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CXC Chemokines in Angiogenesis Related to Pulmonary Fibrosis*
beta gene transfer to the lung induces myofibroblast presence and pulmonary fibrosis. Curr Top Pathol 1999; 93:35– 45 52 Antoniades HN, Bravo M, Avila RE, et al. Platelet-derived growth factor in idiopathic pulmonary fibrosis. J Clin Invest 1990; 86:1055–1064
CXC Chemokines in Angiogenesis Related to Pulmonary Fibrosis* Robert M. Strieter, MD, FCCP; John A. Belperio, MD; and Michael P. Keane, MD, FCCP
Angiogenesis, defined as the growth of new capillaries from preexisting vessels, is a pervasive biological phenomenon that is at the core of many physiologic and pathologic processes. An opposing balance of angiogenic and angiostatic factors regulates angiogenesis. Examples of physiologic processes that depend on angiogenesis include embryogenesis, wound repair, and the ovarian/menstrual cycle. In contrast, chronic inflammation associated with chronic fibroproliferative disorders as well as growth and metastasis of solid tumors are associated with aberrant angiogenesis. CXC chemokines comprise a unique cytokine family that contains members that exhibit on a structural/functional basis either angiogenic or angiostatic biological activity. In this review, we will discuss the role of CXC chemokines and angiogenesis in pulmonary fibrosis. (CHEST 2002; 122:298S–301S) Key words: angiogenesis; chemokine; fibrosis Abbreviations: BALF ⫽ BAL fluid; CXCL ⫽ CXC ligand; CXCR ⫽ CXC receptor; ECM ⫽ extracellular matrix; ENA ⫽ epithelial neutrophil activating protein; GCP ⫽ granulocyte chemotactic protein; GRO ⫽ growth-related gene; IFN ⫽ interferon; IL ⫽ interleukin; IP ⫽ interferon-␥–inducible protein; IPF ⫽ idiopathic pulmonary fibrosis; I-TAC ⫽ interferon-inducible T-cell ␣ chemoattractant; MIG ⫽ monokine induced by IFN-␥; MIP ⫽ macrophage inflammatory protein; MMP ⫽ matrix metalloproteinase; NAP ⫽ neutrophil activating protein
defined as the growth of new capillaries A ngiogenesis, from preexisting vessels, is a pervasive biological phenomenon that is at the core of many physiologic and pathologic processes. Examples of physiologic processes
*From the Departments of Medicine (Drs. Strieter, Belperio and Keane) and Pathology and Laboratory Medicine (Dr. Strieter), Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, Los Angeles, CA. This work was supported, in part, by National Institutes of Health grants HL66027, CA87879, P01 HL67665, and P50 CA90388 (Dr. Strieter); HL04493 and the American Lung Association (Dr. Belperio); and HL03906, P01 HL67665, and the Dalsemer Award from the American Lung Association (Dr. Keane). Correspondence to: Robert M. Strieter, MD, FCCP, Department of Medicine, David Geffen School of Medicine at UCLA, 14-154 Warren Hall, Box 711922, 900 Veteran Ave, Los Angeles, CA 90024-1922; e-mail: [email protected] 298S
that depend on angiogenesis include embryogenesis, wound repair, and the ovarian/menstrual cycle. In contrast, chronic inflammation associated with chronic fibroproliferative disorders, as well as growth and metastasis of solid tumors, are associated with aberrant angiogenesis. Angiogenesis is similar to but distinct from vasculogenesis, which describes the de novo formation of the vascular system from precursor blood islands during embryogenesis. Neovascularization is a term that can be used interchangeably with angiogenesis, but may be more appropriately reserved for describing aberrant angiogenesis that accompanies pathologic processes such as tumorigenesis or chronic inflammation associated with fibroproliferative disorders.
CXC Chemokines, CXC Chemokine Receptors, and Angiogenesis CXC1 chemokines are characteristically heparin-binding proteins. On a structural level, they have four highly conserved cysteine amino acid residues, with the first two cysteines separated by one nonconserved amino acid residue, hence the name CXC.1 Although the CXC motif distinguishes this family from other chemokine families, a second structural domain within this family dictates 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.1 The family members that contain the ELR motif (ELR⫹) are potent promoters of angiogenesis in physiologic concentrations of 1 to 100 nmol.2 In contrast, members of the family that lack the ELR motif (ELR⫺) and, in general, are interferon (IFN) inducible, are potent inhibitors of angiogenesis in physiologic concentrations of 500 pmol to 100 nmol.2 On a structural/ functional level, this suggests that the CXC chemokine family is an unique family of cytokines due to their ability to behave in a disparate manner in the promotion and inhibition of angiogenesis (Table 1).
