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Modern concepts on the role of inflammation in pulmonary fibrosis.

Fibrosis is an important cause of morbidity and mortality in a variety of lung diseases, especially the interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF). IPF is a clinical, physiologic, radiographic, and pathologic entity that has been difficult to characterize as a homogenous "disease." (1,2) The pathologic description of IPF originally was delineated by Leibow and Carrington (3) and termed usual interstitial pneumonia. Of the fibrosing lung disease cases they reviewed, this was the most common, hence the "usual" form. Currently, IPF remains the most common idiopathic interstitial pneumonia. During the last 30 years there has been a refinement in the pathologic description of idiopathic interstitial pneumonias, and the most recent classification represents a consensus of leading experts in the field. (4) However, despite improvements in the diagnostic approach to IPF, its pathogenesis remains poorly understood. This review will explore some newer concepts in the field of pulmonary fibrosis.

The concept that dominated the field in the 1970s and 1980s has been described as the "inflammatory" concept of pulmonary fibrosis. The paradigm was based largely on the observation that bronchoalveolar lavage fluid from patients with IPF had increased numbers of inflammatory cells (mostly neutrophils and eosinophils) relative to individuals with normal pulmonary function.5,6 The concept that permeated the literature in that era was that IPF resulted from an unremitting inflammatory response to an exogenous insult that culminated in progressive fibrosis. By targeting the inflammatory response, the belief was that the fibrosis could be limited or prevented. Unfortunately, it seems that data was more likely explained by structural abnormalities in lung architecture (traction bronchiectasis) such that inflammatory cell trafficking/accumulation was altered. Airway inflammation was likely a result, rather than a cause, of the fibrosis.

In the last 10 years, this version of the inflammatory theory has fallen out of favor. With a more precise histologic definition of this disease, it has become clear that inflammation is relatively slight in nonfibrotic portions of IPF lung, even in the earliest phase that can be clinically identified; that is, early IPF looks like late IPF histologically. There is also a lack of correlation of conventional inflammatory markers with progression of disease, failure of conventional immunosuppressive agents to affect human disease, and inconsistent requirement for an immune response or response to conventional immunosuppression in animal models. As a result, many authors have proposed that inflammation is a secondary feature of this disease at best and that the principal defect in IPF is that of recurrent epithelial injury and/or aberrant wound healing. (7-10)

In support of this hypothesis, literature has accumulated in recent years that strongly suggests that alveolar type II cell injury is an important early feature in the pathogenesis of pulmonary fibrosis. (11) Ultrastructural studies have demonstrated alveolar type II cell injury in lung biopsies from patients with pulmonary fibrosis. (12) Immunohistochemical studies have shown upregulation of various mediators indicative of alveolar cell apoptosis, including Fas, p53 and p21 (the latter a protective mediator), Bax, and caspase-3, whereas Bcl-2 is down-regulated (see Table for definition of caspases and Fas). (13-16) Bronchoalveolar lavage from patients with IPF also show soluble mediators indicative of increased apoptosis. (17) Proof of principle animal experiments using the bleomycin model of lung injury and fibrosis also have suggested that inhibiting epithelial cell apoptosis with various approaches, including inhibiting the Fas-Fas ligand pathway and blocking caspase activation, abrogates the development of experimental fibrosis. (18-21) Direct stimulation of Fas via anti-Fas antibody or injury to type II pneumocytes via a diphtheria toxin approach also leads to pulmonary fibrosis. (22,23)

More recently, the relevance of this theory to human disease has had strong proof of principle support from human genetic syndromes in which mutations in the surfactant proteins themselves lead to pulmonary fibrosis. (24) In a variety of these surfactant protein C mutations, there is activation of the unfolded protein response program leading to cell death. This phenomenon can be replicated in a genetically dominant murine transgenic model (see Table for definition of transgenic and unfolded protein response). Interestingly, patients with these mutations may come to medical attention only in adulthood. The animal data also suggest that, in some cases, the mutation makes the epithelium more sensitive to other injury. Patients with sporadic IPF also have upregulation of the unfolded protein response. (25-27) Additional support of primary cell death as a driver for pulmonary fibrosis comes from the identification of the genetic locus associated with some familial pulmonary fibrosis families as genes involved in a functional telomerase complex (see Table for definition of telomerase). (28,29) Short dysfunctional telomeres activate a DNA damage response that leads to cell death as well as limiting repair capacity. Telomere defects occur in sporadic IPF cases as well. (30,31) It is noteworthy that IPF is a disease of the elderly, with a sharp increase in incidence for those older than 50 years. This fact is also consistent with the hypothesis that there is a repair defect that is slowly overwhelmed as small amounts of injury accumulate in the lung over time.

