View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Kidney International, Vol. 54 (1998), pp. 2152–2159

BASIC SCIENCE - BIOCOMPATIBILITY

Novel roles for chemokines and fibroblasts in interstitial fibrosis

CORY M. HOGABOAM,MATTHEW L. STEINHAUSER,STEPHEN W. CHENSUE, and STEVEN L. KUNKEL

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA

Novel roles for chemokines and fibroblasts in interstitial fibrosis. process is remarkably complex, involving the co-ordination Background. Regardless of its involvement in either wound of numerous types of cells and a myriad of soluble media- healing or excessive fibrosis, the interstitial fibroblast can now be considered an important early participant in inflammatory re- tors. The quest of researchers in this field has been to sponses. Although it is recognized that certain immune cells and characterize the precise chain of events in the fibrotic proinflammatory mediators are intricately linked to fibrotic dis- process in order to determine mechanisms through which ease, little is presently known about the manner in which these this process can be regulated, particularly when it becomes mediators and cells are orchestrated to a fibrotic finale. Experimental studies have shown that interstitial fibroblasts are capable of partic- deleterious as in a number of fibrotic diseases. However, ipating in an inflammatory response by promoting direct fibroblast- this task has been incredibly daunting in light of emerging to-immune cell communication and/or modulating the release of evidence that the fibrotic process is extremely complex and soluble mediators that are mutually recognized by both types of cells. appears to be dictated by a variety of biological networks. It Methods. Primary cultures of murine fibroblasts were recovered from either normal tissue or tissue undergoing a cell-mediated is apparent that excessive fibrosis follows inflammation, but inflammatory response. These stromal cells were assessed for the not all inflammatory events lead to excessive fibrosis. An expression of various cytokines and chemokines indicative of a type inflammatory response to infection or injury within most 1 or type 2 response. In addition, the fibroblasts were co-cultured tissues is typically characterized by the release of a myriad with mononuclear cells to assess the cell-to-cell communication. Results. Fibroblasts recovered from different cell-mediated of newly generated and preformed inflammatory mediators inflammatory responses demonstrated a dramatic alteration in by tissue resident immune (that is, macrophages and mast their cytokine profile. Fibroblasts recovered from the type 2 cells) and non-immune (that is, fibroblasts, epithelial and immune response produced high levels of monocyte chemotactic smooth muscle) cells. Two key functions of the soluble protein-1 (MCP-1), as compared to the normal fibroblasts and fibroblasts recovered from the type 1 lesion. Mononuclear cells mediators are to modulate the function of the resident cells co-cultured with fibroblasts induced a contact-dependent expres- and promote the infiltration of lymphocytes and phago- sion of elevated levels of chemokines, especially the macrophage- cytes. These concerted efforts ideally facilitate the destruc- derived MIP-1 alpha. Thus, both fibroblasts themselves and tion and/or clearance of the inflammatory stimulus. fibroblasts co-cultured with immune-inflammatory cells have the ability to participate in the maintenance of an inflammatory Upon reaching this goal, the final stage of the inflamma- response via the expression of chemokines. tory response involves the restoration of the tissue to its Conclusions. Our laboratory and others have addressed the role of original state. Clearly, the recruitment and tissue reparative chemotactic cytokines or chemokines in the fibrotic process, and stages must be closely regulated in organs such as the have demonstrated that fibroblasts are capable of modulating the activation of various immune cells that have been implicated in kidney, gut, lungs, and heart (and presumably the perito- fibrotic disease. In addition, the interstitial fibroblast is capable of neal membrane), as excessive tissue injury or over-exuber- regulating its own behavior within the interstitial environment via the ant tissue healing can severely disrupt the function of these expression of chemokines and chemokine receptors. Thus, novel organs. Whereas the fibroblast is blamed for the complica- strategies aimed at preventing fibrotic disease will likely need to address the early engagement of inflammatory cells by fibroblasts, tions of end stage or fibrotic disease, growing experimental and possibly modulate the ability of fibroblasts to generate and/or evidence suggest that this tissue resident cell is involved in recognize profibrotic signals supplied by chemokines. the inflammatory process at much earlier stages. For example, a number of experimental studies now suggest that interstitial inflammation and homeostasis is achieved, The fibrotic response is a critical component of tissue in part, by counter-regulatory actions of cytokines and restoration or healing following injury or infection. This chemokines. Therefore, the nature and consequences of the interactions between fibroblasts and immune cells such Key words: wound healing, inflammation, tissue repair, injury, fibrotic as macrophages/monocytes, T cells, mast cells, and eosin- disease, immune response. ophils become important aspects of the normal evolution of © 1998 by the International Society of Nephrology the immune response.