Angiogenic (ELR⫹) CXC Chemokines The angiogenic members of the CXC chemokine family include interleukin (IL)-8 (IL-8/CXC ligand [CXCL]8), epithelial neutrophil activating protein (ENA)-78 (ENA78/CXCL5), growth-related genes (GROs) [GRO-␣, GRO-, and GRO-␥; CXCL1, CXCL2, and CXCL3, respectively], granulocyte chemotactic protein (GCP)-2 (GCP-2/CXCL6), and NH2-terminal truncated forms of platelet basic protein, which include connective tissue activating protein-III, -thromboglobulin, and neutrophil activating protein (NAP)-2 (NAP-2/CXCL7).2 ELR⫹ CXC chemokines have been shown to mediate both in vitro endothelial cell chemotactic and proliferative activity, as well as in vivo angiogenesis in a direct manner using bioassays of angiogenesis.2 These experiments prove that ELR⫹ CXC chemokines have a direct effect on the endothelial cell, and that this angiogenic activity is distinct from their ability to induce inflammation. Furthermore, ELR⫹ CXC chemokines have been found to induce the expression of the matrix metalloproteinases (MMPs), Remodeling and Repair in Respiratory Diseases
Table 1—The ELRⴙ and ELRⴚ CXC Chemokines Are Angiogenic and Angiostatic Factors, Respectively Angiogenic CXC chemokines containing the ELR motif (ELR⫹) IL-8/CXCL8 ENA-78/CXCL5 GRO-␣/CXCL1 GRO-/CXCL2 GRO-␥/CXCL3 GCP-2/CXCL6 NAP-2/CXCL7 Angiostatic IFN-inducible CXC chemokines that lack the ELR motif (ELR⫺) MIG/CXCL9 IP-10/CXCL10 I-TAC/CXCL11
MMP-2 and MMP-9, by tumor cells during tumorigenesis.3 The production of MMP-2 and MMP-9 are not only important for promoting endothelial cell migration in extracellular matrix (ECM) during angiogenesis, but are also involved in enhancing tumor cell migration in ECM leading to metastasis. Therefore, ELR⫹ chemokines not only have a direct effect on endothelial cell chemotaxis and proliferation, but also have an indirect effect in mediating their migration through ECM via the local production of MMPs.
CXC Receptor 2 (CXCR2) Is the Putative Receptor for Angiogenic (ELR⫹) CXC Chemokine-Mediated Angiogenesis Addison and colleagues4 demonstrated that CXC receptor (CXCR)-2 is detected in human microvascular endothelial cells at both the messenger RNA and protein levels. In addition, the expression of CXCR2, not CXCR1, was found to be functional in mediating endothelial cell chemotaxis.4 Moreover, this response was sensitive to Pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in mediating endothelial cell chemotaxis.4 Furthermore, the importance of CXCR2 in mediating ELR⫹ CXC chemokine-induced angiogenesis was demonstrated in vivo using the cornea micropocket assay of angiogenesis in CXCR2 ⫹/⫹ and ⫺/⫺ animals. ELR⫹ CXC chemokine-mediated angiogenesis was inhibited in the corneas of CXCR2 ⫺/⫺ mice, and in the presence of neutralizing antibodies to CXCR2 in the rat corneal micropocket assay (CMP). These studies have been further substantiated using CXCR2 ⫺/⫺ mice in a wound-repair model system.5 Devalaraja and associates5 examined the significance of CXC chemokines in wound healing. In this study, full excisional wounds were created on CXCR2 ⫹/⫹, ⫹/⫺, or ⫺/⫺ mice. Significant delays in woundhealing parameters were found in CXCR2 ⫺/⫺ mice, including decreased neovascularization. These in vitro and in vivo studies establish that CXCR2 is the receptor that mediates ELR⫹ CXC chemokine-dependent angiogenic activity. www.chestjournal.org
IFN-Inducible (ELR⫺) CXC Chemokines Are Inhibitors of Angiogenesis The angiostatic members of the CXC chemokine family include platelet factor-4/CXCL4, monokine induced by IFN-␥ (MIG) [MIG/CXCL9], and IFN-␥–inducible protein (IP)-10 [IP-10/CXCL10].1 IP-10/CXCL10 can be induced by all three interferons (IFN-␣, IFN-, and IFN-␥).1 MIG/CXCL9 is unique in that it is only induced by IFN-␥.1 Recently, a new ELR⫺ member of the CXC chemokine family, IFN-inducible T-cell ␣ chemoattractant (I-TAC) [I-TAC/CXCL11], has been cloned, and its expression appears to be induced primarily by IFN-␥.6 I-TAC/CXCL11, similar to IP-10/CXCL10 and MIG/ CXCL9, inhibits neovascularization in the CMP assay in response to either ELR⫹ CXC chemokines or vascular endothelial growth factor. These findings suggest that all IFN-inducible ELR⫺ CXC chemokines are potent inhibitors of angiogenesis. Moreover, this interrelationship of IFN and IFN-inducible CXC chemokines and their biological function are directly relevant to the function of IL-18 and IL-12, or other molecules that stimulate the expression of IFN. The capability of IL-18 and IL-12 to induce IFN-␥ and subsequently IFN-inducible CXC chemokines may explain their ability to inhibit angiogenesis. Therefore, IL-12 and IL-18, via the induction of IFN-␥, will have a profound effect on the production of IP-10/ CXCL10, MIG/CXCL9, and I-TAC/CXCL11. The subsequent expression of IFN-inducible CXC chemokines may represent the final common pathway and explain the mechanism for the attenuation of angiogenesis related to IFNs. All three IFN-inducible ELR⫺ CXC chemokines specifically bind to the CXC chemokine receptor, CXCR3.1 Recently Romagnani and colleagues7 found that CXCR3 is expressed on endothelial cells in a cell cycle-dependent manner, and this expression mediates the angiostatic activity of IP-10/CXCL10, MIG/CXCL9, and I-TAC/ CXCL11. These findings provide definitive evidence of CXCR3-mediated angiostatic activity by angiostatic IFNinducible ELR⫺ CXC chemokines.