In the models above, basement membrane integrity was not obviously affected, yet fibrosis was markedly influenced. In other settings, the loss of basement membrane integrity is commonly cited as a key additional step in conjunction with cell death to contribute to aberrant remodeling. (32) As a result of this injury, epithelial cells are forced to regrow over an abnormal matrix. This interaction with an abnormal matrix in the setting of various cytokines such as transforming growth factor [beta]1 (TGF-[beta]1) can lead to a process referred to as epithelial mesenchymal transition, in which epithelial cells assume characteristics of mesenchymal cells, with loss of epithelial markers such as E-cadherin and gain of mesenchymal markers such as smooth muscle actin and vimentin and production of collagen. This process appears to involve both the well-known wnt-[beta]-catenin pathway and an [alpha]3 [beta]1 integrin pathway. (33-39) The overall contribution of epithelial mesenchymal transition to human pulmonary fibrosis remains controversial, however.

Even while the epithelial injury/remodeling model has been pursued, another aspect of the inflammatory theory has been retained. Inflammation has been described as an essential component of wound repair for many decades. (40) In particular, it has been known that interference with macrophage function has a negative effect on wound healing, although the extent is not entirely clear. (41,42) Macrophages are well known as the source of many chemoattractants and growth factors that 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. It is important to understand, however, that the type of inflammation seen is not nonspecific, but rather reflects activation of a specialized program.

An important concept in immunology in the last several decades has been the distinction between so-called type I and type II immune responses. During chronic stimulation [CD4.sup.+] T helper ([T.sub.H]) cells can become polarized toward distinct lineages that promote different types of effector activity via production of distinct subsets of cytokines. The development of such T-cell subsets, including [T.sub.H]1, [T.sub.H]2, [T.sub.H]17, and various regulatory T-cell subsets, arises from the cytokine environment established from the initial interaction between the pathogen/immunogen and the innate immune system. (43) The [T.sub.H]1 cells appear important in limiting intracellular infections and prototypically produce interferon (IFN) [gamma]. The [T.sub.H]2 cells appear important in limiting extracellular infections, especially parasites, and are especially important in allergic diseases and prototypically produce interleukin (IL) 4 and IL-13. The [T.sub.H]1 and [T.sub.H]2 pathways are thought to usually inhibit each other.

Corresponding to the distinction between type I and type II immune responses, a growing literature supports the idea that macrophage differentiation also responds to a particular cytokine environment. (44,45) Under these conditions, macrophages undergo differential expression of a variety of factors, which result in markedly different functions. The simplest version of this theory postulates 2 major classes of macrophages corresponding to [T.sub.H]1-stimulated macrophages (M1) and [T.sub.H]2-stimulated macrophages (M2). The M1 macrophages are induced by IFN-[gamma] and identified by expression of IL-12, tumor necrosis factor, and the chemokine CXCL10, and display a phenotype associated with tumor resistance, killing of intracellular parasites, and tissue destruction (see Table for definition and nomenclature of chemokines). The M2 macrophages are induced by IL-4 and/or IL-13 and identified by expression of the hemoglobin scavenger receptor CD163, the IL-1RII decoy receptor, mannose receptors, and increased IL-10, CCl-18, and CCl-22 production, and display a phenotype associated with immunoregulation, tumor promotion and tissue remodeling, fibrosis, and angiogenesis. However, there are additional phenotypes also generally included under the M2 category, including macrophages stimulated with IL-10 and/or TGF-[beta]1 and other, more complicated mixtures. In addition, regulatory T cells may also promote M2 differentiation. (46) These additional categories have varied functions, some of which are shared among the M2 family and some that are specific to their particular phenotype. It is possible that in vivo there is actually a spectrum of differentiation rather than distinct phenotypes. (47) Although there exists good in vitro evidence for these subsets, they are much less well worked out in vivo, where the cytokine environment is much more complicated than the in vitro experiments have investigated. Furthermore, unlike the situation with T cells, there is no suggestion that the cytokine environment results in a terminal differentiation event. Finally, multiple other monocyte/macrophage subsets exist, at least 2 of which have been suggested to correlate with initial pathogen control/tissue injury versus healing/repair. (48) The correlation of these different classification schemes with each other is currently unsettled.

