INTRODUCTION

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Keywords: angiogenesis; chemokine; fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal pulmonary disorder. Prevalence rates of 27 to 29 cases per 100,000 have been reported, and may even be as high as 250 cases per 100,000 individuals > 75 yr of age (1). The incidence of IPF appears to be on the rise (2). Conventional treatment with immunosuppressive therapy has been disappointing, with a median survival of 2-3 yr. The elucidation of mediators that orchestrate this aberrant tissue repair, will allow the development of novel interventions to treat this disorder.

The pathology of IPF demonstrates features of dysregulated and abnormal repair with exaggerated angiogenesis, fibroproliferation, and deposition of extracellular matrix, leading to progressive fibrosis and loss of lung function. The contribution of aberrant vascular remodeling to the progression of fibrosis in IPF has been largely ignored. The existence of neovascularization in IPF was originally identified by Turner-Warwick (3), who examined the lungs of patients with widespread interstitial fibrosis (IPF), and demonstrated neovascularization leading to anastomoses between the systemic and pulmonary microvasculature and evidence of extensive vascular remodeling in areas of fibrosis. Further evidence of neovascularization during the pathogenesis of pulmonary fibrosis has been seen in a rat model of bleomycin-induced pulmonary fibrosis (4).

Our laboratory has shown that members of the CXC chemokine family exert disparate effects in mediating angiogenesis as a function of the presence or absence of three amino acid residues (Glu-Leu-Arg; the "ELR motif") that immediately precede the first cysteine amino acid of the primary structure of these cytokines (5, 6). Epithelial neutrophil-activating peptide 78 (ENA-78) and interleukin 8 (IL-8) both contain the ELR motif, which confers potent angiogenic activity, whereas members such as interferon -inducible protein 10 (IP-10) and monokine induced by interferon-gamma (MIG), which lack the ELR motif, inhibit angiogenesis.

We have previously shown that the CXC chemokines IL-8 and IP-10 regulate angiogenic activity in IPF (7). To demonstrate proof of principle, we extended these studies to a murine model of bleomycin-induced pulmonary fibrosis, and we have shown that neutralization of macrophage inflammatory protein 2 (MIP-2), a murine functional homolog of IL-8, attenuates bleomycin-induced pulmonary fibrosis via inhibition of angiogenesis (8). In contrast, administration of the angiostatic chemokine IP-10 led to a reduction in pulmonary fibrosis that was mediated through the inhibition of angiogenesis.

In this study, we hypothesized that ENA-78 is an additional important regulator of angiogenic activity in IPF. We found that lung tissue from patients with IPF expressed greater levels of ENA-78 as compared with normal control lung tissue. These higher levels of ENA-78 were associated with increased angiogenic activity, as assessed by the corneal micropocket assay, that was significantly attributable to ENA-78. These findings indicate that ENA-78 is an important angiogenic factor in IPF.

	METHODS

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Details are provided in the online data supplement.

Population Studied

The subjects studied consisted of two distinct groups: patient with IPF (n = 91) and subjects (control) undergoing thoracic surgery for reasons other than interstitial lung disease (n = 78). Tissue specimens were obtained from consenting individuals in accordance with institutional review board approval. All had clinical and radiographic findings consistent with the diagnosis of IPF, and all had pathologic confirmation of the diagnosis of usual interstitial pneumonia made by open lung biopsy. None of the patients in either the IPF or control group had been previously treated or were currently being treated with corticosteroids or other immunosuppressive agents. The 78 control lung tissue specimens were from subjects undergoing thoracic surgery for either clinical Stage I or II non-small cell lung cancer (NSCLC). The control group lung tissue was obtained from a site distant from the primary tumor, and was histologically free of neoplasm.

Reagents

Polyclonal anti-human ENA-78-specific antiserum was produced by the immunization of rabbits with recombinant ENA-78 (R&D Systems, Minneapolis, MN) in multiple intradermal sites with Freund's complete adjuvant (9, 10). The specificity of this antibody was assessed by Western blot analysis against a panel of other human recombinant cytokines (9, 10). Antibodies were specific in our sandwich enzyme-linked immunosorbent assay (ELISA) without cross-reactivity to a panel of 12 human recombinant interleukins, including interleukin-1 receptor antagonist protein (IRAP), IL-1, IL-2, IL-4, IL-6, tumor necrosis factor  (TNF-), interferon  (IFN-), and other members of the CXC and CC chemokine families (9, 10). The "antiprotease" buffer for tissue homogenization consisted of 1× phosphate-buffered saline (PBS) with one Complete tablet (Boehringer Mannheim, Indianapolis, IN) per 50 ml.

Cytokine ELISA

Antigenic ENA-78 was quantitated by a modification of a double-ligand method as previously described (9, 10).

Corneal Micropocket Assay of Angiogenesis

Angiogenic activity of lung homogenates was assayed in vivo in the avascular cornea of hooded Long-Evans rat eyes, as previously described (5, 7). All animals were handled in accordance with the unit for laboratory animal medicine.