CXC Chemokines Regulate Angiogenesis in Chronic Fibroproliferative Disorders Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal pulmonary fibroproliferative disorder. The pathogenesis of IPF that ultimately leads to end-stage fibrosis demonstrates features of dysregulated/abnormal repair with exaggerated neovascularization/vascular remodeling, fibroproliferation, and deposition of ECM, leading to progressive fibrosis and loss of lung function. While numerous eloquent studies have examined the biology of fibroblast proliferation and deposition of ECM in interstitial lung disease, few studies have examined the role of angiogenesis/vascular remodeling that promotes fibrogenesis in these disorders. The existence of neovascularization in IPF was originally identified by Turner-Warwick,8 who examined the lungs of patients with IPF and demonstrated neovascularization leading to anastomoses between the systemic and pulmonary microvasculature. Further evidence of neovasCHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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cularization during the pathogenesis of pulmonary fibrosis has been demonstrated in bleomycin-induced pulmonary fibrosis following the perfusion of the vascular tree of rat lungs with methacrylate resin at a time of maximal bleomycin-induced pulmonary fibrosis.9 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,8 and this neovascularization appeared to lead to the formation of systemic-pulmonary anastomoses.9 Although these studies supported the presence of angiogenesis, there have been limited investigations to delineate factors that may be involved in the regulation of this angiogenic activity during pulmonary fibrosis. Our laboratory has demonstrated that in IPF lung tissue, there is an imbalance in the presence of CXC chemokines that behave as either promoters of angiogenesis (IL-8/CXCL8) or inhibitors of angiogenesis (IP-10/ CXCL10).10 This imbalance favors augmented net angiogenic activity.10 Lung tissue from patients with IPF has elevated levels of IL-8/CXCL8, as compared to lung tissue from control subjects, and demonstrates in vivo angiogenic activity that can be significantly attributed to IL-8/ CXCL8.10 Immunolocalization of IL-8/CXCL8 demonstrated that the pulmonary fibroblast was the predominant interstitial cellular source of this chemokine, and areas of IL-8/CXCL8 expression were essentially devoid of neutrophil infiltration.10 This would seem to be discordant with the previous observations of augmented BAL fluid (BALF) IL-8/CXCL8 in IPF, in association with BALF neutrophilia.11 However, this disparity may be explained by the different compartments analyzed in these studies (BALF vs lung interstitium); moreover, BALF neutrophilia may simply be a marker of disease without their involvement in the pathogenesis of IPF. In further support of the role of IL-8/CXCL8 as an angiogenic factor is its association with the regulation of angiogenic activity in rheumatoid arthritis and non-small cell lung cancer.12,13 This supports an alternative biological role for IL-8/ CXCL8 or other ELR⫹ CXC chemokines in the interstitium of IPF lung tissue. In contrast to the increased angiogenic activity attributable to IL-8/CXCL8, we found a deficiency of the production of the angiostatic factor (IP-10/CXCL10) in IPF, as compared to control subjects.10 Interestingly, IFN-␥, a major inducer of IP-10/CXCL10 from a number of cells, is a known inhibitor of wound repair, in part due to its angiostatic properties, and has been shown to attenuate fibrosis in bleomycin-induced pulmonary fibrosis.14 This supports the notion that the distal mediator of the affect of IFN-␥ is IP-10/CXCL10, and an imbalance in the expression of this angiostatic CXC chemokine is found in IPF. These results suggest that attenuation of the angiogenic (IL-8/CXCL8) or augmentation of the angiostatic (IP-10/CXCL10) CXC chemokines may represent a viable therapeutic option for the treatment of IPF. We have recently shown that ENA-78/CXCL5 is an 300S
additional important regulator of angiogenic activity in IPF.15 We found that lung tissue from patients with IPF expressed greater levels of ENA-78/CXCL5 as compared to normal control subject lung tissue. These higher levels of ENA-78/CXCL5 were associated with increased angiogenic activity as assessed by the corneal micropocket assay that was significantly attributable to ENA-78/CXCL5. The predominant cellular sources of ENA-78/CXCL5 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/CXCL8 and suggests that the expression of chemokines with similar biological functions does not necessarily indicate redundancy.10 Furthermore it is further support for the role of nonimmune cells in the pathogenesis of pulmonary fibrosis and may explain the failure of conventional immunosuppressive agents in this disease. The finding that both IL-8/CXCL8 and ENA-78/ CXCL5 have important roles in the pathogenesis of IPF raises the question of the relative roles of IL-8/CXCL8 and ENA-78/CXCL5 in promoting angiogenesis in IPF. In our corneal micropocket model, we have previously shown that neutralizing antibodies to IL-8/CXCL8 significantly inhibit the angiogenic activity of IPF samples; we have now also shown that anti–ENA-78/CXCL5 antibodies significantly inhibit the angiogenic activity of IPF samples. As IL-8/CXCL8 and ENA-78/CXCL5 share the same receptor (CXCR2), one possible explanation is heterologous desensitization of the receptor, whereby neutralization of ENA-78/CXCL5 may overexpose the receptor to IL-8/CXCL8 (and vice versa), thereby resulting in desensitization of the receptor as is seen in chemotaxis assays at high concentrations of ligand.16 Our results do not show that either ENA-78/CXCL5 or IL-8/CXCL8 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⫹ CXC chemokines.4 As both IL-8/CXCL8 and ENA-78/CXCL5 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. To determine whether the imbalance in the expression of these CXC chemokines is relevant to the pathogenesis of pulmonary fibrosis, the expression and biological activity of murine macrophage inflammatory protein (MIP)-2 (MIP-2/CXCL2/3; an angiogenic ELR⫹ CXC chemokine homologous to human GRO-/␥/CXCL2/3) and the angiostatic CXC chemokine (IP-10/CXCL10) were correlated with the extent of fibrosis during bleomycin-induced pulmonary fibrosis in a murine model system.17,18 MIP-2/ CXCL2 and IP-10/CXCL10 were temporally measured during bleomycin-induced pulmonary fibrosis from whole lung tissues, and were found to be directly and inversely correlated, respectively, with total lung hydroxyproline levels, a measure of lung collagen deposition.17,18 Moreover, if either endogenous MIP-2/CXCL2 was depleted by Remodeling and Repair in Respiratory Diseases
passive immunization with neutralizing antibodies, or exogenous IP-10/CXCL10 was administered to the animals during bleomycin exposure, both treatment strategies resulted in marked attenuation of pulmonary fibrosis that was entirely attributable to a reduction in angiogenesis in the lung.17,18 These findings support the notion that angiogenesis is a critical biological event that supports fibroplasia and deposition of ECM in the lung during pulmonary fibrosis, and that angiogenic and angiostatic factors, such as CXC chemokines, play an important role in the pathogenesis of this process. We have recently shown that IL-12 attenuates bleomycin-induced pulmonary fibrosis via induction IFN-␥.19 Moreover, the beneficial effects of IL-12 can be inhibited by simultaneous administration of anti–IFN-␥ antibodies.19 These findings provide further support for IFN-␥ and thereby the IFN-inducible chemokines, IP-10/ CXCL10 and MIG/CXCL9, as inhibitors of fibrosis. With the recent demonstration of the efficacy of IFN-␥ treatment of patients with IPF,20 the above-mentioned studies substantiate that IFN-␥ treatment of IPF may mediate its effect, in part, by shifting the imbalance of the expression of ELR⫹ and ELR⫺ CXC chemokines to favor an angiostatic environment leading to inhibition of dysregulated neovascularization/vascular remodeling, fibroproliferation, and deposition of ECM in patients with IPF.