2152 Hogaboam et al: Chemokines and fibroblasts in fibrosis 2153

CLINICAL INTERSTITIAL FIBROTIC DISEASES cytokines are now commonly referred to as Th1- and Th2-type cytokines, as it is recognized that cells other than Interstitial diseases are inflammatory disorders that are T cells can generate IFN-␥ and IL-4 during immune typically characterized by excessive tissue injury and pro- responses in vivo. For example, natural killer cells are an gressive fibrosis, as exemplified in the pathology of glomer- important source of IFN-␥ while mast cells can generate ulonephritis, idiopathic fibrosis, and end stage sarcoidosis IL-4 [10]. In addition, macrophage derived IL-12 and IL-10 [1]. Although a growing list of inflammatory mediators more recently have been shown to exert prominent cross present in interstitial diseases includes cytokines, growth regulatory effects on Th cell cytokine profiles, and thus factors, prostanoids, eicosanoids and free radicals, little is have also been included in the Th cytokine profiles of currently known about the manner in which these media- Th1-type and Th2-type, respectively. tors initiate and sustain these complex diseases. Clinical With this background knowledge, models of prolonged and laboratory evidence suggest that fibrotic diseases result pulmonary inflammation were generated that predomi- when the tissue is unable to regulate the inflammatory nately exhibit either a Th1- or Th2-type cytokine profile process because of the persistence of one or several inflam- [11]. The intravenous administration of Sephadex beads matory signals [2]. Current clinical strategies directed at coated with purified peptide derivative (PPD) from myco- preventing fibrotic diseases through the nonspecific inhibi- bacteria into PPD-sensitized mice initiates the formation of tion of the inflammatory process with corticosteroid, cyclo- granulomatous inflammation that is driven by Th1-type phosphamide, and azathioprine treatments have been cytokines such as IFN-␥, IL-2 and IL-12 [11]. The Th2-type largely unsuccessful, and are often associated with severe counterpart to this pulmonary inflammation model in side-effects [3]. The failure of the treatment strategies may initiated by the intravenous introduction of the parasite relate to the non-discriminating manner in which these antigen, Schistosoma mansoni egg antigen (SEA), and this drugs inhibit important modulatory factors involved in the model is driven by IL-4 and IL-5 [12]. Thus, the nature of inflammatory response. Accordingly, recent experimental the cytokine profile appears to be critical in the type of data suggest that the development of novel therapeutic inflammatory cells that are involved in the inflammatory strategies for interstitial fibrotic diseases should encompass stage and the magnitude of the tissue reparative stage. The specific modulators of the involvement of the fibroblast in Th1-type model is typified by the presence of mononuclear the inflammatory process. These studies strongly suggest cells such as lymphocytes and monocytes, while the Th2- that the fibroblast is not a bystander during inflammatory type model contains mononuclear cells, fibroblasts, and responses in the different involved tissue, but rather has a eosinophils. The relative importance of IFN-␥ and IL-4 to very specialized role in the recruitment and regulation of the development and the resolution of granuloma forma- immune cells that infiltrate the interstitial space. In addi- tion in the respective models was confirmed using adeno- tion, the balance between normal tissue repair and excess viral-based gene transfer [13] and gene knock-out mice collagen deposition appears to be dictated by the nature of [14]. These studies also confirmed that different Th-type the interaction between immune cells and fibroblasts dur- cytokines have cross-regulatory effects in these models of ing inflammatory conditions. chronic inflammation; however, it was apparent from these experiments that other mediators could facilitate the in- DEVELOPMENT OF ANIMAL MODELS OF flammatory process in these models in the absence of IL-4 INTERSTITIAL FIBROTIC DISEASE or IFN-␥ [14]. The development of animal models which exhibit persis- tent cell-mediated immune reactivity has emerged from CHEMOTACTIC CYTOKINES OR CHEMOKINES observations regarding the participation of cytokines in the The directed migration of inflammatory cells into areas tissue repair stage of the inflammatory process. Interferon- of inflammation is fueled, in part, by chemotactic proteins gamma (IFN-␥), although it is a powerful Th1 inducer of or chemokines. These low molecular weight proteins are cellular immunity, is a strong inhibitor of the processes subdivided into a least four expanding families that are leading to the deposition of extracellular matrix during a characterized by the juxtaposition of cysteine residues in wound healing response [4, 5]. On the opposite end of the the protein’s N-terminus. The C-X-C chemokines were immune spectrum, interleukin-4 (IL-4) is a major Th2 originally described as neutrophil chemotactic and activat- cytokine with potent receptor-mediated activating effects ing factors, and examples include IL-8, interferon inducible on fibroblasts [6]. These effects include enhancement of protein-10 (IP-10), neutrophil activating protein-2 (NAP- extracellular matrix generation [7], increased proliferation 2), KC, MIP-2 and ENA-78. The C-C chemokine family is and chemotaxis [8]. A unique property of Th1 and Th2 comprised of the macrophage inflammatory proteins cytokines is that they possess cross-regulatory actions [9], (MIP-1␣ and MIP-1␤), the monocyte chemoattractant pro- which appear to ensure that an immune response