The type II cytokine hypothesis of fibrosis thus suggests that fibrosis occurs in chronic inflammatory disorders when the cytokine balance shifts in a [T.sub.H]2-cell (type II) direction. (49,50) Although the concept of type I versus type II immune responses came from the study of antigen-specific (acquired) immunity, it has now become apparent that this distinction can be made in nonspecific (innate) immunity as well. Models of wound healing show that at sites of injury, macrophages in vivo have at least partially an M2 phenotype, but that this is not IL-4/13 dependent. (51) In other models, interventions that interfere with this pattern of mediators, including overexpression of type I immune response products or inhibition of type II mediators, have significant effects on the wound healing/fibrotic process. (40,41) Interestingly, corticosteroids promote M2 macrophage differentiation, and thus may in fact do more to promote fibrosis than to alleviate it. (52)

One of the key [T.sub.H]2 cytokines is IL-13. Originally described as a companion cytokine to IL-4, the prototypical [T.sub.H]2 cytokine, from which it arose from a gene duplication event, IL-13 has been implicated in multiple effector mechanisms of [T.sub.H]2 inflammation, including stimulation of immunoglobulin (Ig) E production, enhanced macrophage CD23 (low-affinity IgE receptor) expression, endothelial cell P-selectin and vascular cell adhesion molecule expression, eosinophil activation and recruitment, and mast cell activation. (53) A general rule seems to be that IL-4 is more involved in polarizing the immune response to a [T.sub.H]2 pathway, whereas IL-13 is more involved with end organ effector functions. For example, in animal models of acute allergic airway disease, inhibition of IL-13 was critically important in preventing downstream effects of airway inflammation but not in generation of the response. (54) This is partially explained by lack of receptors for IL-13 on T cells (see below).

In order to characterize the effects of IL-13 in a more chronic (and hence more clinically relevant) model, our group produced mice transgenic for IL-13 under the rat Clara cell secretory protein (also known as Clara cell 10) promoter. This construct, initially characterized by Whitsett and colleagues as discussed by Zhu et al, (55) provides expression on Clara cells in the airway epithelium (roughly 50% of airway epithelial cells of the mouse) with a commonly observed but variable leak in type II pneumocytes. Although the overexpression of a single cytokine may be viewed as artifactual in terms of reproducing the complexity of an entire disease state, it does allow for precise interpretation of upstream and downstream events from a particular mediator in vivo. (56)

Consistent with the known function of this promoter, these mice showed expression of IL-13 along the airways and in type II cells, as shown by mRNA in situ hybridization. (57) These mice show a variety of abnormalities consistent with human allergic airways disease, including airway fibrosis, airway mucus cell metaplasia with accumulation of a macrophage, and eosinophil-rich inflammatory response (note airway fibrosis and macrophage accumulation in Figure 1, B, compared with Figure 1, A). Physiologically, the mice showed airway hyperresponsiveness to methylcholine. (55) These mice proved to have a phenotype more complex than just asthmalike, however. Although fibrosis initially was seen in airways, the mice eventually developed parenchymal fibrosis, both interstitially and in a pattern of organizing pneumonitis (Figure 1, C and D). (58) In addition, they showed type II cell hypertrophy and surfactant accumulation (both surfactant apoproteins and lipids), all features seen in various forms of human interstitial lung disease. Finally, at early time points, protease-dependent parenchymal destruction was also present. (59)

The induction of fibrosis was particularly interesting. Despite the well-known direct activity of IL-13 on stromal cells, (60) we found that in the murine lung its effects depended on the recruitment of a macrophage-rich infiltrate. (61) Monocyte chemotactic protein (MCP) family chemokines are believed to play particularly important roles in allergic disease. This is based on the demonstration that MCP-1 is a potent stimulator of mast cell mediator release, T-cell chemotaxis, basophil chemotaxis, and tissue fibrosis, which also enhances naive T-cell acquisition of a [T.sub.H]2-cell phenotype. It is also based on the demonstration that MCP-1, acting via its major receptor, CCR2, is the major recruiter of macrophages at sites of allergic tissue inflammation, and that MCP-1, MCP-3, and MCP-4 are expressed in a exaggerated fashion in tissues from patients with asthma. CCR2 is also critically important in recruitment of fibrocytes, myeloid lineage cells that produce collagen, and may also be important in fibrosis. (62-64) Other cells, chemokines, and cytokines are also required for IL-13-induced fibrosis. (65-68) Thus we concluded that in our model, IL-13 recruits additional cells and mediators that are required for development of pulmonary fibrosis. Thus, this model shows multiple potential targets for intervention besides IL-13 itself.