Conclusion Angiogenesis is regulated by an opposing balance of angiogenic and angiostatic factors. CXC chemokines comprise a unique cytokine family that contains members that exhibit on a structural/functional basis either angiogenic or angiostatic biological activity. The mentioned studies have demonstrated that as a family, the CXC chemokines appear to be important in the regulation of angiogenesis associated with the pathogenesis of chronic inflammatory/ fibroproliferative disorders. These findings support the notion that therapy directed at either inhibition of angiogenic or augmentation of angiostatic CXC chemokines may be a novel approach in the treatment of chronic fibroproliferative disorders.
References 1 Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity 2000; 12:121–127 2 Strieter RM, Polverini PJ, Kunkel SL, et al. The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol Chem 1995; 270:27348 –27357 3 Inoue K, Slaton JW, Eve BY, et al. Interleukin 8 expression regulates tumorigenicity and metastases in androgen-independent prostate cancer. Clin Cancer Res 2000; 6:2104 –2119 4 Addison CL, Daniel TO, Burdick MD, et al. The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR(⫹) CXC chemokine-induced angiogenic activity. J Immunol 2000; 165:5269 –5277 5 Devalaraja RM, Nanney LB, Qian Q, et al. Delayed wound healing in CXCR2 knockout mice. J Invest Dermatol 2000; 115:234 –244 6 Cole KE, Strick CA, Paradis TJ, et al. Interferon-inducible T cell ␣ chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through www.chestjournal.org
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selective high affinity binding to CXCR3. J Exp Med 1998; 187:2009 –2021 Romagnani P, Annunziato F, Lasagni L, et al. Cell cycledependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J Clin Invest 2001; 107:53– 63 Turner-Warwick M. Precapillary systemic-pulmonary anastomoses. Thorax 1963; 18:225–237 Peao MND, Aguas AP, DeSa CM, et al. Neoformation of blood vessels in association with rat lung fibrosis induced by bleomycin. Anat Rec 1994; 238:57– 67 Keane MP, Arenberg DA, Lynch JP III, et al. The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol 1997; 159:1437– 1443 Lynch JP, Standiford TJ, Kunkel SL, et al. Neutrophilic alveolitis in idiopathic pulmonary fibrosis: the role of interleukin-8. Am Rev Respir Dis 1992; 145:1433–1438 Koch AE, Leibovich SJ, Polverini PJ. Stimulation of neovascularization by human rheumatoid synovial tissue macrophages. Arthritis Rheum 1986; 29:471– 479 Smith DR, Polverini PJ, Kunkel SL, et al. Inhibition of interleukin 8 attenuates angiogenesis in bronchogenic carcinoma. J Exp Med 1994; 179:1409 –1415 Hyde DM, Henderson TS, Giri SN, et al. Effect of murine ␥ interferon on the cellular responses to bleomycin in mice. Exp Lung Res 1988; 14:687– 695 Keane MP, Belperio JA, Burdick MD, et al. ENA-78 is an important angiogenic factor in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2001; 164:2239 –2242 Ben-Baruch A, Michiel DF, Oppenheim JJ. Signals and receptors involved in recruitment of inflammatory cells. J Biol Chem 1995; 270:11703–11706 Keane MP, Belperio JA, Arenberg DA, et al. IFN-␥-inducible protein-10 attenuates bleomycin-induced pulmonary fibrosis via inhibition of angiogenesis. J Immunol 1999; 163:5686 – 5692 Keane MP, Belperio JA, Moore TA, et al. Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis. J Immunol 1999; 162:5511–5518 Keane MP, Belperio JA, Burdick MD, et al. IL-12 attenuates bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2001; 281:L92–L97 Ziesche R, Hofbauer E, Wittmann K, et al. A preliminary study of long-term treatment with interferon ␥-1b and lowdose prednisolone in patients with idiopathic pulmonary fibrosis. N Engl J Med 1999; 341:1264 –1269
Animal Models of Cigarette Smoke-Induced COPD* Joanne L. Wright, MD; and Andrew Churg, MD
Objectives: To review the animal models of COPD, and to compare these data to those found in humans. Results: Smoke-induced animal models can produce emphysema, although the lesions are not generally close mimics of human emphysema, as well as increases in mucous-secreting cells and vascular changes including pulmonary hypertension. There is considerable species-to-species variation in the degree and/or CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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CXC Chemokines in Angiogenesis Related to Pulmonary Fibrosis* Robert M. Strieter, MD, FCCP; John A. Belperio, MD; and Michael P. Keane, MD, FCCP
Angiogenesis, defined as the growth of new capillaries from preexisting vessels, is a pervasive biological phenomenon that is at the core of many physiologic and pathologic processes. An opposing balance of angiogenic and angiostatic factors regulates angiogenesis. Examples of physiologic processes that depend on angiogenesis include embryogenesis, wound repair, and the ovarian/menstrual cycle. In contrast, chronic inflammation associated with chronic fibroproliferative disorders as well as growth and metastasis of solid tumors are associated with aberrant angiogenesis. CXC chemokines comprise a unique cytokine family that contains members that exhibit on a structural/functional basis either angiogenic or angiostatic biological activity. In this review, we will discuss the role of CXC chemokines and angiogenesis in pulmonary fibrosis. (CHEST 2002; 122:298S–301S) Key words: angiogenesis; chemokine; fibrosis Abbreviations: BALF ⫽ BAL fluid; CXCL ⫽ CXC ligand; CXCR ⫽ CXC receptor; ECM ⫽ extracellular matrix; ENA ⫽ epithelial neutrophil activating protein; GCP ⫽ granulocyte chemotactic protein; GRO ⫽ growth-related gene; IFN ⫽ interferon; IL ⫽ interleukin; IP ⫽ interferon-␥–inducible protein; IPF ⫽ idiopathic pulmonary fibrosis; I-TAC ⫽ interferon-inducible T-cell ␣ chemoattractant; MIG ⫽ monokine induced by IFN-␥; MIP ⫽ macrophage inflammatory protein; MMP ⫽ matrix metalloproteinase; NAP ⫽ neutrophil activating protein