is appro- teins (MCP-1,-2, -3, -4, and -5), C10, eotaxin and I-309 [15]. priately balanced to prevent excess tissue injury or repair. It As their names suggest, these chemokines predominately should be noted that these major classifications of Th cell affect the directional movement of mononuclear cells (that 2154 Hogaboam et al: Chemokines and fibroblasts in fibrosis is, T cell, monocytes and macrophages), although notable demonstrated that MCP-1 directly contributes to the pro- exceptions include eotaxin, which is a chemoattractant for duction of IL-4 by antigen-activated T cells in a variety of eosinophils [16]. The list of C-X-C and C-C chemokines is cell-to-cell interactions [21, 22]. Interestingly, the reduced far from complete and the use of bioinformatic techniques lesion size in the Th2-type inflammation model appeared to has revealed numerous other candidates for these and be partly related to the reduced levels of extracellular other distinct families such as the C and C-X-X-X-C matrix in these lesions [23]. chemokines [17]. Interestingly, there are now over 40 different ligands related to this supergene family of cyto- Angiogenesis kines. Another intense area of interest in chemokine The fibroplasia and deposition of extracellular matrix biology pertains to the process by which chemokines en- associated with fibrotic diseases are partly dependent on gage an unique repertoire of G-protein linked cell surface angiogenesis. A number of factors generated during inflam- receptors [18]. The number of receptors for the chemokine mation can precipitate angiogenic responses, but certain ligands continue to be isolated and identified, as there are CXC chemokines containing the Glu-Leu-Arg or ELR now at least 10 different CCR and 4 unique C-X-C motif have recently been shown to exert very potent effects chemokine receptors. While the families of chemokines on neovascularization that are independent of their inflam- bind to their corresponding chemokine receptors referred matory effects. Therefore, whereas enhanced IL-8 produc- to as the C-x-C chemokine receptors (C-x-CR) and CC tion by macrophages was presumed to lead to the develop- chemokine receptors, within the families there does not ment of neutrophilic alveolitis during interstitial fibrosis appear to be receptor binding exclusivity. For example, the [24], more recent studies suggest that IL-8 could be a major chemokine receptor 2 (CCR2) binds a number of C-C contributor to the neovascularizaton in idiopathic fibrotic chemokines including MCP-1 through MCP-5. states [25]. Surprisingly, the major source of IL-8 in idio- pathic fibrosis was the interstitial fibroblast [25]. This study NOVEL ROLES OF CHEMOKINES IN ANIMAL also suggested that an ambitious angiogenic response dur- MODELS OF FIBROTIC DISEASE ing fibrotic disease was a consequence of enhanced angio- Numerous clinical studies have shown that levels of genic levels of IL-8 and decreased levels of angiostatic C-X-C and CC chemokines are increased at various stages IP-10. IP-10 is a non-ELR containing CXC chemokine in interstitial fibrotic diseases. Although the overall contri- produced by a number of cells following activation with bution of these mediators to the onset or development of IFN-␥. Further studies are underway to determine the clinical disease has not been thoroughly investigated, a relative role of angiogenic CXC chemokines during fibrotic number of experimental studies suggest that chemokines responses. may contribute to a number of major events during inter- stitial fibrosis. Some of these events are described below. Chemoattraction of immune cells putatively involved in fibrosis Modulation of cytokine phenotypes Several chemokines have emerged as important che- Additional studies using specific models of fibrosis with moattractants during clinical and experimental fibrotic defined cytokine phenotypes have revealed that chemo- disease. The chemokine “regulated on activation, normal T kines play a significant immunoregulatory role that is cell expressed and secreted” (RANTES) is a potent che- related, in part, to their effects on the generation of Th1- moattractant for T cells and eosinophils, and is markedly and Th2-type cytokines. Neutralization of the CC chemo- elevated in interstitial fibrosis [26]. Little is currently known kine, monocyte chemotactic protein-1 (MCP-1), with a about the role of RANTES in the interstitial fibrotic polyclonal rabbit anti-mouse antibody in the Th2-type process. MCP-1 and MIP-1␣ are also abundantly expressed model was associated with significantly reduced IL-4 and during many stages of interstitial fibrotic disease [27–29]. In IL-5 generation by draining lymph nodes from the area of addition to macrophages, epithelial cells and smooth mus- inflamed tissue and markedly smaller inflammatory lesions cle cells, fibroblasts isolated from patients with idiopathic [19]. Treatment with neutralizing antibody directed against fibrosis exhibit enhanced MCP-1 and MIP-1␣ generation MCP-1 in the Th1-type model had no effect on cytokine [28]. Experimental models of fibrosis in the lung [30, 31] production or inflammatory lesion size. An additional and kidney [32] are also characterized by increased MCP-1 interesting observation from this study was that MCP-1 had and MIP-1␣ generation. The role of MIP-1␣ has been a clear inhibitory effect on the ability of isolated peritoneal examined in a bleomycin model of pulmonary fibrosis, and macrophages to generate IL-12 following an inflammatory it appears that it exerts a prominent role in the recruitment stimulus [19]. In fact, a number of studies are emerging that of mononuclear and polymorphonuclear cells to the lung suggest that MCP-1 is