Regulation of Fibrosis by the Immune System

Regulation of Fibrosis by the Immune System Mark L. Lupher, Jr., and W. Michael Gallatin* ICOS Corporation, Bothell, Washington*Current address: Frazier Healthcare Ventures, Seattle, Washington.

1. 2. 3. 4. 5. 6. 7.

Abstract............................................................................................................. Introduction ....................................................................................................... Fibrotic Disease Pathogenesis................................................................................ Cellular Mediators of Fibrosis ............................................................................... Inflammatory Chemokines that Regulate Fibrosis...................................................... The Role of Integrins in Regulating the Fibrotic Response ......................................... Other Potential Targets for Anti‐Fibrotic Therapy ..................................................... Conclusions........................................................................................................ References .........................................................................................................

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Abstract Inflammation and fibrosis are two inter‐related conditions with many overlapping mechanisms. Three specific cell types, macrophages, T helper cells, and myofibroblasts, each play important roles in regulating both processes. Following tissue injury, an inflammatory stimulus is often necessary to initiate tissue repair, where cytokines released from resident and infiltrating leukocytes stimulate proliferation and activation of myofibroblasts. However, in many cases this drive stimulates an inappropriate pro‐fibrotic response. In addition, activated myofibroblasts can take on the role of traditional APCs, secrete pro‐ inflammatory cytokines, and recruit inflammatory cells to fibrotic foci, amplifying the fibrotic response in a vicious cycle. Moreover, inflammatory cells have been shown to play contradictory roles in initiation, amplification, and resolution of fibrotic disease processes. The central role of the macrophage in contributing to the fibrotic response and fibrotic resolution is only beginning to be fully appreciated. In the following review, we discuss the fibrotic disease process from the context of the immune response to injury. We review the major cellular and soluble factors controlling these responses and suggest ways in which more specific and, hopefully, more effective therapies may be derived. 1. Introduction Inflammation and fibrosis are two inter‐related processes with many overlapping mechanisms. An inflammatory stimulus is often necessary to initiate *

Current address: Frazier Healthcare Ventures, Seattle, Washington.

245 advances in immunology, vol. 89 # 2006 Elsevier Inc. All rights reserved.

0065-2776/06 $35.00 DOI: 10.1016/S0065-2776(05)89006-6

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wound closure, where cytokines released from resident and infiltrating leukocytes stimulate proliferation and activation of myofibroblasts. However, in many cases this drive stimulates an inappropriate pro‐fibrotic response. In addition, activated myofibroblasts can take on the role of traditional APCs, secrete pro‐inflammatory cytokines, and recruit inflammatory cells to fibrotic foci, amplifying the fibrotic response in a vicious cycle. Furthermore, some myofibroblasts appear to be of bone marrow origin, and can differentiate directly from circulating monocytes (i.e., fibrocytes). Moreover, inflammatory cells have been shown to play contradictory roles in initiation, amplification, and resolution of fibrotic disease processes. The central role of the macrophage in contributing to the fibrotic response and fibrotic resolution is only beginning to be fully appreciated. Therefore, the lines between the hematopoetic/immune system and the wound healing/fibrotic system are continually blurring. Given this, it is perhaps not surprising that broadly immunosuppressive drugs that may impact both pro‐ and anti‐fibrotic aspects of the immune system have been relatively ineffective in controlling fibrotic disease progression. However, there is hope that with a better understanding of the precise cellular and biochemical processes that inter‐relate inflammatory and fibrotic disease, more specific and effective therapies can be derived. In the following review we will first attempt to convey an understanding of the general processes involved in fibrotic disease initiation, progression, and resolution. Activation of the myofibroblast is critical for most (if not all) aspects of the fibrotic process. Second, we will discuss how the innate immune system contributes to and perhaps regulates these processes. Third, we will describe how the adaptive immune system can direct the innate response down pathways of resolution or fibrosis through production of TH1 or TH2 cytokines, respectively. Finally, we will look at therapeutic modalities in development and attempt to suggest potential new areas to explore based upon our current understanding of the inter‐relatedness of the inflammatory and fibrotic responses. 2. Fibrotic Disease Pathogenesis Fibrosis is a leading cause of morbidity and mortality and a key component of multiple diseases affecting millions of people worldwide including: liver cirrhosis; idiopathic pulmonary fibrosis; scleroderma; diabetic retinopathy and age‐related macular degeneration; diabetic nephropathy; glomerulosclerosis and IgA nephropathy; and congestive heart failure. There are currently no approved treatments that directly target the process of fibrosis despite this large unmet medical need. Fibrosis can best be described as an improperly regulated wound healing response, and is a normal reaction of tissues to injury (reviewed in [Martin,

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1997; Mutsaers et al., 1997]). This series of events is similar in most tissues including liver, lung, kidney, heart, and skin. Leakage of blood components at the injury site exposes platelets to extracellular matrix, triggering aggregation, clot formation, and haemostasis. In addition, the surrounding blood vessels constrict, reducing hemorrhage. Next, components derived from platelet degranulation cause vasodilation and increased permeability of nearby blood vessels; whereas, cleavage of fibrinogen by thrombin during the clotting cascade, together with fibronectin, holds the damaged tissues together in a provisional matrix. Inflammatory cells and later fibroblasts are recruited across this provisional matrix and migrate by chemotaxis to the site of injury. At early stages of wound healing, neutrophils are the most abundant inflammatory cell. As they degranulate and die, macrophages are recruited from circulation and the surrounding tissue and accumulate at the site of injury. Macrophages are vital for wound resolution and if their infiltration is blocked, healing is impaired (Leibovich and Ross, 1975). Macrophages and neutrophils act together to phagocytose debris and any invading microorganisms and are the source of many chemoattractants and growth factors (discussed later) which regulate the wound healing response. These factors are mitogenic and chemotactic for endothelial cells which surround the injury and form new blood vessels as they migrate towards its center as well as for T cells which become activated and secrete profibrotic cytokines. Additionally, fibroblasts migrate along the fibrin lattice into the wound and become activated into myofibroblasts. Fibroblasts can be derived from local mesenchymal pericytes, or be recruited from the bone marrow. Epithelial cells can also undergo epithelial‐mesenchymal transition (EMT), providing a continuous source of new fibroblasts (see Fig. 1). This complex mix of densely populated macrophages, myofibroblasts, T cells, and neovasculature embedded within a relatively loose matrix of hyaluronic acid, collagen, and fibronectin is called the granulation tissue. In the next phase, the now activated myofibroblasts produce and deposit large quantities of matrix proteins, predominantly types I and III collagen, which increases the tensile strength of the wound. Over time, the myofibroblasts contract the collagen lattice, reducing the size of the wound and bringing the wound margins closer together. During this process there is rapid synthesis and degradation of the matrix proteins called remodeling. In the remodeling phase the synthesis of new collagen exceeds the rate at which it is degraded such that the total amount of collagen continues to increase, resulting in scar formation. At the earliest stages, collagen III predominates, but at later stages it is replaced by collagen I. Although the wound may appear healed at this intermediate stage, chemical and structural changes are still occurring. Collagen fibrils become tightly packed and stabilized by the formation of inter‐ and intra‐molecular crosslinks.

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Figure 1 Mechanisms of fibrogenesis. During initial stages of tissue injury, polymorphonuclear leukocytes (PMN) and monocytes are recruited to the injury site. Cytokines such as TH1‐derived INF‐g, stimulate the differentiation of monocytes into M1 macrophages. PMN and M1 macrophages generate superoxides (O2) and TNF‐a, respectively, which can trigger apoptosis of surrounding tissue. Phagocytosis of apoptotic bodies (Apop) by monocytes or stimulation of monocytes by TH2‐derived IL‐13 or IL‐4 stimulates their differentiation into M2 macrophages. Similarly phagocytosis of apoptotic bodies by resident pericytes stimulates their differentiation into myofibroblasts. Pericytes can also be stimulated by M2 macrophage‐derived TGF‐b to differentiate into myofibroblasts. M2 macrophage‐derived TGF‐b may also stimulate endothelial cells to undergo endothelial‐to‐mesenchymal transition (EMT) to form additional myofibroblasts. Regardless of their origin, these activated myofibroblasts secrete large amounts of extracellular matrix including collagen 1. Integrin‐mediated contraction of this collagen matrix by the myofibroblasts leads to mechanical tension which by itself can also stimulate activation of fibroblasts (Fibro) to differentiate into myofibroblasts, thus amplifying the fibrogenic response.