defined as the growth of new capillaries A ngiogenesis, from preexisting vessels, is a pervasive biological phenomenon that is at the core of many physiologic and pathologic processes. Examples of physiologic processes
*From the Departments of Medicine (Drs. Strieter, Belperio and Keane) and Pathology and Laboratory Medicine (Dr. Strieter), Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, Los Angeles, CA. This work was supported, in part, by National Institutes of Health grants HL66027, CA87879, P01 HL67665, and P50 CA90388 (Dr. Strieter); HL04493 and the American Lung Association (Dr. Belperio); and HL03906, P01 HL67665, and the Dalsemer Award from the American Lung Association (Dr. Keane). Correspondence to: Robert M. Strieter, MD, FCCP, Department of Medicine, David Geffen School of Medicine at UCLA, 14-154 Warren Hall, Box 711922, 900 Veteran Ave, Los Angeles, CA 90024-1922; e-mail: [email protected] 298S
that depend on angiogenesis include embryogenesis, wound repair, and the ovarian/menstrual cycle. In contrast, chronic inflammation associated with chronic fibroproliferative disorders, as well as growth and metastasis of solid tumors, are associated with aberrant angiogenesis. Angiogenesis is similar to but distinct from vasculogenesis, which describes the de novo formation of the vascular system from precursor blood islands during embryogenesis. Neovascularization is a term that can be used interchangeably with angiogenesis, but may be more appropriately reserved for describing aberrant angiogenesis that accompanies pathologic processes such as tumorigenesis or chronic inflammation associated with fibroproliferative disorders.
CXC Chemokines, CXC Chemokine Receptors, and Angiogenesis CXC1 chemokines are characteristically heparin-binding proteins. On a structural level, they have four highly conserved cysteine amino acid residues, with the first two cysteines separated by one nonconserved amino acid residue, hence the name CXC.1 Although the CXC motif distinguishes this family from other chemokine families, a second structural domain within this family dictates 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.1 The family members that contain the ELR motif (ELR⫹) are potent promoters of angiogenesis in physiologic concentrations of 1 to 100 nmol.2 In contrast, members of the family that lack the ELR motif (ELR⫺) and, in general, are interferon (IFN) inducible, are potent inhibitors of angiogenesis in physiologic concentrations of 500 pmol to 100 nmol.2 On a structural/ functional level, this suggests that the CXC chemokine family is an unique family of cytokines due to their ability to behave in a disparate manner in the promotion and inhibition of angiogenesis (Table 1).
Angiogenic (ELR⫹) CXC Chemokines The angiogenic members of the CXC chemokine family include interleukin (IL)-8 (IL-8/CXC ligand [CXCL]8), epithelial neutrophil activating protein (ENA)-78 (ENA78/CXCL5), growth-related genes (GROs) [GRO-␣, GRO-, and GRO-␥; CXCL1, CXCL2, and CXCL3, respectively], granulocyte chemotactic protein (GCP)-2 (GCP-2/CXCL6), and NH2-terminal truncated forms of platelet basic protein, which include connective tissue activating protein-III, -thromboglobulin, and neutrophil activating protein (NAP)-2 (NAP-2/CXCL7).2 ELR⫹ CXC chemokines have been shown to mediate both in vitro endothelial cell chemotactic and proliferative activity, as well as in vivo angiogenesis in a direct manner using bioassays of angiogenesis.2 These experiments prove that ELR⫹ CXC chemokines have a direct effect on the endothelial cell, and that this angiogenic activity is distinct from their ability to induce inflammation. Furthermore, ELR⫹ CXC chemokines have been found to induce the expression of the matrix metalloproteinases (MMPs), Remodeling and Repair in Respiratory Diseases
Table 1—The ELRⴙ and ELRⴚ CXC Chemokines Are Angiogenic and Angiostatic Factors, Respectively Angiogenic CXC chemokines containing the ELR motif (ELR⫹) IL-8/CXCL8 ENA-78/CXCL5 GRO-␣/CXCL1 GRO-/CXCL2 GRO-␥/CXCL3 GCP-2/CXCL6 NAP-2/CXCL7 Angiostatic IFN-inducible CXC chemokines that lack the ELR motif (ELR⫺) MIG/CXCL9 IP-10/CXCL10 I-TAC/CXCL11
MMP-2 and MMP-9, by tumor cells during tumorigenesis.3 The production of MMP-2 and MMP-9 are not only important for promoting endothelial cell migration in extracellular matrix (ECM) during angiogenesis, but are also involved in enhancing tumor cell migration in ECM leading to metastasis. Therefore, ELR⫹ chemokines not only have a direct effect on endothelial cell chemotaxis and proliferation, but also have an indirect effect in mediating their migration through ECM via the local production of MMPs.