intricately involved in the regulation [33]. Immunoneutralization of MCP-1 in a bleomycin of Th1- and Th2-type responses. Karpus and co-workers model of lung fibrosis resulted in an approximately 30% have shown that T cell activation in the presence of MCP-1 reduction in the number of recruited mononuclear cells enhances IL-4 generation [20]. Along similar lines we have [33]. In studies examining the development of fibrosis Hogaboam et al: Chemokines and fibroblasts in fibrosis 2155 during experimental crescentic nephritis, Lloyd et al used of neutrophils, monocytes and macrophages [39]. Other immunoneutralization techniques to demonstrate that interesting findings regarding fibroblasts pertain to their MCP-1, but not RANTES or MIP-1␣, contributes to regulation of chemokine receptor expression. Recent stud- crescent formation and renal fibrosis (that is, type I pro- ies in this laboratory demonstrate that fibroblasts cultured collagen deposition) [32]. However, the role of MCP-1 in from a fibrotic lesions have markedly elevated expression of the renal fibrotic response in this model could not be CCR2 compared to fibroblasts cultured from non-fibrotic entirely defined by the effect of MCP-1 on the recruitment lesions. These findings are of potential therapeutic signifi- of T cells and macrophages to the inflamed glomerulus cance, since it may be possible to selectively inhibit the [32]. fibroblast from generating or responding to a number of chemokines that appear to be directly involved in the Augmented collagen generation by fibroblasts interstitial fibrotic process. Another putative explanation for the findings presented by Lloyd et al [32] is derived from recent studies by INTERSTITIAL FIBROBLASTS CAN ENGAGE Gharaee-Kermani, Denholm and Phan [34], who have INFLAMMATORY AND IMMUNE CELLS shown that MCP-1 indirectly contributes to de novo colla- The recruitment of inflammatory and immune cells such gen production by fibroblasts. MCP-1-activated fibroblasts as monocytes, T cells, mast cells and eosinophils into the markedly increased transforming growth factor-␤ (TGF-␤) interstitial space presumably represents an important initial production, which in turn was the major effector molecule step in the cascade of events leading to end-stage fibrosis. in the induction of type I collagen in these fibroblasts [34]. Numerous studies now indicate that these recruited cells TGF-␤ is one of the most potent profibrotic cytokines yet are activated and/or regulated in a fashion that is depen- described [35]. Whereas targeting TGF-␤ in fibrotic re- dent upon their interactions with tissue resident cells such sponses may be fraught with serious problems similar to as the fibroblast. Below are examples of cell-to-cell inter- those observed in TGF-␤ gene disruption studies [36], actions that are postulated to be involved in sustaining modulation of MCP-1 production during interstitial fibrotic inflammatory or immune events that ultimately lead to responses may provide a viable treatment option in inter- inappropriate tissue repair. stitial fibrotic disease. Thus, these studies, as in the studies assessing glomerulonephritis, suggest that C-C chemokines Macrophages and recruited monocytes not only recruit immune cells that generate profibrotic These cells appear to have a central role in the initiation mediators, but are also involved in the direct activation of and exacerbation of inflammatory tissue injury, and are the fibroblast leading to extracellular matrix generation. hypothesized to be responsible for the eventual develop- ment of various types of idiopathic fibrosis [1]. Macro- INTERSTITIAL FIBROBLASTS ARE A DYNAMIC phages are among the first immune cells found in increased SOURCE OF CHEMOKINE GENERATION numbers at the early stages of fibrosis, and because these Chemokines and their respective receptors were primar- resident cells are uniquely situated within the interstitum, ily considered to be the products of immune cells, but the their released mediators activate numerous structural cell literature now strongly suggests otherwise [37]. The fibro- types including epithelial cells, smooth muscle cells and blast is among the many non-immune cells that readily fibroblasts. Platelet-derived growth factor was one of the generate chemokines and express chemokine receptors first profibrotic mediators identified to be spontaneously following cytokine activation. Fibroblasts from many tissue released by macrophages in patients with fibrotic disease sites (including the peritoneal cavity) and species are a [40, 41]. Macrophages may further contribute to tissue major source of constitutive and cytokine-induced C-C and injury through their synthesis of IL-8. One putative mech- C-X-C chemokines such as MCP-1, MIP-1␣, RANTES, anism for sustained IL-8 production in fibrosis may relate IP-10, KC and eotaxin [38]. Xia and colleagues have to the destructive effect of activated neutrophils on the recently supplied very interesting data that suggests that matrix components of the tissue interstitum. Although this fibroblasts are differently regulated in their chemokine process is necessary in normal tissue regeneration or heal- generation than bone-marrow-derived cells [39]. An impor- ing responses, in fibrosis it appears that hyaluronan frag- tant regulator of chemokine production by fibroblasts ments of extracellular matrix may exacerbate or maintain appears to be RelB, a transcription factor that is rapidly the production of IL-8 by macrophages through a CD44- increased following the activation of fibroblasts with in- dependent mechanism [42, 43]. As explained above, IL-8 flammatory stimuli such as IL-1␤, TNF␣ and LPS. In the may also play a major role in the angiogenic response absence of RelB expression in fibroblasts, but not in during tissue fibrosis. Additional immunohistochemical ev- macrophages, NF-␬B activity remains unchecked and che- idence suggests that the lung fibroblast may also contribute mokine generation continues unabated. Interestingly, RelB to the changes in PDGF and IL-8 levels in idiopathic knock-out mice exhibit a syndrome of multi-organ inflam- pulmonary fibrosis (IPF) [25], but whether these changes in mation characterized by a predominant interstitial infiltrate the synthetic capacity of the fibroblast are related to direct 2156 Hogaboam et al: Chemokines and fibroblasts in fibrosis