Immunohistochemistry of ENA-78

Paraffin-embedded tissue from IPF or control lung was processed for immunohistochemical localization of ENA-78, using a method previously described (7, 9).

Statistical Analysis

Data were analyzed on a Dell PC computer, using the Statview 4.5 statistical package (Abacus Concepts, Berkeley, CA). ELISA data were compared by the nonparametric Mann-Whitney test. A p value of 0.05 or less was considered significant.

	RESULTS

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Lung tissue from patients with IPF constitutively expresses more ENA-78 than does control lung tissue. We obtained open lung biopsies from patients undergoing thoracic surgery for reasons other than interstitial lung disease (control lung) (n = 78) and from patients with IPF (n = 91), and measured ENA-78 by specific ELISAs standardized to 6-mm punch biopsy. Open lung biopsies from patients with IPF, as compared with control lung tissue, demonstrated greater levels of ENA-78 (37.7 ± 9.67 vs. 12.2 ± 2.89 ng/ml) (p = 0.005) (Figure 1).

View larger version (11K): [in this window] [in a new window]   Figure 1.   Box-plot summary of the mean lung tissue ENA-78 levels (ng/ml) from normal (n = 78) as compared with IPF (n = 91) lung tissue. Horizontal lines represent the median, the box encompasses the 25th to 75th percentile, and the error bars encompass the 10th to 90th percentile. *p = 0.005 by Mann-Whitney U test.

Immunolocalization of ENA-78 is predominantly associated with hyperplastic Type II epithelial cells and macrophages in lung tissue. The leading edge of fibrosis in IPF is characterized by fibroblastic foci and hyperplastic Type II pneumocytes (Figure 2A). Because ENA-78 was elevated in IPF lung tissue we next assessed the predominant cellular source of ENA-78 in IPF lung tissue. Using immunohistochemistry, we found that the predominant cells in IPF lung tissue that expressed ENA-78 were hyperplastic Type II pneumocytes and macrophages (Figure 2B and 2C). Interestingly, the areas associated with ENA-78 immunolocalization in IPF were essentially devoid of infiltrating neutrophils, suggesting an alternative role for ENA-78 other than in neutrophil recruitment.

View larger version (135K): [in this window] [in a new window]   Figure 2.   Photomicrograph showing immunolocalization of ENA-78 from IPF lung tissue specimens. (A) Hematoxylin and eosin stain of IPF lung tissue, demonstrating a fibroblastic focus (FF  ) in close proximity to hyperplastic epithelium (arrow). (B) IPF lung section stained with control antibodies demonstrating the lack of nonspecific staining. (C ) IPF lung section stained with anti-ENA-78 antibodies demonstrating immunolocalization to hyperplastic Type II pneumocytes (open arrows) and macrophages (solid arrow). Original magnification: (A-C ) ×66.

Lung tissue from patients with IPF induces greater angiogenic activity than control lung, and this angiogenic activity is significantly attributable to ENA-78. We have previously shown that IPF lung tissue demonstrates significant vascular remodeling as evidenced by the immunolocalization of Factor VIII-related antigen (7). To substantiate that ENA-78 may be modulating lung tissue-derived angiogenic activity, we next assessed the ex vivo angiogenic activity of six random samples of either control or IPF lung tissue, in the presence or absence of either preimmune (control) or neutralizing ENA-78 antibodies, utilizing the rat corneal micropocket model of neovascularization (Figure 3 and Table 1). These antibodies did not contain significant quantities of lipopolysaccharide (LPS) contamination as assessed by Limulus assay, and all samples were normalized to total protein. We found that IPF lung tissue (Figure 3B) induced a greater angiogenic response, as compared with control lung tissue (Figure 3A) (n = 6 for each manipulation). Neutralizing antibodies to ENA-78 significantly attenuated the angiogenic activity of IPF lung tissue (Figure 3C). These findings suggest that ENA-78 is a significant angiogenic factor in IPF.

View larger version (88K): [in this window] [in a new window]   Figure 3.   Representative photograph of corneal neovascularization in response to lung tissue specimens. (A) Control lung tissue. (B) IPF lung tissue in the presence of control antibodies. (C ) IPF lung tissue in the presence of neutralizing antibodies to ENA-78. n = 6 for each group. Original magnification: (A-C ) ×25.

	DISCUSSION

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Whereas angiogenesis has been shown to play a role in the evolution of tissue repair and fibroplasia associated with acute lung injury and sarcoidosis (12, 13), the contribution of neovascularization to the pathogenesis of fibrosis in IPF has been largely ignored. The existence of morphological neovascularization in IPF was originally identified by Turner-Warwick (3), who examined the lungs of patients with widespread interstitial fibrosis (IPF), and demonstrated neovascularization/ vascular remodeling that was often associated with anastomoses between the systemic and pulmonary microvasculature.