CXC Receptor 2 (CXCR2) Is the Putative Receptor for Angiogenic (ELR⫹) CXC Chemokine-Mediated Angiogenesis Addison and colleagues4 demonstrated that CXC receptor (CXCR)-2 is detected in human microvascular endothelial cells at both the messenger RNA and protein levels. In addition, the expression of CXCR2, not CXCR1, was found to be functional in mediating endothelial cell chemotaxis.4 Moreover, this response was sensitive to Pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in mediating endothelial cell chemotaxis.4 Furthermore, the importance of CXCR2 in mediating ELR⫹ CXC chemokine-induced angiogenesis was demonstrated in vivo using the cornea micropocket assay of angiogenesis in CXCR2 ⫹/⫹ and ⫺/⫺ animals. ELR⫹ CXC chemokine-mediated angiogenesis was inhibited in the corneas of CXCR2 ⫺/⫺ mice, and in the presence of neutralizing antibodies to CXCR2 in the rat corneal micropocket assay (CMP). These studies have been further substantiated using CXCR2 ⫺/⫺ mice in a wound-repair model system.5 Devalaraja and associates5 examined the significance of CXC chemokines in wound healing. In this study, full excisional wounds were created on CXCR2 ⫹/⫹, ⫹/⫺, or ⫺/⫺ mice. Significant delays in woundhealing parameters were found in CXCR2 ⫺/⫺ mice, including decreased neovascularization. These in vitro and in vivo studies establish that CXCR2 is the receptor that mediates ELR⫹ CXC chemokine-dependent angiogenic activity. www.chestjournal.org
IFN-Inducible (ELR⫺) CXC Chemokines Are Inhibitors of Angiogenesis The angiostatic members of the CXC chemokine family include platelet factor-4/CXCL4, monokine induced by IFN-␥ (MIG) [MIG/CXCL9], and IFN-␥–inducible protein (IP)-10 [IP-10/CXCL10].1 IP-10/CXCL10 can be induced by all three interferons (IFN-␣, IFN-, and IFN-␥).1 MIG/CXCL9 is unique in that it is only induced by IFN-␥.1 Recently, a new ELR⫺ member of the CXC chemokine family, IFN-inducible T-cell ␣ chemoattractant (I-TAC) [I-TAC/CXCL11], has been cloned, and its expression appears to be induced primarily by IFN-␥.6 I-TAC/CXCL11, similar to IP-10/CXCL10 and MIG/ CXCL9, inhibits neovascularization in the CMP assay in response to either ELR⫹ CXC chemokines or vascular endothelial growth factor. These findings suggest that all IFN-inducible ELR⫺ CXC chemokines are potent inhibitors of angiogenesis. Moreover, this interrelationship of IFN and IFN-inducible CXC chemokines and their biological function are directly relevant to the function of IL-18 and IL-12, or other molecules that stimulate the expression of IFN. The capability of IL-18 and IL-12 to induce IFN-␥ and subsequently IFN-inducible CXC chemokines may explain their ability to inhibit angiogenesis. Therefore, IL-12 and IL-18, via the induction of IFN-␥, will have a profound effect on the production of IP-10/ CXCL10, MIG/CXCL9, and I-TAC/CXCL11. The subsequent expression of IFN-inducible CXC chemokines may represent the final common pathway and explain the mechanism for the attenuation of angiogenesis related to IFNs. All three IFN-inducible ELR⫺ CXC chemokines specifically bind to the CXC chemokine receptor, CXCR3.1 Recently Romagnani and colleagues7 found that CXCR3 is expressed on endothelial cells in a cell cycle-dependent manner, and this expression mediates the angiostatic activity of IP-10/CXCL10, MIG/CXCL9, and I-TAC/ CXCL11. These findings provide definitive evidence of CXCR3-mediated angiostatic activity by angiostatic IFNinducible ELR⫺ CXC chemokines.