IL-4, which will recognize specific receptors expressed by fibroblasts [6], cause these cells to proliferate [47] and to undergo directed movement toward inflammatory stimuli [8]. IL-4 activation of fibroblasts also promotes the biogen- esis of extracellular matrix proteins in normal repair pro- cesses and during pathologic fibrotic responses in which T cells are involved [7]. However, the interaction between T cells and fibroblasts is not unidirectional. Rather, there is emerging evidence that the fibroblast can modulate the activation state of T cells. Bombara et al have shown that direct contact between T cells and fibroblasts promotes the induction of adhesion molecules and cytokine production by the T cells [48]. Other studies have shown MCP-1 induced binding of T cells to fibronectin and endothelium- Fig. 1. Macrophage/fibroblast co-culture induces contact-dependent derived extracellular matrix through the activation of MIP-1␣ production. Primary peritoneal macrophage (PM) cultures VLA-4 and-5 on the T cell surface. Interestingly, MCP-1 (100,000/well) were layered onto a primary lung fibroblast (F) monolayer. Peritoneal macrophage controls were layered onto a fixed fibroblast did not augment T cell interactions with ICAM-1 or (FIXED F) monolayer (10 min with 4% paraformaldehyde). In the F ϩ vascular cell adhesion molecule-1 (VCAM-1) [49]. In stud- PM(TW) group, the two cell populations were physically separated by a ies conducted in our laboratory, the generation of MCP-1 membrane which allows the passage of culture media plus any soluble factors. MIP-1␣ concentrations were evaluated by ELISA after 24 hours of by untreated and cytokine-pretreated fibroblasts was nec- co-incubation and expressed as mean ϩ SE. N ϭ 3 to 6. MIP-1␣ was only essary for the generation of IL-4 by antigen-rechallenged detected in co-cultures containing F ϩ PM. * P Ͻ 0.05. CD4ϩ T cells. This effect was demonstrated through both the specific immunoneutralization of MCP-1 using a poly- clonal antibody and the inhibition of MCP-1 synthesis using interactions between these cells and alveolar macrophages MCP-1 antisense oligonucleotides [22]. However, the ab- has yet to be determined. sence of MCP-1 during the co-culture of CD4ϩ T cells and The increased trafficking of monocytes into the inflamed fibroblasts was also associated with significantly enhanced pulmonary interstitum during IPF is presumed to occur, in CD4ϩ T cell proliferation. Thus, the data at present part, through the increased production of C-C chemokines suggests that bi-directional interactions between T cells and such MIP-1␣. As mentioned earlier, MIP-1␣ levels are fibroblasts presumably serve to modulate the activity of increased in IPF due to the exaggerated production of this both cells. MCP-1 regulation of T cells during chronic mediator by alveolar macrophages and epithelial cells [29]. inflammatory responses may have great implications for the We have examined the role of fibroblasts in the generation progression of interstitial fibrosis. of MIP-1␣ by a macrophage cell line (Raw 264.7) in a cell-to-cell co-culture system. MIP-1␣ production by the Mast cells co-cultured macrophages was markedly elevated after one Mast cell numbers and histamine levels have been shown hour of direct contact between these cells and lung fibro- to be increased in samples taken from patients with fibrosis blasts. Direct contact between these cells was required for [50]. As in the case of T cells, mast cells are found in fibrotic MIP-1␣ generation by macrophages since the transwell lesions [35, 51], but the exact role of this tissue resident cell separation of these cells inhibited this response (Fig. 1). is unknown. Recent data suggest that mast cells may have a Further experiments revealed that MIP-1␣ production was deleterious role to play in the fibrotic process. In particular, dependent upon the TNF␣-induced expression of ICAM-1 interactions between mast cells and fibroblasts facilitates by the fibroblast [44]. Taken together, these data suggest the de novo production of eotaxin, a potent eosinophil that the interaction between fibroblasts and macrophages chemattractant [16, 52, 53]. In normal fibroblasts, eotaxin may be an important early event in the recruitment of production approaching 1 ng/ml could be detected follow- monocytes and may facilitate a cytokine network that ing exposure of these cells to IL-4, but not TNF␣ or IFN-␥, maintains the activation of tissue resident alveolar macro- for 24 hours. However, the highest production of eotaxin phages. was observed in 24 hour co-cultures of mast cells and untreated fibroblasts. In this system, mast cells and fibro- T cells blasts cultured alone produced Ͻ 100 pg/ml of eotaxin, but Chronological analysis of cells present in the tissue from in combination these cells generated levels of eotaxin patients with fibrosis indicated that there was an initial exceeding 20 ng/ml. Interestingly, the expression of eotaxin infiltration of different T helper cells [45]. Th2 cells can in the co-culture system was detected