Scar resolution is the final process in normal wound healing. This process occurs through a combination of reduced collagen synthesis and increased collagen degradation, coupled to regeneration of epithelial and endothelial layers over the resolving wound. The degradation of wound collagen and other matrix proteins is controlled by a variety of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by the

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Figure 2 Mechanisms of fibrosis resolution. Infiltrating monocytes are stimulated to differentiate into M1 macrophages by TH1‐derived cytokines such as INF‐g. M1 macrophages secrete matrix metalloproteinases (MMPs) that degrade extracellular matrix and signal myofibroblasts to down‐ regulate tissue inhibitor of matrix metalloproteinases (TIMPs) as well as to undergo apoptosis (Apop). Decreased TIMP expression and myofibroblast apoptosis are required for resolution of fibrotic injury; however, the precise signaling events induced by M1 macrophages resulting in this response is currently unknown, but may involve TNF‐a, BMP‐7, HGF, and/or down‐regulation of avb3 binding.

granulocytes, macrophages, epidermal cells, and myofibroblasts recruited to the injury site. Thus, the wound healing process involves shifts in metabolic equilibrium, with an early increase in proteolytic activity, followed by a stimulation of deposition and then a resolution of the scar matrix (see Fig. 2). Any disruption in this equilibrium may result in excessive deposition of matrix components resulting in a destruction of normal tissue architecture and a compromise in tissue function; this disruption is termed fibrosis. Chronic inflammation is a common instigator, where repeated injury and repair can prevent resolution and drive the healing response to fibrosis. 3. Cellular Mediators of Fibrosis 3.1. The Origin and Role of the Myofibroblast in Fibrosis Myofibroblasts are generally believed to be the principal cell responsible for pathogenic deposition of extracellular matrix proteins within fibrotic tissue and the contraction of collagen matrix contributing to increased vascular pressure

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in the tissue and its associated morbidity in fibrotic disease. The ‘myofibroblast’ phenotype was first described by Gabbiani and coworkers in 1971 (Gabbiani et al., 1971) as fibroblastic cells located within granulation tissue which exhibited cytoplasmic microfilamentous processes. These microfilaments were later demonstrated to contain myosin and a‐smooth muscle (SM) actin, and a‐SM actin has become the most reliable marker of myofibroblastic cells (Gabbiani, 2003). A range of fibroblast‐to‐myofibroblast phenotypes exist in fibrotic lesions, from normal fibroblasts to those resembling SM cells. This range of phenotypes likely reflects different stages of transition between inactivated and ‘activated’ myofibroblasts in response to the combined action of three different pro‐fibrotic signals: (1) mechanical tension (Tomasek et al., 2002); (2) profibrotic cytokines such as transforming growth factor‐b (TGF‐b), interleukin‐13 (IL‐13), and IL‐4 (Desmouliere et al., 1993; Ronnov‐Jessen and Petersen, 1993; Wynn, 2004); and (3) EIII‐A fibronectin (George et al., 2000; Jarnagin et al., 1994; Serini et al., 1998). Presence of the differentiated myofibroblast is characteristic of nearly all fibrotic diseases. The myofibroblast population present in fibrotic lesions can be derived from multiple sources that may be inter‐related or tissue‐specific. The three main sources of myofibroblasts are (1) activation of circulating bone marrow‐derived ‘fibrocytes,’ potentially in all tissues (Abe et al., 2001); (2) resident epithelial‐ mesenchymal transition (EMT), mainly characterized in the kidney (Iwano et al., 2002), but likely occurring in other tissues as well (Kalluri and Neilson, 2003); and (3) ‘activation’ of resident pericytes such as stellate cells within the liver and pancreas and mesangial cells in the kidney (Bachem et al., 1998; Bissell, 1998; Demirci et al., 1996; Leyland et al., 1996; Reeves et al., 1996). Although there is the potential for a wide range of heterogeneity of myofibroblast phenotype due to differing lineages of the originating tissue fibroblast, it appears that the wound healing response has evolved to use common mechanisms of myofibroblast induction, and this has produced a relatively common phenotype in the resulting myofibroblast. This is fortuitous for anti‐fibrotic drug development as it implies that common targets may be identifiable which could yield drugs with application across multiple fibrotic diseases. For the purpose of reviewing their generalities, we will refer to all activated fibroblast populations as myofibroblasts for the remainder of this review, regardless of their tissue of origin (e.g., hepatic or pancreatic stellate cells, fibrocytes, mesangial cells, etc.). However, the reader is directed to some excellent reviews of the specific roll of EMT in kidney fibrosis (Kalluri and Neilson, 2003; Liu, 2004) and hepatic stellate cell activation in fibrotic liver disease (Bataller and Brenner, 2005; Bissell, 1998; Friedman, 1993) for a more thorough description of their subtle differences. The important role of the myofibroblast in fibrotic disease is emphasized by the fact that the majority of factors and functions specific to fibrogenesis and

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wound healing can be produced or controlled by the activated myofibroblast. For example, the most prominent feature of fibrosis is the fibrotic scar, due to excessive deposition of extracellular matrix components. Activation of myofibroblasts increases their secretion of types I, III, and IV collagens, fibronectin, laminin, and proteoglycans; some of which are increased by greater than 50‐ fold (Maher and McGuire, 1990). Contraction of collagen gels is also a prominent feature of activated myofibroblasts, which in vivo, likely contributes to hypertension within the tissue and its associated morbidity. For example, portal hypertension within the liver is a complication of cirrhosis likely controlled by the contraction of myofibroblasts and is a significant risk factor for esophageal varices (Rockey, 2003). Remodeling of the extracellular matrix due to its concomitant production and degradation during fibrogenesis is also a prominent feature of fibrotic disease that may be controlled by activated myofibroblasts (Arthur, 2000). During progression of fibrosis there is increased expression of MMPs (MMP‐2, MMP‐3, and MMP‐9) and to a larger extent, TIMPs (TIMP‐1 and TIMP‐2). Activated myofibroblasts express virtually all the key components required for matrix degradation including MMP‐2, MMP‐ 3, TIMP‐1, and TIMP‐2 (Arthur, 2000). Furthermore, resolving wound healing is associated with removal of activated myofibroblasts through apoptosis ((Saile et al., 1997); reviewed in (Gressner, 1998)). For example, myofibroblast apoptosis associated with reduced TIMP‐1 expression has been demonstrated during the recovery phase of experimentally induced liver injury (Iredale et al., 1998). Myofibroblasts may even be considered an active part of the innate immune system and display many functions and receptors in common with macrophages. Similar to macrophages, myofibroblasts can phagocytose apoptotic cells from wounded tissue and this process activates expression of TGF‐b and collagen I (Canbay et al., 2003). Activated myofibroblasts express MHC class I and II antigens, CD1b and CD1c, as well as co‐stimulatory molecules such as intracellular adhesion molecule 1 (ICAM‐1), CD40, and CD80 and can function as antigen presenting cells to T cells (Brennan et al., 1990; Hellerbrand et al., 1996; Knittel et al., 1999; Schwabe et al., 2001; Vinas et al., 2003). They also respond to pro‐inflammatory cytokines TNF‐a and IFN‐g by releasing the chemokine MCP‐1 (CCL2) (Marra et al., 1993). Moreover, myofibroblasts express innate immune system surveillance receptors such as mannose receptor, TLR‐2, TLR‐ 4, and CD14 and are stimulated by lipopolysaccharide (LPS) (Otte et al., 2003; Paik et al., 2003; Sprenger et al., 1997). CD40 in particular may play a major role in myofibroblast activation and recruitment of inflammatory amplification. Fibroblasts respond to CD40 ligation by increasing expression of ICAM‐1 and vascular cell adhesion molecule‐1 (VCAM‐1) and producing additional inflammatory mediators such as IL‐1, IL‐6, IL‐8, prostaglandins, and hyaluronate (Schwabe et al., 2001; Sempowski

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et al., 1997; Yellin et al., 1995). Also, blockade of CD40–CD40L interactions can protect mice from radiation pneumonitis and fibrosis and dramatically reduce lung pathology as evidenced by a limited influx of inflammatory cells, minimal collagen deposition, and reduced septal thickening (Adawi et al., 1998). Contraction of the collagen matrix may also be regulated by the myofibroblast. Myofibroblasts express the major collagen receptor a1b1 integrin in vivo and both a1b1 and a2b1 integrins upon in vitro culture (Racine‐Samson et al., 1997). Myofibroblasts from multiple tissues are capable of collagen gel contraction in vitro in an a1b1‐dependent fashion (Cook et al., 2002; Kagami et al., 2002; Racine‐Samson et al., 1997) (see also Section 5). Therefore, all three major features of active fibrotic disease, namely matrix production, remodeling, and contraction, can be regulated by activated myofibroblasts. In addition, myofibroblasts have the ability to recruit and stimulate the innate and adaptive immune response to amplify wound healing and fibrosis. For these reasons, the activated myofibroblast has become a prime target for therapeutic research. Study of the pathways leading to activation and regulation of myofibroblast function has revealed the prominent role that the immune system has in this process and identified additional potential therapeutic targets. 3.2. The Central Role of the Macrophage in Fibrosis The macrophage plays a very prominent role in fibrotic disease and regulates both pro‐ and anti‐fibrotic processes. Resident and/or infiltrating macrophages play a critical part in initiation of myofibroblast activation from precursor fibroblasts, stellate cells, and endothelial cells. Initial inflammatory macrophage recruitment to a wound site functions in debridement of necrotic and apoptotic tissue (Mutsaers et al., 1997). However, phagocytosis of apoptotic cells by inflammatory and/or resident macrophages results in a change in their phenotype from pro‐inflammatory to pro‐fibrotic by down‐regulating inflammatory cytokines and upregulating TGF‐b (Fadok et al., 1998). This TGF‐b production by resident and infiltrating macrophages may serve as the initiating event in myofibroblast activation from resident pericytes, activation of infiltrating fibrocytes, as well as stimulating EMT from resident epithelial cells. 3.2.1. TGF‐b In mammals, TGF‐b exists in three isotypes, TGF‐b1, ‐b2, and ‐b‐3, which have similar biological activities (Gorelik and Flavell, 2002). However, tissue fibrosis is mainly attributed to the TGF‐b1 isoform, and macrophages, both circulating and tissue‐derived, are the main cellular source (Bissell et al., 1995;