CXC Chemokines Regulate Angiogenesis in Chronic Fibroproliferative Disorders Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal pulmonary fibroproliferative disorder. The pathogenesis of IPF that ultimately leads to end-stage fibrosis demonstrates features of dysregulated/abnormal repair with exaggerated neovascularization/vascular remodeling, fibroproliferation, and deposition of ECM, leading to progressive fibrosis and loss of lung function. While numerous eloquent studies have examined the biology of fibroblast proliferation and deposition of ECM in interstitial lung disease, few studies have examined the role of angiogenesis/vascular remodeling that promotes fibrogenesis in these disorders. The existence of neovascularization in IPF was originally identified by Turner-Warwick,8 who examined the lungs of patients with IPF and demonstrated neovascularization leading to anastomoses between the systemic and pulmonary microvasculature. Further evidence of neovasCHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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cularization during the pathogenesis of pulmonary fibrosis has been demonstrated in bleomycin-induced pulmonary fibrosis following the perfusion of the vascular tree of rat lungs with methacrylate resin at a time of maximal bleomycin-induced pulmonary fibrosis.9 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,8 and this neovascularization appeared to lead to the formation of systemic-pulmonary anastomoses.9 Although these studies supported the presence of angiogenesis, there have been limited investigations to delineate factors that may be involved in the regulation of this angiogenic activity during pulmonary fibrosis. Our laboratory has demonstrated that in IPF lung tissue, there is an imbalance in the presence of CXC chemokines that behave as either promoters of angiogenesis (IL-8/CXCL8) or inhibitors of angiogenesis (IP-10/ CXCL10).10 This imbalance favors augmented net angiogenic activity.10 Lung tissue from patients with IPF has elevated levels of IL-8/CXCL8, as compared to lung tissue from control subjects, and demonstrates in vivo angiogenic activity that can be significantly attributed to IL-8/ CXCL8.10 Immunolocalization of IL-8/CXCL8 demonstrated that the pulmonary fibroblast was the predominant interstitial cellular source of this chemokine, and areas of IL-8/CXCL8 expression were essentially devoid of neutrophil infiltration.10 This would seem to be discordant with the previous observations of augmented BAL fluid (BALF) IL-8/CXCL8 in IPF, in association with BALF neutrophilia.11 However, this disparity may be explained by the different compartments analyzed in these studies (BALF vs lung interstitium); moreover, BALF neutrophilia may simply be a marker of disease without their involvement in the pathogenesis of IPF. In further support of the role of IL-8/CXCL8 as an angiogenic factor is its association with the regulation of angiogenic activity in rheumatoid arthritis and non-small cell lung cancer.12,13 This supports an alternative biological role for IL-8/ CXCL8 or other ELR⫹ CXC chemokines in the interstitium of IPF lung tissue. In contrast to the increased angiogenic activity attributable to IL-8/CXCL8, we found a deficiency of the production of the angiostatic factor (IP-10/CXCL10) in IPF, as compared to control subjects.10 Interestingly, IFN-␥, a major inducer of IP-10/CXCL10 from a number of cells, is a known inhibitor of wound repair, in part due to its angiostatic properties, and has been shown to attenuate fibrosis in bleomycin-induced pulmonary fibrosis.14 This supports the notion that the distal mediator of the affect of IFN-␥ is IP-10/CXCL10, and an imbalance in the expression of this angiostatic CXC chemokine is found in IPF. These results suggest that attenuation of the angiogenic (IL-8/CXCL8) or augmentation of the angiostatic (IP-10/CXCL10) CXC chemokines may represent a viable therapeutic option for the treatment of IPF. We have recently shown that ENA-78/CXCL5 is an 300S
additional important regulator of angiogenic activity in IPF.15 We found that lung tissue from patients with IPF expressed greater levels of ENA-78/CXCL5 as compared to normal control subject lung tissue. These higher levels of ENA-78/CXCL5 were associated with increased angiogenic activity as assessed by the corneal micropocket assay that was significantly attributable to ENA-78/CXCL5. The predominant cellular sources of ENA-78/CXCL5 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/CXCL8 and suggests that the expression of chemokines with similar biological functions does not necessarily indicate redundancy.10 Furthermore it is further support for the role of nonimmune cells in the pathogenesis of pulmonary fibrosis and may explain the failure of conventional immunosuppressive agents in this disease. The finding that both IL-8/CXCL8 and ENA-78/ CXCL5 have important roles in the pathogenesis of IPF raises the question of the relative roles of IL-8/CXCL8 and ENA-78/CXCL5 in promoting angiogenesis in IPF. In our corneal micropocket model, we have previously shown that neutralizing antibodies to IL-8/CXCL8 significantly inhibit the angiogenic activity of IPF samples; we have now also shown that anti–ENA-78/CXCL5 antibodies significantly inhibit the angiogenic activity of IPF samples. As IL-8/CXCL8 and ENA-78/CXCL5 share the same receptor (CXCR2), one possible explanation is heterologous desensitization of the receptor, whereby neutralization of ENA-78/CXCL5 may overexpose the receptor to IL-8/CXCL8 (and vice versa), thereby resulting in desensitization of the receptor as is seen in chemotaxis assays at high concentrations of ligand.16 Our results do not show that either ENA-78/CXCL5 or IL-8/CXCL8 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⫹ CXC chemokines.4 As both IL-8/CXCL8 and ENA-78/CXCL5 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. To determine whether the imbalance in the expression of these CXC chemokines is relevant to the pathogenesis of pulmonary fibrosis, the expression and biological activity of murine macrophage inflammatory protein (MIP)-2 (MIP-2/CXCL2/3; an angiogenic ELR⫹ CXC chemokine homologous to human GRO-/␥/CXCL2/3) and the angiostatic CXC chemokine (IP-10/CXCL10) were correlated with the extent of fibrosis during bleomycin-induced pulmonary fibrosis in a murine model system.17,18 MIP-2/ CXCL2 and IP-10/CXCL10 were temporally measured during bleomycin-induced pulmonary fibrosis from whole lung tissues, and were found to be directly and inversely correlated, respectively, with total lung hydroxyproline levels, a measure of lung collagen deposition.17,18 Moreover, if either endogenous MIP-2/CXCL2 was depleted by Remodeling and Repair in Respiratory Diseases
passive immunization with neutralizing antibodies, or exogenous IP-10/CXCL10 was administered to the animals during bleomycin exposure, both treatment strategies resulted in marked attenuation of pulmonary fibrosis that was entirely attributable to a reduction in angiogenesis in the lung.17,18 These findings support the notion that angiogenesis is a critical biological event that supports fibroplasia and deposition of ECM in the lung during pulmonary fibrosis, and that angiogenic and angiostatic factors, such as CXC chemokines, play an important role in the pathogenesis of this process. We have recently shown that IL-12 attenuates bleomycin-induced pulmonary fibrosis via induction IFN-␥.19 Moreover, the beneficial effects of IL-12 can be inhibited by simultaneous administration of anti–IFN-␥ antibodies.19 These findings provide further support for IFN-␥ and thereby the IFN-inducible chemokines, IP-10/ CXCL10 and MIG/CXCL9, as inhibitors of fibrosis. With the recent demonstration of the efficacy of IFN-␥ treatment of patients with IPF,20 the above-mentioned studies substantiate that IFN-␥ treatment of IPF may mediate its effect, in part, by shifting the imbalance of the expression of ELR⫹ and ELR⫺ CXC chemokines to favor an angiostatic environment leading to inhibition of dysregulated neovascularization/vascular remodeling, fibroproliferation, and deposition of ECM in patients with IPF.