in both cell types, and modulate the proliferation and biosynthetic capacity of was dependent upon the cell surface expression of stem cell fibroblasts [46], largely because of their ability to generate factor by the fibroblast. Thus, these recent findings supply Hogaboam et al: Chemokines and fibroblasts in fibrosis 2157 evidence that lung fibroblasts, via its expression of a cell modulation in the lung interstitum, it would certainly seem surface ligand that mast cells recognize, can enhance the that it is an ideal target in the treatment of fibrotic diseases. expression of a potent chemoattractant for eosinophils. Selective regulation of the chemokine synthetic capacity of the fibroblast is certainly feasible in light of recent obser- Eosinophils vations that chemokine generation in fibroblasts is regu- Eosinophils have only recently been identified as culprits lated differently from immune cells such as macrophages. in fibrotic responses in the lung [54–56] and it is generally While the precise clinical contribution of chemokines to the recognized that eosinophilia is present in many disorders fibrotic process needs further exploration, the modulation characterized by tissue fibrosis [57, 58]. A strong associa- of chemokine production and/or binding of these mediators tion between the presence of eosinophils in tissue and a to chemokine receptors may supply some novel therapeutic poor disease prognosis or a resistance to anti-inflammatory alternatives in the treatment of interstitial fibrosis not only therapy is now recognized in fibrotic disease, especially in the lung, but elsewhere. interstitial pulmonary diseases [57]. Experimental fibrosis induced via bleomycin is also associated with profound Reprint requests to Steven L. Kunkel, Ph.D., Department of Pathology, eosinophil infiltration [31] and a direct role for eosinophils University of Michigan Medical School, 5214 Med Sci I, 1301 Catherine Rd, in the fibrotic process [59]. These observations have Ann Arbor, Michigan 48109-0602, USA. E-mail: [email protected] spawned numerous studies examining the nature of the interaction between fibroblasts and eosinophils. Eosino- phils readily bind to fibroblasts and many studies have REFERENCES shown that this leads to the release of mitogens that 1. WEISSLER JC: Idiopathic pulmonary fibrosis: Cellular and molecular augment fibroblasts proliferation [60] and collagen produc- pathogenesis. Am J Med Sci 297:91–104, 1989 tion [61]. For example, eosinophils appear to be a major 2. MEDURI GU, HEADLEY S, KOHLER G, STENTZ F, TOLLEY E, UM- source of MCP-1 and TGF-␤ during the later proliferative BERGER R, LEEPER K: Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Plasma IL-1 and IL-6 levels are stages of the bleomycin fibrosis model [31, 33]. There is also consistent and efficient predictors of outcome over time. Chest abundant evidence that the interaction between fibroblasts 107:1062–1073, 1995 and eosinophils is bi-directional, since the survival of 3. SCHWARTZ MI: Interstitial pulmonary fibrosis, in Textbook of Internal Medicine, edited by KELLEY WN, Philadelphia, J.P. Lippincott, 1989, eosinophils appears to be dependent on the lung fibroblast pp 1902–1905 through its production of granulocyte macrophage-colony 4. GOLDRING MB, SANDELL LJ, STEPHENSON ML: Immune interferon stimulating factor [62]. Thus, the findings currently point to suppresses levels of procollagen mRNA and type Ii collagen synthesis in articular and costal chondrocytes. J Immunol 261:9049–9056, 1986 a mutual interaction between eosinophils and fibroblasts 5. DUNCAN MR, BERMAN B: Gamma interferon is the lymphokine and that has the potential to recapitulate their interactions beta interferon is the monokine responsible for the inhibition of through the sustained activation of eosinophils and the fibroblast collagen production and late but not early fibroblast prolif- eration. J Exp Med 162:516–527, 1985 activation of the lung fibroblast to that of a biosynthetic 6. SEMPOWSKI GD, BECKMANN MP, DERDAK S, PHIPPS RP: Subsets of cell. murine lung fibroblasts express membrane-bound and soluble IL-4 receptors. Role of IL-4 in enhancing fibroblast proliferation and CONCLUSIONS collagen synthesis. J Immunol 152:3606–3614, 1994 7. POSTLETHWAITE AE, HOLNESS MA, KATAI H, RAGHOW R: Human Previously, the fibroblast was considered to be a passive fibroblasts synthesize elevated levels of extracellular matrix proteins in participant in the fibrotic process through its end stage response to interleukin-4. J Clin Invest 90:1479–1485, 1992 8. POSTLETHWAITE AE, SEYER JM: Fibroblast chemotaxis induction by contribution of extracellular matrix. However, emerging human recombinant interleukin-4. Identification by synthetic peptide evidence from many laboratories now points to a more analysis of two chemotactic domains residing in amino acid sequences active role for fibroblasts in the inflammatory process 70–88 and 89–122. J Clin Invest 87:2147–2152, 1991 9. MOSMANN TR, MOORE KW: The role of IL-10 in crossregulation of leading up to end stage disease. In the context of the Th1 and Th2 responses. Immunol Today 12:49–53, 1991 peritoneal cavity interstitial fibroblast activation may play a 10. PLAUT M, PIERCE JH, WATSON CJ, HANLEY-HYDE J, NORDAN RP, key role in peritoneal inflammatory processes and thus PAUL WE: Mast cell lines produce lymphokines in response to crosslinking FcER1 or to calcium ionophores. (abstract) Nature might contribute to the development of peritoneal fibrosis 339:64, 1989 [63, 64]. Similarly, pulmonary fibroblasts are capable of 11. CHENSUE SW, WARMINGTON K, RUTH J, LINCOLN P, KUO M-C, generating a number of chemokines that are directly or KUNKEL SL: Cytokine responses