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Khalil et al., 1996; Letterio and Roberts, 1998). Indeed, it has been proposed that alveolar macrophages produce most of the active TGF‐b that is involved in the pathologic deposition of ECM in the bleomycin‐induced lung fibrosis model (Khalil et al., 1996). However, endothelial cells, epithelial cells, and myofibroblasts themselves can also produce TGF‐b (Bissell et al., 1995; Khalil et al., 1996; Letterio and Roberts, 1998), and such autocrine TGF‐b production may be sufficient for later stages of fibrosis progression. TGF‐b is one of the most pro‐fibrogenic cytokines known and is both necessary and sufficient for fibrotic disease induction and progression in many preclinical models. In animal models of lung fibrosis, collagen accumulation is preceded by increased TGF‐b expression (Coker et al., 1997; Santana et al., 1995; Sime et al., 1997; Westergren‐Thorsson et al., 1993). In addition, transgenic TGF‐b over‐expression in the liver resulted in fibrotic disease characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern (Sanderson et al., 1995). Similarly, transient overexpression of active, but not latent, TGF‐b in the lung resulted in prolonged and severe interstitial and pleural fibrosis demonstrated by extensive deposition of collagen, fibronectin, and elastin, and by a significant increase in the number of lung myofibroblasts (Sime and O’Reilly, 2001; Sime et al., 1997). Conversely, when TGF‐b is inhibited in the rat bile duct ligation model or CCL4 model of fibrotic liver disease using a soluble TGF‐bR type II protein (sTGF‐bR), subsequent fibrosis is reduced (George et al., 1999; Yata et al., 2002). Using this system, collagen protein deposition in the liver was decreased by up to 55% even when the sTGF‐bR was administered four days after injury induction. Additionally, similar results were obtained in kidney and lung fibrotic disease models when TGF‐b is inhibited (Border et al., 1992; Fukasawa et al., 2004; Giri et al., 1993; McCormick et al., 1999; Sato et al., 2003; Wang et al., 1999). Importantly, TGF‐b inhibition was effective even when administered after disease induction (Fukasawa et al., 2004). Macrophages control the level of active TGF‐b mainly through regulation of both secretion and activation of latent TGF‐b, rather than at the level of transcription. In the cell, disulfide‐linked TGF‐b homodimers are kept in an inactive complex with latency‐associated protein (LAP). LAP is formed by cleavage of the amino terminus of the TGF‐b and normally forms a homodimer which non‐covalently associates with a homodimer of the mature, active TGF‐b. In this form, LAP prevents mature TGF‐b from binding its receptors and inducing active signaling. Dissociation of LAP is necessary for binding of TGF‐b to its receptors, and this dissociation process is catalyzed in vivo by a number of factors, including MMPs, avb6 integrin, plasmin, cathepsins, calpain, and thrombospondin (Gorelik and Flavell, 2002; Letterio and Roberts,

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1998; Munger et al., 1999). Following dissociation, TGF‐b signals are transduced by transmembrane serine/threonine kinase type I and type II receptors and intracellular mediators known as Smads (Massague, 2000). TGF‐b stimulation results in serine phosphorylation of Smad‐2 and Smad‐3 by the type 1 receptor, inducing formation of Smad‐2/3 heterodimers. The Smad‐2/3 heterodimers then associate with Smad‐4 and translocate to the nucleus, where they control the transcription of TGF‐b‐responsive genes such as collagen I and III and integrin‐linked kinase (ILK) (Li et al., 2003; Roberts et al., 2003). In vivo, both irradiation‐induced dermal fibrosis and unilateral ureteral obstruction (UUO)‐induced renal fibrosis are significantly reduced in Smad‐3 null mice (Flanders et al., 2002; Sato et al., 2003). Thus, one mechanism, whereby macrophages promote fibrosis is through production of TGF‐b which directly activates fibroblasts, epithelial cells, and pericytes to differentiate into collagen‐producing myofibroblasts. 3.2.2. PDGF Platelet‐derived growth factor (PDGF) produced mainly by macrophages is one of the most potent myofibroblast mitogens described (Bonner, 2004; Friedman and Arthur, 1989; Pinzani et al., 1989; Wong et al., 1994), and also functions as a chemoattractant and stimulant for collagen production and cell adhesion in myofibroblasts (Bonner, 2004). PDGF is a dimer of two chains selected from A, B, C, and D, which results in five potential isoforms, PDGF‐ AA, PDGF‐AB, PDGF‐BB, PDGF‐CC, and PDGF‐DD. PDGF‐AA and PDGF‐CC bind selectively to the PDGFRa, while PDGF‐AB and ‐BB isoforms bind and dimerize both PDGFRa and PDGFRb, and PDGF‐DD isoforms bind PDGFRb (Bonner, 2004). Both PDGF‐A, PDGF‐B and their receptors are induced in vivo during injury and fibrotic disease of the liver (Pinzani et al., 1994; Wong et al., 1994), lung (Martinet et al., 1986, 1987; Yi et al., 1996), kidney (Abboud, 1995; Iida et al., 1991), synovium (Rubin et al., 1988a), skin (Gay et al., 1989; Klareskog et al., 1990), and vasculature (Rubin et al., 1988b). Less is known about PDGF‐C and PDGF‐D, although the latter has been implicated in human obstructive nephropathy (Taneda et al., 2003). Since PDGF and its receptors are major mediators of myofibroblast growth and survival, it is predicted that PDGF or PDGF receptor inhibitors may be effective treatments of fibrotic disease. Indeed, treatment of rats with a PDGF‐B DNA‐aptamer antagonist or antibody prevented glomerulosclerosis and tubulointerstitial damage in a progressive mesangioproliferative glomerulonephritis model (Johnson et al., 1992; Ostendorf et al., 2001). Pirfenidone is an anti‐fibrotic agent with unknown biochemical target; however, it significantly inhibits PDGF‐stimulated hepatic myofibroblast proliferation in vitro and reduces hepatic fibrosis (Di Sario et al., 2002) and bleomycin‐induced

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lung fibrosis in vivo (Gurujeyalakshmi et al., 1999). Furthermore, small molecule inhibitors of PDGFR tyrosine kinase activity have shown significant activity in both lung (Rice et al., 1999) and liver fibrosis disease models (Yoshiji et al., 2005). 3.3. M1 and M2 Macrop hages Identification of the pro‐fibrotic phenotype of subsets of macrophages within fibrotic tissue has changed the way we perceive the role of macrophages in inflammation and fibrotic disease (Duffield, 2003; Goerdt and Orfanos, 1999). Macrophages can be functionally distinguished into two separate pools based upon cell surface phenotyping and route of activation. Classically activated macrophages (M1) are induced by TH1 lymphokines (INF‐g), bacterial and fungal cell wall components, or degraded matrix (Duffield, 2003; Goerdt and Orfanos, 1999; Pierce et al., 1996; Zhang et al., 1998). These same stimuli downregulate surface expression of the hemoglobin scavenger receptor CD163 (Buechler et al., 2000; Hogger et al., 1998). In contrast, alternatively activated macrophages (M2) are induced by TH2 lymphokines (IL‐4, IL‐13, IL‐10, and TGF‐b), phagocytosis of apoptotic cells, and corticosteroids (Goerdt and Orfanos, 1999; Stein et al., 1992; Duffield, 2003). M2 cells preferentially express the foreign antigen receptors of innate immunity, such as the macrophage mannose receptor, scavenger receptor type I, and CD163 (Geng and Hansson, 1992; Hogger et al., 1998; Mosser and Handman, 1992; Stein et al., 1992). M1 and M2 cells mediate contrasting and complementary functions in tissue fibrosis. M1 cells may play an obligatory role in initiation of the fibrotic response. First, M1 cells can induce apoptosis of surrounding tissue. As described above, subsequent phagocytosis of these apoptotic cells is a powerful stimulator of myofibroblast activation (Canbay et al., 2003). In the kidney, both mesangial and tubular cell apoptosis can be induced by macrophage membrane‐bound TNF‐a (Duffield et al., 2000, 2001). Second, M1 cells play important roles in matrix degradation through the direct and indirect production of MMPs. M1 cells display increased expression MMP‐9, MMP‐2 (gelatinases), MMP‐12 (metalloelastase), and MMP‐7 (matrilysin) (Gibbs et al., 1999; Song et al., 2000). M1 cells can also induce lung myofibroblasts to generate MMP‐13 (Mariani et al., 1998) and kidney myofibroblasts to produce MMP‐3 (Kitamura, 1998). Since the process of EMT is thought to be stimulated in part through the initial loss of basement membrane extracellular matrix contacts (Kalluri and Neilson, 2003; Liu, 2004), increased production of MMP‐9 and MMP‐2 by M1 cells may stimulate EMT during the initial inflammatory response.