Conclusion Angiogenesis is regulated by an opposing balance of angiogenic and angiostatic factors. CXC chemokines comprise a unique cytokine family that contains members that exhibit on a structural/functional basis either angiogenic or angiostatic biological activity. The mentioned studies have demonstrated that as a family, the CXC chemokines appear to be important in the regulation of angiogenesis associated with the pathogenesis of chronic inflammatory/ fibroproliferative disorders. These findings support the notion that therapy directed at either inhibition of angiogenic or augmentation of angiostatic CXC chemokines may be a novel approach in the treatment of chronic fibroproliferative disorders.
References 1 Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity 2000; 12:121–127 2 Strieter RM, Polverini PJ, Kunkel SL, et al. The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol Chem 1995; 270:27348 –27357 3 Inoue K, Slaton JW, Eve BY, et al. Interleukin 8 expression regulates tumorigenicity and metastases in androgen-independent prostate cancer. Clin Cancer Res 2000; 6:2104 –2119 4 Addison CL, Daniel TO, Burdick MD, et al. The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR(⫹) CXC chemokine-induced angiogenic activity. J Immunol 2000; 165:5269 –5277 5 Devalaraja RM, Nanney LB, Qian Q, et al. Delayed wound healing in CXCR2 knockout mice. J Invest Dermatol 2000; 115:234 –244 6 Cole KE, Strick CA, Paradis TJ, et al. Interferon-inducible T cell ␣ chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through www.chestjournal.org
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selective high affinity binding to CXCR3. J Exp Med 1998; 187:2009 –2021 Romagnani P, Annunziato F, Lasagni L, et al. Cell cycledependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J Clin Invest 2001; 107:53– 63 Turner-Warwick M. Precapillary systemic-pulmonary anastomoses. Thorax 1963; 18:225–237 Peao MND, Aguas AP, DeSa CM, et al. Neoformation of blood vessels in association with rat lung fibrosis induced by bleomycin. Anat Rec 1994; 238:57– 67 Keane MP, Arenberg DA, Lynch JP III, et al. The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol 1997; 159:1437– 1443 Lynch JP, Standiford TJ, Kunkel SL, et al. Neutrophilic alveolitis in idiopathic pulmonary fibrosis: the role of interleukin-8. Am Rev Respir Dis 1992; 145:1433–1438 Koch AE, Leibovich SJ, Polverini PJ. Stimulation of neovascularization by human rheumatoid synovial tissue macrophages. Arthritis Rheum 1986; 29:471– 479 Smith DR, Polverini PJ, Kunkel SL, et al. Inhibition of interleukin 8 attenuates angiogenesis in bronchogenic carcinoma. J Exp Med 1994; 179:1409 –1415 Hyde DM, Henderson TS, Giri SN, et al. Effect of murine ␥ interferon on the cellular responses to bleomycin in mice. Exp Lung Res 1988; 14:687– 695 Keane MP, Belperio JA, Burdick MD, et al. ENA-78 is an important angiogenic factor in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2001; 164:2239 –2242 Ben-Baruch A, Michiel DF, Oppenheim JJ. Signals and receptors involved in recruitment of inflammatory cells. J Biol Chem 1995; 270:11703–11706 Keane MP, Belperio JA, Arenberg DA, et al. IFN-␥-inducible protein-10 attenuates bleomycin-induced pulmonary fibrosis via inhibition of angiogenesis. J Immunol 1999; 163:5686 – 5692 Keane MP, Belperio JA, Moore TA, et al. Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis. J Immunol 1999; 162:5511–5518 Keane MP, Belperio JA, Burdick MD, et al. IL-12 attenuates bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2001; 281:L92–L97 Ziesche R, Hofbauer E, Wittmann K, et al. A preliminary study of long-term treatment with interferon ␥-1b and lowdose prednisolone in patients with idiopathic pulmonary fibrosis. N Engl J Med 1999; 341:1264 –1269
Animal Models of Cigarette Smoke-Induced COPD* Joanne L. Wright, MD; and Andrew Churg, MD
Objectives: To review the animal models of COPD, and to compare these data to those found in humans. Results: Smoke-induced animal models can produce emphysema, although the lesions are not generally close mimics of human emphysema, as well as increases in mucous-secreting cells and vascular changes including pulmonary hypertension. There is considerable species-to-species variation in the degree and/or CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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