Future evidence of neovascularization during the pathogenesis of pulmonary fibrosis has been seen in a rat model of bleomycin-induced pulmonary fibrosis (4). Peao and associates perfused the vascular tree of rat lungs with methacrylate resin at a time of maximal pulmonary fibrosis (4). Using scanning electron microscopy, these investigators demonstrated major vascular modifications that included neovascularization of an elaborate network of microvasculature located in the peribronchial regions of the lungs, and distortion of the architecture of the alveolar capillaries. The location of neovascularization was closely associated with regions of pulmonary fibrosis, similar to the findings for human lungs (3), and this neovascularization appeared to lead to the formation of systemic-pulmonary anastomoses (4). Angiogenesis has been shown to develop in the mouse lung within 1 wk in response to ischemia, with the new vessels arising entirely from vessels between the parietal and visceral pleura (14).

We have shown that the CXC chemokines IL-8 and IP-10 are important factors that regulate angiogenic activity in IPF and that an imbalance exists in their expression that favors net angiogenesis in this disease (7). We found that levels of IL-8 were greater from tissue specimens of patients with IPF, as compared with control subjects. In contrast, IP-10 levels were higher from tissue specimens obtained from control subjects, as compared with patients with IPF. These findings support the notion that IL-8 and IP-10 are important factors that regulate angiogenic activity in IPF. We have further extended these studies to the murine model of bleomycin-induced pulmonary fibrosis and have shown that fibrosis can be attenuated either by the inhibition of the angiogenic chemokine MIP-2 or augmentation of the angiostatic chemokine IP-10 (8, 15). These findings provide further support for the important role of angiogenesis in the pathogenesis of pulmonary fibrosis.

In the current study, we have shown that the angiogenic CXC chemokine ENA-78 is elevated in IPF lung tissue and is associated with increased angiogenic activity. ENA-78 has been shown to be associated with other acute and chronic inflammatory diseases such as rheumatoid arthritis, hepatic ischemia reperfusion injury, and allergic airway inflammation (16). The predominant cellular sources of ENA-78 were hyperplastic Type II cells and macrophages. These hyperplastic Type II cells are associated with areas of active inflammation and are often found in proximity to fibroblastic foci. This is in contrast to our previous findings that pulmonary fibroblasts were the predominant cellular source of IL-8 and suggests that the expression of chemokines with similar biological functions does not necessarily indicate redundancy (7). Furthermore, it provides further support for the role of nonimmune cells in the pathogenesis of pulmonary fibrosis and may explain the failure of conventional immunosuppressive agents in the treatment of this disease.

Although ENA-78 is an important neutrophil chemotactic agent, immunolocalization demonstrated that areas of ENA-78 expression were essentially devoid of infiltrating neutrophils, suggesting that ENA-78 is playing a role other than neutrophil recruitment in IPF. This is consistent with our previous findings of elevated levels of IL-8 in IPF that regulated angiogenic activity (7). Although IL-8 has been shown to be associated with bronchoalveolar lavage (BAL) neutrophilia in IPF it is not associated with neutrophils in the interstitium and the neutrophil is generally not felt to play an important role in IPF (20). Persistent BAL neutrophilia after treatment with immunosuppressive agents is not necessarily associated with clinical deterioration (21). Therefore, BAL neutrophilia may merely be a marker of disease without being involved in the pathogenesis. Furthermore, we have shown that ENA-78 is an important angiogenic factor in NSCLC, and in the synovium of rheumatoid arthritis, a chronic inflammatory disease in which the neutrophil is not felt to play a prominent role (22, 23).

We have previously shown that IL-8 has an important role in angiogenesis associated with IPF (7). This raises the question of the relative roles of IL-8 and ENA-78 in promoting angiogenesis in IPF. In our corneal micropocket model, we have previously shown that neutralizing antibodies to IL-8 significantly inhibit the angiogenic activity of IPF samples; we now show that anti-ENA-78 significantly inhibits the angiogenic activity of IPF samples. This is similar to previous findings in rheumatoid arthritis (23). As IL-8 and ENA-78 share the same receptor (CXCR2), one possible explanation is heterologous desensitization of the receptor, whereby neutralization of ENA-78 may overexpose the receptor to IL-8 (and vice versa) thereby resulting in desensitization of the receptor as is seen in chemotaxis assays at high concentrations of ligand (24). Our results do not show that either ENA-78 or IL-8 is more important but merely that they both play an important role in angiogenic activity in IPF. Furthermore, we cannot exclude that other angiogenic factors might be involved. Our laboratory has described CXCR2 as the receptor that mediates the angiogenic activity of the ELR-positive CXC chemokines (25). As both IL-8 and ENA-78 bind to CXCR2 this may represent an attractive therapeutic target with respect to the inhibition of angiogenesis, thereby inhibiting or retarding the progression of IPF. Future studies will define the role of CXCR2 in the regulation of angiogenesis in IPF.