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M1 cells may also play a role in resolution of fibrosis. Stimulation of the production of MMPs by M1 cells during later stages of fibrosis may shift the equilibrium towards degradation and play an important role in resolution of disease. Indeed, expression of the collagenases MMP‐1 and MMP‐13 predominate during the resolution phase of liver fibrosis (Iredale et al., 1998). Also, stimulation of apoptosis in myofibroblasts by M1 cells may contribute to their reduction during resolution of liver fibrosis (Iredale et al., 1998). In contrast, M2 cells appear to be highly pro‐fibrogenic and contribute to the remodeling phase of fibrotic disease where increased extracellular matrix deposition predominates. Indeed, M2 cells produce large amounts of TGF‐b, and complex matrix deposition is promoted when myofibroblasts are co‐ cultured with M2 cells (Erwig et al., 1998; Fadok et al., 1998; Mantovani et al., 2002; Song et al., 2000). In addition, M2 cells can induce myofibroblast proliferation through production of platelet‐derived growth factor (PDGF) and insulin‐like growth factor (IGF) (Pierce et al., 1989; Song et al., 2000). Moreover, M2 cells in healing wounds express high levels of transglutaminase, which crosslinks extracellular matrix proteins such as collagen, fibronectin, fibrinogen, and laminin, rendering them resistant to breakdown by proteases (Haroon et al., 1999a,b), and this may impede resolution of fibrotic injury (Issa et al., 2004). Further control of the process of matrix deposition during fibrosis by M1 and M2 cells may occur at the level of control of the pool of available free L‐ proline. Collagen synthesis is strictly dependent upon the availability of L‐ proline. M1 cells possess cytotoxic and antimicrobial effector functions based upon their expression of inducible nitric‐oxide synthase (iNOS) and its ability to produce nitric oxide (NO) (MacMicking et al., 1997). iNOS oxidizes the substrate L‐arginine to form NO and L‐citrulline. In contrast, M2 cells express the arginase‐1 (ARG1) enzyme which metabolizes L‐arginine into L‐ornithine and urea, and expression of iNOS vs. ARG1 appears to be mutually exclusive (Corraliza et al., 1995; Modolell et al., 1995; Munder et al., 1998). L‐ornithine in turn is the substrate for two additional enzymes, ornithine decarboxylase (ODC) and ornithine amino transferase (OAT). ODC generates polyamines which are necessary for cell growth and OAT generates L‐proline which is critical for collagen synthesis. Therefore, the presence of M1 vs. M2 cells at a site of fibrosis may affect the level of L‐proline available for collagen synthesis by myofibroblasts. Interestingly, fibroblasts themselves demonstrate similar patterns of iNOS and ARG1 expression when stimulated with TH1 vs. TH2 cytokines, respectively (Witte et al., 2002). The role of the macrophage in fibrogenesis has also been suggested by several studies in vivo. Early studies demonstrated that macrophage depletion using anti‐macrophage serum in a healing wound model resulted in reduced

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matrix production and fibrosis (Leibovich and Ross, 1975). In a more recent study of fibrotic kidney disease, tubular atrophy and interstitial fibrosis were significantly diminished when selectin‐dependent migration of macrophages into the kidney was blocked (Lange‐Sperandio et al., 2002). Additionally, depletion of alveolar macrophages greatly decreases collagen deposition in the lung following liposome‐encapsulated dichloromethylene diphosphonate administration (Zhang‐Hoover et al., 2000). In contrast, in the rat Thy1.1 glomerulonephritis model, the presence of macrophages within the kidney correlated with fewer myofibroblasts and resolution of the disease (De Heer et al., 1998; Westerhuis et al., 2000). However, these studies did not distinguish between M1 and M2 macrophage contributions and the stage at which they are acting. For example, blockade of M2 macrophages should decrease the fibrotic response, whereas the increased presence of M1 macrophages should correlate with disease resolution. An elegant and definitive study has recently confirmed the necessary role of M2 and M1 cells in the propagation and resolution, respectively, of liver fibrosis in vivo (Duffield et al., 2005). Duffield et al., has created a transgenic mouse model in which the selective depletion of macrophages can be temporally regulated. The investigators then used CCl4 to establish resolving liver fibrosis in the transgenic animals and determined the effect of macrophage depletion during the remodeling phase when injury and collagen deposition are still actively occurring, or during the resolution phase when further injury has been removed through cessation of CCl4 administration, and collagen levels are decreasing. Interestingly, removal of macrophages at these two time points had completely opposite effects on matrix deposition. In the first case, removal of macrophages during active remodeling reduced the level of matrix deposition, whereas in the second case removal of macrophages during resolution exacerbated matrix deposition. These results could be explained by the phenotype of the macrophages present within the fibrotic tissue at each time point. During the remodeling phase the macrophages present in the scar displayed an M2 phenotype whereas during the resolution phase the macrophages present in the scar displayed an M1 phenotype. These results are fully consistent with the conclusions above that M2 cells are profibrotic and M1 cells are involved in resolution of fibrotic injury. Left unanswered by this study is whether the two populations of macrophages are derived from the same source by transition of the early M2 cells into an M1 phenotype during resolution, or are the result of two different sources whereby the M2 cells leave (or die) and the M1 cells newly enter or become activated. In future studies it will be important to identify the signals necessary for the M1 cell transition and/or recruitment in order to resolve the fibrotic lesion.

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Taken together the data above are consistent with the concept that the macrophage/myofibroblast system is a tightly interrelated innate fibrotic response system to tissue injury. This system links innate inflammatory responses to activation of myofibroblasts and tissue healing. The ability of both these cell types to provide inter‐ and intra‐cellular amplification of initiating pro‐fibrotic events may help explain why certain anti‐inflammatory therapies have proven ineffective at controlling established fibrotic disease. In particular, corticosteroids which augment macrophage conversion to the M2 cell phenotype (Duffield, 2003) and stimulate macrophage production of PDGF‐B (Haynes and Shaw, 1992) may in fact do more to promote fibrosis than alleviate it. 3.4. Regulation of Fibrosis by T Helper 1 (TH1) and 2 (TH2) CD4þ T Cells Much as the adaptive immune system has evolved to take advantage of the innate immune system for many of its effector functions, an adaptive fibrotic response system also appears to have evolved to take advantage of the innate fibrotic response system described above. In many respects the traditional TH2 CD4þ T cell cytokine system plays a larger role as an initiator and amplifier of the innate fibrotic response system than as a suppressor of the TH1 CD4þ T cell response originally described (reviewed in [Chtanova and Mackay, 2001]). A variety of studies indicate that shifting the cytokine balance to a TH2 CD4þ T cell response involving IL‐4, IL‐5, and IL‐13 is strongly linked to the promotion of fibrotic disease (reviewed in [Wynn, 2004]). For example, TH2 inflammation is involved in the pathogenesis of fibrotic disorders such as hepatic fibrosis (Chiaramonte et al., 1999, 2001; Fallon et al., 2000; Hoffmann et al., 2000; Shi et al., 1997), pulmonary fibrosis (Gharaee‐Kermani and Phan, 1997; Majumdar et al., 1999; Wallace et al., 1995; Westermann et al., 1999), and systemic sclerosis (Hasegawa et al., 1997; Majumdar et al., 1999). T cells polarized in vitro to TH2 cells also induce lung fibrosis when passively transferred and activated in vivo (Wangoo et al., 2001). 3.4.1. IL‐4 Initially, IL‐4 was felt to be the primary pro‐fibrogenic cytokine produced by TH2 cells. Elevated levels of IL‐4 are found in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis, in the peripheral blood mononuclear cells of patients with periportal fibrosis, and in the bronchoalveolar lavage fluids of patients with idiopathic pulmonary fibrosis (IPF) (Booth et al., 2004; Emura et al., 1990; Wallace et al., 1995). In vitro, IL‐4 stimulates production of collagen types I and III and fibronectin from liver, lung, and scleroderma‐ derived fibroblasts (Doucet et al., 1998; Fertin et al., 1991; Tiggelman et al., 1995). Early in vivo studies seemed to confirm these in vitro results, as IL‐4

