regulating vascular remodeling and trafficking of circulating mesenchymal progenitor cells (also known as fibrocytes) in pulmonary fibrosis.

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.

Cytokines in innate host defense in the lung.

Historically the lung has been perceived as an organ primarily involved in gas exchange. However, due to its unique relationship with the environment, the lung must defend itself from infection by numerous inhaled micro-organisms. Various innate defenses protect the lung from infection, including the cough reflex, mucociliary clearance (see Knowles and Boucher, this Perspective series, ref. 1), and antimicrobial properties of the mucosal surface (see Ganz, this series, ref. 2; and McCormack and Whitsett, this series, ref. 3). In addition, an extensive alveolar-capillary membrane containing immune and nonimmune cells is exposed to microbial challenges. Consequently, pulmonary tissues generate a brisk innate host response to both inhaled and hematogenous pathogens in order to clear the offending micro-organism and preserve gas exchange. The successful execution of the innate defense response in the lung is critical to the eventual transition and development of the adaptive immunity. Once containment of the micro-organism has occurred, the response resolves, leading to repair or tissue remodeling. Loss of these later responses leaves the lung susceptible to tissue injury caused by excessive local or distant innate responses. In conditions such as acute respiratory distress syndrome, overexuberant tissue inflammation in response to micro-organisms may lead to irreversible lung injury and mortality. While a variety of factors — arachidonic acid metabolites, coagulation factors, complement, acute-phase proteins, and antimicrobial peptides among them — are involved in the innate response, cytokines constitute the largest and most pleiotropic group of such mediators and will be the focus of this Perspective. The initiation, maintenance, and resolution of pulmonary innate responses depend upon cellular communication via cytokines. Along with other soluble factors, as well as adhesion molecules, the cytokines contribute to the recognition of pathogens, the recruitment of neutrophils and mononuclear cells, and the removal of the invading micro-organism. Cytokine signaling occurs through receptor-ligand interactions on specific immune or nonimmune target cell populations. These populations differ not only in their complement of cytokine receptors, but also in their capacities to elaborate and secrete specific cytokines in response to particular stimuli. Interactions among various cell populations have led to the concept of cytokine networking, in which one population of cells may respond directly to specific exogenous or endogenous stimuli, leading to the elaboration of a particular cytokine that exerts distinct effects upon another population of cells. The targets respond by producing cytokines, which may serve as feedback signals to the initiating cell, or, alternatively, by releasing signaling molecules that affect yet another array of target cells. Inflammatory effector cells, such as neutrophils and monocytes, may be locally recruited and activated in response to specific chemotactic signals, resulting in further amplification of a cytokine cascade by nonimmune resident cells.

Pleuropulmonary manifestations of systemic lupus erythematosus

The pleuropulmonary manifestation of systemic lupus erythematous (SLE) are pleuritis, acute lupus pneumonitis, chronic interstitial lung disease with fibrosis, alveolar hemorrhage, respiratory muscle and diaphragmatic dysfunction, atelectasis, bronchiolitis obliterans, pulmonary vascular disease with pulmonary hypertension, and pulmonary embolism. This article reviews these specific pleuropulmonary consequences of SLE while focusing on clinical, pathologic, and therapeutic considerations.