during mycobacterial and schistoso- mal antigen-induced pulmonary granuloma formation. Production of indirectly involved in the fibrotic process, such as IL-8, Th1and Th2 cytokines and relative contribution of tumor necrosis RANTES, MCP-1 and MIP-1␣. In addition, fibroblasts are factor. Am J Pathol 145:1105–1113, 1994 uniquely positioned and are capable of modulating T cell 12. CHENSUE SW, TEREBUH PD, WARMINGTON KS, HERSHEY SD, EVANOFF HL, KUNKEL SL, HIGASHI GI: Role of IL-4 and IFN in lymphokine generation, mast cell activation leading to the Schistosoma mansoni egg-induced hypersensitivity granuloma forma- elaboration of the eosinophil chemoattractant eotaxin, and tion. Orchestration, relative contribution, and relationship to macro- activation of eosinophils by producing eosinophil survival phage function. J Immunol 148:900–906, 1992 13. LUKACS NW, ADDISON C, GAULDIE J, GRAHAM F, SIMPSON K, factors. Given the numerous ways in which the fibroblast STRIETER RM, WARMINGTON K, CHENSUE SW, KUNKEL SL: Trans- may be involved in many aspects of immune activation and gene-induced production of IL-4 alters the development and collagen 2158 Hogaboam et al: Chemokines and fibroblasts in fibrosis

expression of T helper cell 1-type pulmonary granulomas. J Immunol crescent formation and interstitial fibrosis. J Exp Med 185:1371–1380, 158:4478–4484, 1997 1997 14. CHENSUE SW, WARMINGTON K, RUTH JH, LUKACS N, KUNKEL SL: 33. SMITH RE, STRIETER RM, PHAN SH, KUNKEL SL: C-C chemokines: Mycobacterial and Schistosomal antigen-elicited granuloma forma- Novel mediators of the profibrotic inflammatory response to bleomy- tion in IFNgamma and IL-4 knockout mice. Analysis of local and cin challenge. Am J Respir Cell Mol Biol 15:693–702, 1996 regional cytokine and chemokine networks. J Immunol 159:3563– 34. GHARAEE-KERMANI M, DENHOLM EM, PHAN SH: Costimulation of 3573, 1997 fibroblast collagen and transforming growth factor beta1 gene expres- 15. KUNKEL SL, LUKACS N, STRIETER RM: Chemokines and their role in sion by monocyte chemoattractant protein-1 via specific receptors. human disease. Agents Actions 46(Suppl):11–22, 1995 J Biol Chem 271:17779–17784, 1996 16. JOSE PJ, GRIFFITHS-JOHNSON DA, WALSH DT, MOQBEL R, TOTTY NF, 35. JORDANA M: Mast cells and fibrosis - Who’s on first? Am J Respir Cell TRUONG O, HSUAN JJ, WILLIAMS TJ: Eotaxin: A potent eosinophil Mol Biol 8:7–8, 1993 chemoattractant cytokine detected in a guinea-pig model of allergic 36. SHULL MM, ORMSBY I, KIER AB, PAWLOWSKI S, DIEBOLD RJ, YIN M, airways inflammation. J Exp Med 179:881–887, 1994 ALLEN R, SIDMAN C, PROETZL G, CALVIN D, ANNUNZIATA N, 17. HEDRICK JA, ZLOTNICK A: Identification and characterization of a DOETSCHMAN T: Targeted disruption of the mouse transforming novel Beta chemokine containing six conserved cysteines. J Immunol growth factor-beta1 gene results in multifocal inflammatory disease. 159:1589–1593, 1997 Nature 359:693–699, 1992 18. PREMACK BA, SCHALL TJ: Chemokine receptors: Gateways to inflam- 37. TAUB DD, OPPENHEIM JJ: Chemokines, inflammation and the im- mation and infection. Nature Med 2:1174–1178, 1996 mune system. Ther Immunol 1:229–246, 1994 19. CHENSUE SW, WARMINGTON KS, RUTH JH, SANGHI PS, LINCOLN PM, 38. LUKACS NW, KUNKEL SL, ALLEN R, EVANOFF HL, SHAKLEE CL, KUNKEL SL: Role of monocyte chemoattractant protein-1 in Th1 SHERMAN JS, BURDICK MD, STRIETER RM: Stimulus and cell-specific (Mycobacterial) and Th2 (Schistosomal) antigen-induced granuloma expression of C-X-C and C-C chemokines by pulmonary stromal cell formation: Relationship to local inflammation, Th cell expression and populations. Am J Physiol 268(5 Pt 1):L856–L861, 1995 IL-12 production. J Immunol 157:4602–4608, 1996 39. XIA Y, PAUZA ME, FENG L, LO D: RelB regulation of chemokine 20. KARPUS WJ, LUKACS NW, KENNEDY KJ, SMITH WS, HURST SD, expression modulates local inflammation. Am J Pathol 151:375–387, BARRETT TA: Differential CC chemokine-induced enhancement of T 1997 helper cell cytokine production. J Immunol 158:4129–4136, 1997 40. NAGAOKA I, TRAPNELL BC, CRYSTAL RG: Up-regulation of platelet- 21. LUKACS NW, CHENSUE SW, KARPUS WJ, LINCOLN P, KEEFER C, derived growth factor-A and -B gene expression in alveolar macro- STRIETER RM, KUNKEL SL: C-C chemokines differentially alter phages of individuals with idiopathic pulmonary fibrosis. J Clin Invest interleukin-4 production from lymphocytes. Am J Pathol 150:1861– 85:2023–2027, 1990 1868, 1997 41. ANTONIADES HN, BRAVO MA, AVILA RE, GALANOPOULOS T, NEV- 22. HOGABOAM CM, LUKACS NW, CHENSUE SW, STRIETER RM, KUNKEL ILLE-GOLDEN J, MAXWELL M, SELMAN M: Platelet-derived growth SL: Monocyte chemoattractant protein-1 synthesis by murine lung factor in idiopathic pulmonary fibrosis. J Clin Invest 86:1055–1064, fibroblasts modulates CD4ϩ T cell activation. J Immunol 160:4606– 1990 4614, 1998 42. MCKEE CM, PENNO MB, COWMAN M, BURDICK MD, STRIETER RM, 23. LU B, RUTLEDGE BJ, GU L, FIORILLO J, LUKACS NW, KUNKEL SL, BAO C, NOBLE PW: Hyaluronan (HA) fragments induce chemokine NORTH R, GERARD C, ROLLINS BJ: Abnormalities in monocyte gene expression in alveolar macrophages. The role of HA size and recruitment and cytokine expression in monocyte chemoattractant CD44. J Clin Invest 98:2403–2413, 1996 protein-1-deficient mice. J Exp Med 187:601–608, 1998 43. CARRE PC, MORTENSON RL, KING TE, NOBLE PW, SABLE CL, RICHES 24. LYNCH JP, STANDIFORD TJ, ROLFE MW, KUNKEL SL, STRIETER RM: DW: Increased expression of the interleukin-8 gene by alveolar Neutrophilic alveolitis in idiopathic pulmonary fibrosis. The role of macrophages in idiopathic pulmonary fibrosis. J Clin Invest 88:1802– IL-8. Am Rev Respir Dis 145:1433–1439, 1992 1810, 1991 25. KEANE MP, ARENBERG DA, LYNCH JPR, WHYTE RI, IANNETTONI 