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blockade in models of liver and skin fibrosis inhibited deposition of extracellular matrix (Cheever et al., 1994; Le Moine et al., 1999; Ong et al., 1998). However, interpretation of these studies was complicated by the fact that IL‐ 13 levels are reduced when IL‐4 is inhibited (Cheever et al., 1994). Subsequent studies directly comparing the contribution of IL‐13 and IL‐4 in models of lung and liver fibrosis have demonstrated that IL‐13 may in fact be the dominant pro‐fibrotic TH2 cytokine (Chiaramonte et al., 1999; Fallon et al., 2000; Kolodsick et al., 2004). These studies compared IL‐4 null, IL‐13 null, and IL‐4/IL‐13 double‐null mice, and demonstrated that IL‐13 but not IL‐4 was required for the fibrotic response. Furthermore, IL‐4 over‐expression in the lung does not induce pulmonary fibrosis (Rankin et al., 1996), therefore IL‐4 is neither necessary nor sufficient for fibrosis in preclinical models in vivo. 3.4.2. IL‐13 Many of the pro‐fibrogenic effects of TH2 CD4þ T cells can be attributed to their production of IL‐13 (Wynn, 2004). In addition to its stimulation of macrophages to an M2 phenotype described above (Duffield, 2003), IL‐13 is also a potent stimulator of myofibroblast proliferation and collagen production in vitro (Chiaramonte et al., 1999; Doucet et al., 1998; Oriente et al., 2000). IL‐ 13 may play an additional role in fibrosis by indirectly activating TGF‐b through the upregulation of MMP‐9 and MMP‐12 which cleave the LAP‐ TGF‐b complex, thus releasing active TGF‐b (Lanone et al., 2002; Lee et al., 2001). In vivo, transgenic over‐expression of IL‐13 in murine airway causes subepithelial airway fibrosis (Zhu et al., 1999), and blockade of IL‐13 function inhibits development of hepatic fibrosis in murine schistosomiasis (Chiaramonte et al., 1999; Fallon et al., 2000) and development of lung fibrosis in both a murine fluorescein isothiocyanate model (Kolodsick et al., 2004) and bleomyocin model (Belperio et al., 2002). Moreover, IL‐13 has been implicated in the human pathogenesis of hepatic and lung fibrosis, systemic sclerosis, and nodular sclerosing Hodgkin’s disease (de Lalla et al., 2004; Hancock et al., 1998; Hasegawa et al., 1997; Ohshima et al., 2001). However, mechanistically it is unclear why IL‐13 would have greater fibrogenic activity than IL‐4. Both IL‐4 and IL‐13 can use the same IL‐4 receptor a chain and its associated signal transducer and activator of transcription protein 6 (STAT6) signaling pathway (Zurawski et al., 1993). Additionally, both cytokines induce fibroblasts from multiple tissues to produce collagen (Chiaramonte et al., 1999; Oriente et al., 2000; Saito et al., 2003). One possibility may simply be prevalence of expression, as IL‐13 levels often exceed IL‐4 levels in vivo by significant amounts (Wynn, 2004). Interestingly, active production of lung fibrosis can proceed in the absence of IL‐4Ra or STAT6 under certain

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conditions (Blease et al., 2002; Webb et al., 2003). In the latter case, subsequent deletion of IL‐13 responsive cells in STAT6 null mice blocked airway hyper‐responsiveness, but not pulmonary fibrosis (Blease et al., 2002). The mechanism whereby IL‐13 mediates its pro‐fibrotic effects is also still under debate and may vary by tissue. IL‐13 directly stimulates production of TGF‐b from M2 macrophages in fibrotic lung over‐expressing IL‐13, and fibrosis in this model was dependent upon TGF‐b and MMP9 (Lee et al., 2001). Notably, TGF‐b production and fibrosis in the IL‐13 transgenic mice could also be blocked by a null mutation in the IL‐11Ra chain (Chen et al., 2005), suggesting that IL‐13 stimulates IL‐11 production locally and this in turn drives fibrosis in the lung through TGF‐b. However, the above studies depend upon transgenic over‐expression of IL‐13. In contrast, when hepatic fibrosis is initiated by schistosomiasis, no effect on disease progression or collagen deposition was observed with blockade of TGF‐b, MMP9, or Smad‐ 3 (Kaviratne et al., 2004). IL‐13 injection in TGF‐b null animals markedly induced several genes involved in the fibrotic process including interstitial collagens, TIMP‐1, fibrillin, tanascin, and several MMPs (Kaviratne et al., 2004). Therefore, IL‐13 appears to have the capability of inducing TGF‐b‐ independent mechanisms of fibrosis. In this regard, it has also been shown that IL‐13 stimulates the expression of CD154 (CD40 ligand) on human lung fibroblasts (Kaufman et al., 2004), and the level of CD154 expression in human fibrotic lung tissue correlated strongly with the extent of fibrosis. Since lung myofibroblasts also express CD40 and are activated by its ligation (Schwabe et al., 2001; Sempowski et al., 1997; Yellin et al., 1995), amplification of the CD40/CD154 interaction may be another mechanism of IL‐13‐ mediated pro‐fibrotic responses. Also, IL‐13 is a potent inducer of several CC‐chemokines such as CCL2 (MCP‐1), CCL3 (MIP‐1a), CCL4 (MIP‐1b), CCL6 (C10), CCL11, CCL20 (MIP‐3a), and CCL22 (macrophage‐derived chemokine) (Belperio et al., 2002; Ma et al., 2004; Zhu et al., 2002). Blockade of CCL6 or chemokine receptor CCR2 diminishes fibrosis induction within the lung of IL‐13 transgenic mice (Ma et al., 2004; Zhu et al., 2002), suggesting additional ways in which IL‐13 may amplify inflammatory infiltration into a fibrotic foci (see also Section 4). 3.4.3. IFN‐g In contrast to TH2 responses, shifting the cytokine balance to a TH1 CD4þ T cell response involving interferon‐g (IFN‐g) or IL‐12 appears to be protective from fibrosis progression. IFN‐g downregulates the genes for TGF‐b and collagen 1 and inhibits the proliferation of fibroblasts (Narayanan et al., 1992). Indeed, IFN‐g was shown to be anti‐fibrotic in experimental models of hepatic, pulmonary. and renal fibrotic disease (Gurujeyalakshmi and Giri

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1995; Hoffmann et al., 2000; Oldroyd et al., 1999; Sime and O’Reilly, 2001), and similar responses have been observed with IL‐12 treatment (Keane et al., 2001; Wynn et al., 1995). These studies indicate that chronic inflammation does not always lead to fibrosis and that the magnitude of the fibrotic response can be strongly influenced by the nature of the T helper cell induced. However, application of this approach to human pulmonary fibrosis has met with mixed results so far (Raghu et al., 2004; Ziesche et al., 1999). In the study by Ziesche et al., 18 patients with IPF received either IFN‐g and prednisolone or prednisolone alone (Ziesche et al., 1999). Total lung capacity increased, the partial pressure of arterial oxygen improved, and the gene expression for TGF‐b and connective‐tissue growth factor (CTGF) decreased substantially in the IFN‐g treated patients, but not in the prednisolone‐alone group. However, a subsequent larger multicenter study by Raghu and coworkers in 330 IPF patients demonstrated no significant effect on the primary endpoints of improved pulmonary function, gas exchange, or the quality of life in the patients receiving IFN‐g (Raghu et al., 2004). Surprisingly, the authors did observe increased survival among patients who were compliant with the IFN‐g treatment, but given the lack of pulmonary function improvements, the exact mechanism behind the increased survival is unclear. It is possible that concurrent administration of prednisolone during these studies diminished the potential benefit of IFN‐g due to the steroid’s potential to further promote M2 macrophage differentiation. 4. Inflammatory Chemokines that Regulate Fibrosis Chemokines are potent chemoattractant peptides that may play important roles in the fibrogenic process by recruiting leukocytes and myofibroblasts to the site of injury. Several chemokines and their receptors have been shown to have critical roles in the development of fibrosis in specific animal models. In general, blockade of the TH2‐associated CC chemokines is protective from fibrosis, whereas blockade of the TH1‐associated CXC chemokines exacerbates fibrotic disease. However, there are also non‐TH1‐associated CXC chemokines (i.e., non‐IFN‐g inducible) which are pro‐angiogenic and pro‐fibrogenic (Strieter et al., 2002). 4.1. CC Chemokines A variety of TH2‐associated chemokines has been implicated in development of fibrotic disease. CCL2 is produced by activated myofibroblasts and is chemotactic for monocytes (Marra et al., 1993). Neutralization of CCL2 in a crescentic nephritis model of kidney fibrosis resulted in a dramatic decrease in

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both glomerular crescent formation and deposition of type I collagen (Lloyd et al., 1997). CCL3 is produced by macrophages and epithelial cells and is also chemotactic for monocytes. In the bleomycin‐induced lung fibrosis model, intratracheal challenge of CBA/J mice with bleomycin resulted in a significant time‐dependent increase in CCL3 protein levels both in whole‐lung homogenates and bronchoalveolar lavage fluid (Smith et al., 1994). Similar to CCL2 blockade above, passive immunization of bleomycin‐challenged mice with anti‐CCL3 antibodies significantly reduced pulmonary macrophage accumulation and fibrosis (Smith et al., 1994). Neutralization of CCL6 diminishes pulmonary fibrosis in IL‐13 over‐expressing transgenic mice (Ma et al., 2004), as well as in the bleomycin‐induced lung fibrosis model (Belperio et al., 2002). CCL17 is another TH2‐associated chemokine that is chemotactic for macrophages, and both CCL17 and its receptor CCR4 are overexpressed in the lung in the bleomycin‐induced lung fibrosis model (Belperio et al., 2004). Blockade of CCL17 in this model led to a significant reduction in pulmonary fibrosis (Belperio et al., 2004). In a complementary fashion, blockade of specific CC chemokine receptors also reduces fibrotic responses. For example, blockade of CCR1 (a receptor for CCL3) has been shown to reduce fibrosis in models of bleomycin‐induced lung fibrosis (Tokuda et al., 2000), chronic fungal allergic airway disease (Blease et al., 2000), and renal fibrosis following UUO (Anders et al., 2002; Eis et al., 2004), lupus nephritis (Anders et al., 2004), or adriamycin‐induced focal segmental glomerulosclerosis (Vielhauer et al., 2004). Blockade of CCR2 (a receptor for CCL2) has similarly been shown to reduce fibrosis in models of pulmonary fibrosis (Moore et al., 2001; Zhu et al. 2002). Interestingly, one mechanism by which CCR2 blockade may function in pulmonary fibrosis is by preventing specific recruitment of bone marrow‐derived fibrocytes via CCL2 (Moore et al., 2005). 4.2. CXC Chemokines The CXC chemokines also play significant roles in fibrosis development, through a mechanism that may involve control of angiogenesis. CXC chemokines can be divided into two groups based upon the presence or absence of a three amino acid Glu‐Leu‐Arg (ELR) motif at their amino‐terminus. The non‐ IFN‐g‐inducible CXC chemokines are all ELRþ and this group includes CXCL1 (GRO‐a), CXCL2 (GRO‐b), CXCL3 (GRO‐g), CXCL5 (ENA‐78), CXCL6 (GCP‐2), CXCL7 (NAP‐2), and CXCL8 (IL‐8). The TH1‐associated CXC chemokines (i.e., IFN‐g inducible) are all ELR
and include CXCL9 (MIG), CXCL10 (IP‐10), and CXCL11 (I‐TAC) (Strieter et al., 2002). In addition to their roles in inflammation (Zlotnik and Yoshie, 2000), ELRþ

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CXC chemokines are potent promoters of angiogenesis, whereas ELR
CXC chemokines are potent inhibitors of angiogenesis (Strieter et al., 1995). ELRþ CXC chemokines stimulate endothelial cell chemotaxis and proliferation in vitro, and stimulate angiogenesis in a cornea micropocket (CMP) assay in a CXCR2‐dependent manner (Addison et al., 2000). In vivo incisional wound healing is delayed in CXCR2 null mice and this correlates with decreased neovascularization (Devalaraja et al., 2000). In contrast, the ELR
CXC chemokines all inhibit neovascularization in the CMP assay in response to either ELRþ CXC chemokines or vascular endothelial growth factor (VEGF) in a manner dependent upon endothelial expression of CXCR3 (Romagnani et al., 2001). Neovascularization has been demonstrated within the lungs of rats during bleomycin‐induced lung fibrosis in close association with areas of pulmonary fibrosis (Peao et al., 1994). This has been correlated in human IPF lung tissue where there were elevated levels of CXCL8 localized to pulmonary fibroblasts, elevated levels of CXCL5 localized to hyperplastic type II cells and macrophages, but decreased levels of CXCL10 relative to control subjects, suggesting an imbalance that favors net angiogenic activity (Keane et al., 1997, 2001). In addition, neutralizing antibodies to either CXCL8 or CXCL5 blocked angiogenesis in the CMP model stimulated by IPF samples (Keane et al., 1997, 2001). Similarly, CXCL2 and CXCL10 levels were found to be directly and inversely correlated with total lung hydroxyproline levels in the murine bleomycin‐induced lung fibrosis model (Keane et al., 1999a,b). Furthermore, blockade of CXCL2 by passive immunization with neutralizing antibodies or treatment with CXCL10 resulted in marked decreases in pulmonary fibrosis which was attributed to reductions in angiogenesis in the lung (Keane et al., 1999a). Together these data strongly suggest that lung fibrosis involves neovascularization regulated by CXC chemokine production. However, this may not tell the whole story, as a recent study investigating the effect of deletion of CXCR3 (the receptor for the IFN‐g‐inducible CXC chemokines CXCL9, CXCL10 and CXCL11) suggests additional mechanisms of action for TH1‐associated CXC chemokines in fibrotic lung disease (Jiang et al., 2004). First, the lung pathology in the CXCR3 null mice administered bleomycin was distinct from that observed following treatment of WT mice, and was very similar to the cystic honeycomb pattern observed in human IPF. Second, the progressive interstitial fibrosis present in the CXCR3 null mice occurred without changes in neutrophil or monocyte levels in the lungs, but with a selective decrease in CD8þ T cells and NK cells (Jiang et al., 2004). Increased fibrosis was associated with reduced early burst of IFN‐g production and decreased expression of CXCL10 after lung injury in CXCR3 null animals relative to controls. In addition, the increased fibrosis in CXCR3 null mice was significantly reversed by treatment with exogenous IFN‐g (Jiang et al., 2004).

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If IFN‐g was acting in this model to produce ELR
CXC chemokines in order to suppress angiogenesis as suggested by Strieter and coworkers (Strieter et al., 2002), then treatment with IFN‐g in CXCR3 null mice should not have had any effect. However, since IFN‐g can rescue the phenotype induced by CXCR3 deficiency (Jiang et al., 2004), these data suggest that either other receptors for IFN‐g inducible CXC chemokines exist which can mediate the anti‐angiogenic effects of ELR
CXC chemokines, or NK cell recruitment, rather than angiogenesis suppression, may in fact be the primary role of TH1‐associated, IFN‐g‐inducible CXC chemokines in pulmonary fibrosis. 5. The Role of Integrins in Regulating the Fibrotic Response Integrins are a large family of heterodimeric transmembrane proteins involved in cell–cell and cell–extracellular matrix interaction originally identified as antigens that increased expression following prolonged activation of T cells (Hemler, 1990). Each integrin molecule is composed of a single a subunit and a single b subunit. There are currently 18 identified a subunits, 8 specific b subunits, and 24 distinct integrin heterodimers, as certain a and b subunits allow for promiscuous heterodimerization. Integrins play central roles in modulating virtually every aspect of cell behavior, including migration, establishment of polarity, growth, survival, and differentiation. Integrin cytoplasmic domains bind directly to adaptor proteins which bind directly to cellular actin, allowing integrins to connect changes in cell shape or mechanical tension to signaling events that modify cellular behavior (Tomasek et al., 2002). Indeed, cell adhesion to extracellular matrix profoundly influences myofibroblast activation, differentiation, and proliferation in vitro (Bhowmick et al., 2001; Davis, 1988; Friedman et al., 1989; Gaca et al., 2003; Thannickal et al., 2003). As such, several integrin molecules have been directly and indirectly implicated in the regulation of wound healing and fibrotic disease (Sheppard, 2003). 5.1. a1b1 a1b1 integrin (also known as very late antigen‐1, VLA‐1) is one member of a family of four b1 integrin molecules that have been shown to bind to the extracellular matrix proteins collagen and laminin (Hemler and Lobb, 1995). The other b1 integrin collagen receptors include a2b1, a10b1, and a11b1. These four collagen receptors share overlapping but distinct expression profiles. They also appear to have distinct ligand preferences in vitro. For example, a1b1 has been shown to bind more effectively to type IV collagen than type I collagen while a2b1 binds to type I collagen better than to type IV collagen (Dickeson et al., 1999). Interestingly, a1b1 can signal via Shc into the MAP

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kinase pathway and thus can regulate cell proliferation following collagen ligation (Pozzi et al., 1998). Furthermore, b1 signaling may be required for TGF‐b‐mediated activation of the MAP kinase pathway leading to EMT (Bhowmick et al., 2001). a1b1 is expressed on several cell populations relevant to fibrotic disease, including microvascular endothelial cells, fibroblasts, and myofibroblasts (Racine‐Samson et al., 1997). It is also expressed on certain activated cells of the immune system including T cells, macrophages, and NK cells, but not on normal peripheral blood mononuclear cells (PBMC) (de Fougerolles et al., 2000). Despite this broad expression profile, a1 null mice are viable and fertile and initial characterization showed no overt phenotype, demonstrating that the molecule is not required for development (Gardner et al., 1996). However, subsequent studies have demonstrated a slight defect in the proliferation rate of dermal fibroblasts from a1 null mice resulting in hypocellularity within the skin (Pozzi et al., 1998). Two studies have investigated the effects of loss of a1b1 in wound healing. Gardner and coworkers followed up their earlier observations in the skin (above) by investigating full thickness incisional wound healing in wt and a1 null mice (Gardner et al., 1999). Although no differences were observed at day 7 after injury, by day 12, the distribution of collagen fibrils showed a variegated, nodular pattern in contrast to the even distribution in the wt animals. The second study by Ekholm and coworkers tested the regeneration of a fractured long bone in wt and a1 null animals (Ekholm et al., 2002). Although a1 null mice were able to heal, a significant reduction was detected in their capacity to make cartilaginous callus. These results correlated with a decreased proliferation rate in mesenchymal stem cells although proliferation of chondrocyte‐like cells was unaltered. Collectively, these results are consistent with a role for a1b1 in wound healing. a1b1 may also play a major role in liver fibrosis. For example, in vitro, an a1 mAb blocks liver myofibroblast adhesion to collagen and endothelin stimulation of myofibroblast contraction of collagen lattices (Bissell, 1998; Racine‐ Samson et al., 1997). a1b1 is also required for liver myofibroblast migration induced by TGF‐b1, EGF, or collagen I (Yang et al., 2003). In addition, a1b1 is the primary integrin expressed by liver myofibroblasts in vivo (Bissell, 1998; Racine‐Samson et al., 1997) and a1b1 can regulate matrix metalloproteinase (MMP) expression (Gardner et al., 1996; Lochter et al., 1999; Pozzi et al., 2000, 2002), thereby affecting collagen degradation. Thus, a1b1 has the potential to impact fibrotic disease progression in liver at multiple steps. a1b1 may also play a role in renal fibrotic disease. Alport syndrome is a genetic disorder characterized by progressive glomerulonephritis resulting in fibrosis of the kidneys and, ultimately, kidney failure (Cosgrove et al., 2000).