44. STEINHAUSER ML, KUNKEL SL, HOGABOAM CM, EVANOFF H, STRI- MD, BURDICK MD, WILKE CA, MORRIS SB, GLASS MC, DIGIOVINE B, ETER RM, LUKACS NW: Macrophage/fibroblast co-culture induces KUNKEL SL, STRIETER RM: The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immu- macrophage inflammatory protein-1alpha production mediated by nol 159:1437–1443, 1997 intercellular adhesion molecule-1 and oxygen radicals. J Leuk Biol (in 26. PETREK M, PANTELIDIS P, SOUTHCOTT AM, LYMPANY P, SAFRANEK P, press) ATH LEXANDER ULMER BLACK CM, KOLEK V, WEIGL E, DU BOIS RM: The source and role of 45. V RR, A CB, F JD: The lymphocytic infiltrative RANTES in interstitial lung disease. Eur Repir J 10:1207–1216, 1994 lung diseases. Clin Chest Med 3:619–634, 1982 27. ANTONIADES HN, NEVILLE-GOLDEN J, GALANOPOULOS T, KRADIN 46. POSTLETHWAITE AE, SMITH GN, MAINARDI CL, SEYER JM, KANG RL, VALENTE AJ, GRAVES DT: Expression of monocyte chemoattrac- AH: Lymphocyte modulation of fibroblast in vitro: Stimulation and tant protein-1 mRNA in human idiopathic pulmonary fibrosis. Proc inhibition of collagen production by different effector molecules. Natl Acad Sci USA 89:5371–5375, 1992 J Immunol 132:2470–2477, 1984 28. STANDIFORD TJ, ROLFE MR, KUNKEL SL, LYNCH JPD, BECKER FS, 47. MONROE JG, HALDAR S, PRYSTOWSKY MB, LAMMIE P: Lymphokine ORRINGER MB, PHAN S, STRIETER RM: Altered production and regulation of inflammatory processes: Interleukin-4 stimulates fibro- regulation of monocyte chemoattractant protein-1 from pulmonary blast proliferation. Clin Immunol Immunopathol 49:292–298, 1988 fibroblasts isolated from patients with idiopathic pulmonary fibrosis. 48. BOMBARA MP, WEBB DL, CONRAD P, MARLOR CW, SARR T, RANGES Chest 103(Suppl 2):121S, 1993 GE, AUNE TM, GREVE JM, BLUE ML: Cell contact between T cells 29. STANDIFORD TJ, ROLFE MW, KUNKEL SL, LYNCH JP, BURDICK M, and synovial fibroblasts causes induction of adhesion molecules and GILBERT AR, ORRINGER MB, WHYTE RI, STRIETER RM: Macrophage cytokines. J Leuk Biol 54:399–406, 1993 inflammatory protein-1alpha expression in interstitial lung disease. 49. CARR MW, ALON R, SPRINGER TA: The C-C chemokine MCP-1 J Immunol 151:2852–2863, 1993 differentially modulates the avidity of beta 1 and beta 2 integrins on T 30. BRIELAND JK, JONES ML, FLORY CM, MILLER GR, WARREN JS, PHAN lymphocytes. Immunity 4:179–187, 1996 SH, FANTONE JC: Expression of monocyte chemoattractant protein-1 50. AGIUS RM, GODFREY RC, HOLGATE ST: Mast cell and histamine (MCP-1) by rat alveolar macrophages during chronic lung injury. Am J content of human bronchoalveolar lavage fluid. Thorax 40:760–767, Respir Cell Mol Biol 9:300–305, 1993 1985 31. ZHANG K, GHARAEE-KERMANI M, JONES ML, WARREN JS, PHAN SH: 51. CLAMAN HN: Mast cells and fibrosis. Rheum Dis Clin North Am Lung monocyte chemoattractant protein-1 gene expression in bleo- 16:141–151, 1990 mycin-induced pulmonary fibrosis. J Immunol 153:4733–4741, 1994 52. GRIFFITHS-JOHNSON DA, COLLINS PD, ROSSI AG, JOSE PJ, WILLIAMS 32. LLOYD CM, MINTO AW, DORF ME, PROUDFOOT A, WELLS TN, TJ: The chemokine, eotaxin, activates guinea-pig eosinophils in vitro SALANT DJ, GUTIERREZ-RAMOS JC: RANTES and monocyte che- and causes their accumulation in vivo. Biochem Biophys Res Commun moattractant protein-1 (MCP-1) play an important role in the inflam- 197:1167–1172, 1993 matory phase of crescentic nephritis, but only MCP-1 is involved in 53. ROTHENBERG ME, LUSTER AD, LEDER P: Murine eotaxin: An eosinophil Hogaboam et al: Chemokines and fibroblasts in fibrosis 2159

chemoattractant inducible in endothelial cells and in interleukin 4-in- 60. SHOCK A, RABE KF, DENT G, CHAMBERS RC, GRAY AJ, CHUNG KF, duced tumor suppression. Proc Natl Acad Sci USA 92:8960–8964, 1995 BARNES PJ, LAURENT GJ: Eosinophils adhere to and stimulate replica- 54. HALLGREN R, BJERMER L, LUNDGREN R, VENGE P: The eosinophil tion of lung fibroblasts in vitro. Clin Exp Immunol 86:185–190, 1991 component of the alveolitis in idiopathic pulmonary fibrosis. Signs of 61. BIRKLAND TP, CHEAVENS MD, PINCUS SH: Human eosinophils stim- eosinophil activation in the lung is associated with impaired lung ulate DNA synthesis and matrix production in dermal fibroblasts. Arch function. Am Rev Resp Dis 139:373–383, 1989 Dermatol Res 286:312–318, 1994 55. NOGUCHI H, KEPHART GM, COLBY TV, GLEICH GJ: Tissue eosino- 62. VANCHERI C, GAULDIE J, BIENENSTOCK J, COX G, SCICCHITANO R, philia and eosinophil degranulation in syndromes associated with STANISZ A, JORDANA M: Human lung fibroblast-derived granulocyte fibrosis. Am J Pathol 140:521–528, 1992 macrophage colony stimulating factor (GM-CSF) mediates eosinophil 56. FUJIMOTO K, KUBO K, YAMAGUCHI S, HONDA T, MATSUZAWA Y: survival in vitro. Am J Respir Cell Mol Biol 1:289–295, 1989 Eosinophil activation in patients with pulmonary fibrosis. Chest 108: 63. LANG JM, HOPPE J, WILLIAMS JD, TOPLEY N.: Human peritoneal 48–54, 1995 fibroblast proliferation in 3-dimensional culture: Modulation by cyto- 57. PETERSON MW, MONICK M, HUNNINGHAKE GW: Prognostic role of kines, growth factors and peritoneal dialysis effluent. Kidney Int eosinophils in pulmonary fibrosis. Chest 92:51–56, 1987 51:205–215, 1997 58. ENRIGHT T, CHUA S, LIM DT: Pulmonary eosinophilic syndromes. 64. J¨ORRES A, LUDAT K, LANG J, SANDER K, GAHL, GM, FREI U, Ann Allergy 62:277–283, 1989 DEJONGE K, WILLIAMS JD TOPLEY N: Establishment and functional 59. ZHANG K, GHARAEE-KERMANI M, MCGARRY B, REMICK D, PHAN SH: characterisation of human peritoneal fibroblasts in culture: Induction TNF-alpha-mediated lung cytokine networking and eosinophil re- of IL-6 synthesis by macrophage-derived cytokines J Am Soc Nephrol cruitment in pulmonary fibrosis. J Immunol 158:954–959, 1997 7:2192–2201, 1996