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Alport syndrome affects approximately 1 in 5000 people and is caused by mutations in the type IV collagen genes. This condition has been mimicked in mice by knocking out the gene of the alpha3 chain of type IV collagen (Alport mouse). Concomitant knockout of the a1b1 integrin in the ‘‘Alport mouse’’ delays onset of fibrosis (Cosgrove et al., 2000). In addition, inhibition of TGF‐b1 with a soluble receptor construct had a synergistic effect with the inactivation of a1 in slowing the onset and severity of glomerular disease. These results correlated with a dramatic decrease in the accumulation of myofibroblasts and macrophages in the tubular interstitium of double knockout mice (Sampson et al., 2001). Another report studying the effects of a1 expression in transfected glomerular mesangial cells showed that a1b1 expression levels influenced the cell growth, cell size, and collagen matrix remodeling ability of these cells (Kagami et al., 2000). Moreover, two studies demonstrate that anti‐a1b1 mAb can directly affect pathologic mesangial cell remodeling of extracellular matrix in progressive kidney disease in mice (Cook et al., 2002; Kagami et al., 2002). Collectively, these two studies demonstrated that blocking a1b1 mAb significantly decreased mesangial cell‐mediated collagen deposition, collagen gel contraction, production of serum creatinine, and increased survival, even when administered after the onset of measurable interstitial fibrosis. However, differing results have been noted in a recent study using adriamycin (ADR)‐induced nephropathy in a1‐deficient mice (Chen et al., 2004). In this study the authors observed an increase in collagen IV (but not collagen 1) production, and increased reactive oxygen generation in a1 null animals, resulting in increased mesangial cell death, although the distinctions between a1 null and a1 wt were minor at most time points. Collectively, these data are consistent with the concept that a1b1 may impact fibrotic disease progression at multiple stages. a1b1 expression on macrophages and T cells may influence M1 and M2 macrophage retention in the peripheral tissue, or subsequent expression of cytokines. a1b1 expression on myofibroblasts appears to play a critical role in their contractile activity in vitro and may represent their main collagen receptor for mediating collagen contraction in vivo, thereby impacting tissue architecture and local vascular tone. Finally, a1b1 regulation of MMP expression could impact the collagen remodeling ability of myofibroblasts and macrophages at sites of active fibrogenesis. 5.2. avb6 avb6 is another integrin molecule strongly implicated in the development and control of fibrotic disease (Sheppard, 2001). avb6 expression is restricted to epithelial cells and it functions in binding to the extracellular matrix proteins fibronectin, tenascin‐C, and vitronectin through the linear tripeptide sequence

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Arg‐Gly‐Asp (RGD) common to all av integrins. Initial studies analyzing the effects of heterologous expression of avb6 determined that it enhanced proliferation of cells in three‐dimensional culture and induced expression of MMP‐ 9 (Sheppard, 2001). However, significant interest in the role of avb6 in fibrotic disease stemmed from the unexpected phenotype in b6 null mice of significant inflammation in the lungs and skin (Huang et al., 1996), but a complete lack of pulmonary fibrosis in these animals upon challenge with bleomycin (Munger et al., 1999). Although the phenotype of the b6 null mice was reminiscent of the enhanced inflammation observed in TGF‐b1 null animals (Shull et al., 1992), no difference in expression of TGF‐b was observed between wt and b6 null animals after bleomycin treatment nor at baseline (Munger et al., 1999). Instead, the authors determined that similar to other av integrins (Munger et al., 1998), avb6 bound to the RGD sequence within the TGF‐b‐inactivating LAP protein. Unlike avb1 or avb5 binding, avb6 binding was a strong activator of TGF‐b function in cell culture. However, the conformational change in the TGF‐b/LAP complex induced by avb6 binding does not appear to release free active TGF‐b, thus providing a means for spatially restricted presentation of active TGF‐b (Munger et al., 1999). A useful secondary application of the b6 null mice has been in a comprehensive profile of the global gene expression in the lungs following bleomycin treatment in b6 null and wt mice (Kaminski et al., 2000). This study has allowed the distinction of genes involved in the inflammatory response to bleomycin from the genes that specifically contribute to the fibrotic response in a time‐dependent manner. 5.3. avb3 Just as integrins are implicated in controlling activation and propagation of the fibrotic process, they may also play a role in fibrosis resolution. Spontaneous resolution of liver fibrosis in rats coincides with decreased TIMP‐1 expression, enhanced MMP activity, and apoptosis of the activated myofibroblasts (Iredale et al., 1998; Issa et al., 2001), all of which may be regulated to a degree by integrin engagement. Although the natural trigger for hepatic myofibroblast apoptosis is currently unknown, endothelial cell survival in vitro and in vivo requires ligation of the integrin avb3 (Eliceiri and Cheresh, 2000). A recent study demonstrated that avb3 is expressed by rat and human liver myofibroblasts in vitro (Zhou et al., 2004). Blockade of avb3 function in these cells resulted in decreased proliferation, increased apoptosis, decreased TIMP‐1 expression, and increased MMP‐9 expression (Zhou et al. 2004). Therefore, these data suggest that avb3 may play a sensory role in recognizing degradation of extracellular matrix during resolution thus triggering apoptosis of myofibroblasts.

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5.4. ILK Integrin‐linked kinase (ILK) has recently been associated with the fibrotic process in the kidney downstream of TGF‐b (Li et al., 2003), and its expression appears to be both necessary and sufficient for the process of EMT leading to production of myofibroblasts from tubular epithelial cells. EMT appears to proceed in a step‐wise fashion involving four crucial events (Yang and Liu, 2001): (1) loss of epithelial adhesion properties mediated through E‐cadherin; (2) de novo expression of aSMA and actin reorganization; (3) disruption of tubular basement membrane (TBM); and (4) enhanced cell migration and invasion. ILK is a cytoplasmic serine/threonine protein kinase that interacts with the intracellular domains of integrins and numerous cytoskeleton‐ associated proteins (Wu and Dedhar, 2001). In an elegant series of experiments, Li and coworkers have demonstrated that ILK plays a major role in several of the events resulting in EMT (Li et al., 2003). First, ILK expression coincided with EMT induction in vivo in two models of chronic renal fibrosis, UUO, and diabetic injury. Second, ILK expression is induced by TGF‐b in tubular epithelial cells in vitro. Third, several of the events leading to EMT in vitro were induced by ILK over‐expression in epithelial cells, such as loss of E‐ cadherin expression, induction of fibronectin and MMP‐2 expression, and enhanced cell migration and invasion. Fourth, blockade of ILK function through expression of a dominant‐negative, kinase‐dead ILK prevented TGF‐b‐induced EMT. Fifth, inhibition of EMT in vitro and in vivo by hepatocyte growth hormone (HGF, see below) correlated with inhibition of ILK expression (Li et al., 2003). These studies are complemented by demonstration of ILK over‐expression in human patients with congenital nephritic syndrome associated with proteinuria (Kretzler et al., 2001), and in diabetic nephropathy patients at sites of mesangial expansion within the glomeruli in close association with fibronectin matrix deposition (Guo et al., 2001). Therefore, both in vitro and in vivo data suggest that ILK may serve as a very interesting new target for fibrotic disease therapy. 6. Other Potential Targets for Anti‐Fibrotic Therapy 6.1. CTGF Connective tissue growth factor (CTGF) is a cytokine that may act downstream of TGF‐b to regulate matrix metabolism and was first identified as a product of human umbilical vein endothelial cells that was chemotactic and mitogenic for fibroblasts (Bradham et al., 1991). CTGF is expressed by fibroblasts in the lesions but not normal adjacent tissue of patients with progressive systemic sclerosis, localized scleroderma, and keloids (Igarashi et al., 1996). CTGF

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mRNA is upregulated in lesions of crescentic glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis, and diabetic nephropathy (Ito et al., 1998; Riser et al., 2000). Normal human skin fibroblasts express CTGF in response to TGF‐b (Igarashi et al., 1993). Similar to TGF‐b, CTGF injected under the skin of mice induces a rapid and marked increase in connective tissue cells and ECM proteins (Frazier et al., 1996). Mesangial cells treated with human CTGF significantly increased fibronectin and collagen type I production (Riser et al., 2000). Importantly, TGF‐b‐stimulated fibroblast proliferation (Kothapalli et al., 1997, 1998) and collagen synthesis (Duncan et al., 1999) can be blocked with anti‐CTGF antibodies or by inhibition of CTGF synthesis, indicating that CTGF acts as a downstream mediator of certain TGF‐b effects. Finally, when TGF‐b transgenic mice were subtotally nephrectomized and treated with CTGF antisense oligodeoxynucleotide, they showed a reduction in mRNA levels of matrix molecules as well as proteinase inhibitors plasminogen activator inhibitor‐1 (PAI‐1) and TIMP‐1, as well as suppression of renal interstitial fibrosis (Okada et al., 2005). 6.2. HGF Hepatocyte growth factor (HGF) plays an important role in early kidney development and the conversion of nephrogenic mesenchyme to epithelial cells (Horster et al., 1999). EMT is essentially the reverse of this process. In the rat remnant kidney model, an increase in renal and systemic production of HGF coupled with an increase in renal c‐met (the HGF receptor) was observed (Liu et al., 2000). Administration of an anti‐HGF antibody in the model resulted in a rapid decrease in glomerular filtration rate and increased renal fibrosis. A marked increase in ECM accumulation and in aSMA‐positive cells was observed in both the interstitium and tubular epithelium of the antibody‐treated rats. In vitro studies demonstrated that rather than increase ECM synthetic rate in HCK cells, HGF markedly increased MMP‐9 protein expression and decreased the expression of tissue inhibitors of matrix metalloproteinase‐1 (TIMP‐ 1) and TIMP‐2 (Liu et al., 2000). These data suggested that HGF was involved in extracellular matrix degradation and may play a role in preservation of epithelial phenotypes perhaps by inhibiting the process of EMT. Subsequent studies demonstrated that HGF reversed virtually all phenotypic conversion stimulated by TGF‐b such that it restored E‐cadherin expression and suppressed aSMA, vimentin, and fibronectin expression (Yang and Liu, 2002). Additionally, treatment with recombinant HGF protein or its gene effectively blocked EMT in vivo and significantly reduced renal interstitial fibrosis in an obstructive nephropathy model (Yang and Liu, 2002; Yang et al., 2001). Interestingly, administration of HGF after injury induction effectively

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blocked further fibrotic injury, but did not reverse it (Yang and Liu, 2003). Mechanistically, HGF acts by inducing expression of the SnoN transcriptional corepressor of Smad, which in turn interacts with Smad‐2 and blocks the transactivation of Smad‐regulated genes, including ILK (Li et al., 2003). 6.3. BMP‐7 Bone morphogenic protein 7 (BMP‐7, also called OP‐1) is a member of the TGF‐b superfamily and an endogenous antagonist of TGF‐b induced signaling, including EMT in the kidney and other tissues (Zeisberg et al., 2003a,b). BMP‐7 signaling induces a subset of Smad proteins (Smad5) that antagonize the Smad2/3 heterodimers activated by TGF‐b, resulting in re‐expression of E‐ cadherin on tubular epithelial cells induced to undergo EMT. Importantly, Zeisberg et al., have demonstrated that systemic administration of recombinant BMP‐7 in mice with kidney fibrosis following UUO resulted in reversal of EMT and repair of damaged tubular structures with repopulation of healthy tubular epithelial cells associated with a return of renal function (Zeisberg et al., 2003b). The same laboratory also demonstrated that BMP‐7 can provide renal protection in models of diabetic nephropathy (Zeisberg et al., 2003b). 7. Conclusions Inflammation and fibrosis are two inter‐related conditions with many overlapping mechanisms. As we have observed above, an inflammatory stimulus is often necessary to initiate wound closure; however, in many cases this drive stimulates an inappropriate pro‐fibrotic response. In addition, activated myofibroblasts can take on the role of traditional APCs, secrete pro‐inflammatory cytokines, and recruit inflammatory cells to fibrotic foci, amplifying this process. M2 macrophages, TH2 T cells, and activated myofibroblasts have critical roles in fibrosis progression. In contrast, M1 macrophages and TH1 T cells play equally critical roles in fibrosis initiation and even more importantly in fibrosis resolution. The central role of the macrophage in contributing to the fibrotic response and fibrotic resolution is only beginning to being fully appreciated. To date, broadly immunosuppressive drugs (e.g., corticosteroids) have been largely ineffective in treating fibrotic disease. This may be due to the fact that these drugs impact both pro‐ and anti‐fibrotic leukocyte subsets. There is hope that with a better understanding of the precise cellular and biochemical processes that inter‐relate inflammatory and fibrotic responses, more specific and effective therapies can be derived. During the course of this review, we have attempted to provide a useful framework with which to discuss the inter‐relatedness of the immune system

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and fibrotic/wound‐healing system, in a relatively unbiased fashion. Cells of the immune system and soluble effector molecules produced by them play important roles in regulating initiation, propagation, and resolution of many types of fibrotic disease (see Table 1). However, it is important to note that the nature of the immune cell population changes during the course of fibrotic disease. These changes, such as macrophage transition from M1 to M2 and back to M1 phenotypes, are necessary for normal healing and disregulated during fibrotic disease. Therefore, a common opinion held by physicians who Table 1 Pro‐Fibrotic Mediators and the Cells that Express Them Pro‐fibrotic mediators: Myofibroblasts

M2 Macrophages

M1 Macrophages

TH2 Cells

TH1 Cells

Cytokines: IL‐4 IL‐5 IL‐13

X X X

Chemokines: CCL‐2 CCL‐3 CCL‐6 CCL‐17 CXCL2

X X ? ? ?

Growth Factors: TGF‐b PDGF IGF CTGF

X

X X X X

X X X X

X

Matrix Regulation: L‐Proline Collagen 1 TIMP‐1 TIMP‐2 MMP‐2 MMP‐9

X X

Integrins: a1b1 anb6 anb3

X X X

X

X

X

X

?

X, indicates expression of the given protein by the cell type. ?, indicates inferred or implied expression of the given protein by the cell type. For further descriptions of each protein, see text.

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treat fibrotic disease that inflammation plays a minor role during propagation and resolution of fibrotic disease may need to evolve in light of the more recent data cited here. We propose that immune cell populations (both macrophages and T‐cells) and the cytokines, growth factors, and chemokines they produce play a critical role in all aspects of the fibrotic process. Early studies using corticosteroids to imply that immune cells do not play a role in fibrotic disease ignore the positive activating effect these molecules have on the induction of the M2 macrophage phenotype. Therefore, in contemplating novel approaches to treat fibrotic disease we must be cognizant of the stage of disease the individual pathology presents to us and the likely role the immune system plays at that stage of disease in order to design appropriate targeted therapies. If used inappropriately, broad‐based anti‐inflammatory treatments are likely to suppress the very mediators of fibrosis resolution that we would like to promote (see Table 2). Although TGF‐b is an obvious target for inhibition in fibrotic disease, and several companies are actively pursuing both small molecule and biological inhibitors (de Gouville et al., 2005; Yata et al., 2002), we must keep in mind Table 2 Anti‐Fibrotic Mediators and the Cells that Express Them Anti‐fibrotic mediators: Myofibroblasts

M2 Macrophages

M1 Macrophages

TH2 Cells

TH1 Cells

Cytokines: INF‐g IL‐12 TNF‐a

X X X

Growth Factors: HGF BMP‐7

?

X ?

?

Matrix Remodeling: MMP‐2 MMP‐3 MMP‐7 MMP‐12 MMP‐13

X X

X X X

X

Chemokines: CCL‐2 CXCL‐10

X ?

X, indicates expression of the given protein by the cell type. ?, indicates inferred or implied expression of the given protein by the cell type. For further descriptions of each protein, see text.

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that TGF‐b is also one of the major anti‐inflammatory mediators controlling autoimmunity in highly antigen‐exposed tissues. For example, TGF‐b plays an important role in non‐disease settings preventing persistent inflammation in the lung (Letterio and Roberts, 1998; Roberts and Sporn, 1993). Thus, severe perivascular inflammation and death results when TGF‐b activity is blocked in the mouse lung (Gorelik and Flavell, 2000, 2002). Therefore, it is imperative that efforts targeting this pathway consider ways in which to separate out TGF‐b anti‐inflammatory activity from its pro‐fibrotic activity, perhaps by specifically targeting more downstream signaling mediators than the TGF‐b receptor tyrosine kinase itself. Some alternatives may be ILK (Li et al., 2003), ROCK (Nagatoya et al., 2002), or CTGF (Okada et al., 2005). Alternatively, TGF‐b signaling could be disrupted by using recombinant HGF or BMP‐7. However, caution should always be used, lest we inadvertently activate autoimmunity. New therapeutics that specifically target M2 vs. M1 macrophages may represent a more selective approach, thus, inhibiting the M2 macrophage during active fibrosis propagation or attempting to convert the M2 phenotype to the M1 phenotype in order to promote fibrosis resolution. One such method may be through specific inhibition of Arg1 activity; additional work in this area is warranted. Alternatively, specific inhibition of IL‐13 or the use of caspase inhibitors to block apoptotic cell production (Canbay et al., 2004; Valentino et al., 2003) may reduce the number and activity of M2 macrophages. Finally, a few words regarding the challenges of clinical development in this area are appropriate. Our current ability to clinically evaluate novel anti‐ fibrotic agents is impeded by a lack of validated surrogate clinical endpoints for fibrotic disease progression. Hardline endpoints currently required for drug approval by the FDA such as long‐term survival and 48‐week serial tissue biopsy are impractical for the earlier stage phase II clinical studies so critical to new therapy advancement. Although progress is being made in serum biomarker development and new non‐invasive imaging studies, more work is needed. Furthermore, their broad application across multiple fibrotic diseases is questionable. As we work towards our individual research goals, we must strive to never lose sight of the end goal of new effective therapy approval. The patients are desperately waiting, and many don’t have the luxury of time. References Abboud, H. E. (1995). Role of platelet‐derived growth factor in renal injury. Annu. Rev. Physiol. 57, 297–309. Abe, R., Donnelly, S. C., Peng, T., Bucala, R., and Metz, C. N. (2001). Peripheral blood fibrocytes: Differentiation pathway and migration to wound sites. J. Immunol. 166(12), 7556–7562.

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