[FIGURE 1 OMITTED]

One immediate question is the relevance of the IL-13 overexpression model to other models of lung fibrosis. Remarkably, in all other models of murine pulmonary fibrosis that have been examined, IL-13 has been shown to be critically dependent. This includes models in which fibrosis is induced by granulomatous inflammation, (69) by fluorescein isocyanate (a non-T-cell-dependent model in which IL-4 deficiency is ineffective in blocking fibrosis), (70) and by adenosine deaminase deficiency. (71,72) Furthermore, murine [gamma]-herpesvirus 68 will induce lung fibrosis only in the absence of IFN-[gamma], where there is thus an excess type II cytokine environment. (73) IL-13 has also been shown to be important in a surprising variety of extrapulmonary fibrosis models, including non-immune-based systems such as steatohepatitis and trinitrobenzene sulfonic acid-induced colitis. (74-76)

Far and away, however, the most prevalent model of experimental lung fibrosis is that induced by bleomycin, an antineoplastic agent that induces DNA damage via oxidative injury. Remarkably, bleomycin also stimulates IL-13 production, which peaks on day 12 by both mRNA and protein analysis. A variety of methods of inhibiting IL-13 signaling are effective in blocking bleomycin-induced fibrosis. (77-79) It is striking that this supposedly nonspecific agent induces a gene expression profile that is relatively more closely related to [T.sub.H]2 models than to models of bacterial infection. (80) A very recent article suggests that bleomycin involves an IL-17A-dependent pathway rather than IL-13. (81) The discrepancy between these results is not yet explained. Mediators potentially important in lung fibrosis are also regulated by IL-4/13. For example, found in inflammatory zone (FIZZ) 1, which is induced in alveolar type II epithelial cells in bleomycin-induced lung fibrosis, stimulates myofibroblast differentiation in vitro. Of all cytokines examined, only IL-4 and IL-13 were effective in stimulating FIZZ1 expression in alveolar type II epithelial cells. In vivo, blocking IL-4 and/or IL-13 or STAT6 (a transcription factor specific for IL-4/13 signal transduction) blocks FIZZ1 expression in the bleomycin model. (82) The ability of the [T.sub.H]1/[T.sub.H]2 dichotomy to explain bleomycin-induced fibrosis is also supported by the observation that [T.sub.H]1 deficiency via loss of CXCR3 leads to increased bleomycin-induced fibrosis via loss of natural killer cells, which produce IFN-[gamma]. (83) Finally, our own work demonstrates that modulation of macrophage function with the pentraxin protein serum amyloid P restores the M1/M2 balance and reduces the fibrosis and remodeling caused by bleomycin. (84)

Given the significance of IL-13 in murine models of lung and other organ fibrosis, it was critically important to determine if a similar [T.sub.H]2 (or more properly, a type II) cytokine profile is present in lungs of humans with IPF. Several studies have shown increased IL-13 expression in human IPF. (85-88) Moreover, IL-13 tissue levels correlate well with aspects of overall disease severity and progression. (89)

Given the results above, it is logical to ask for the cellular source of IL-13 under those conditions. Although Hancock et al (88) showed production by a population enriched in alveolar macrophages, production at the single-cell level was not shown; thus, some contaminating T cells could not be excluded. More recently, it is clear that a variety of non-T-cell hematopoietic cells, including dendritic cells, macrophages, mast cells, basophils, eosinophils, and natural killer cells, are sources of IL-13. (54,90,91) Epithelial cells have also been reported to show IL-13 expression, which in one study was shown to enhance epithelial repair via epidermal growth factor family members. (92-94)

With respect to bleomycin specifically, it appears to activate macrophages via one of the toll-like receptors, TLR2. (95) TLR2 activation in turn has been reported to lead to macrophage polarization. (96) Consistent with this model, it has been shown that bleomycin-induced skin fibrosis is IL-13 dependent but T-cell independent. (97-99)

IL-13 mediates its actions via a complex receptor system that includes the IL-4R[alpha] chain and at least 2 IL-13-binding proteins that have been designated as IL-13R[alpha]1 and IL13R[alpha]2. The IL-13R[alpha]1 chain pairs with the IL-4R[alpha] chain to produce the type IIIL-4 receptor. In addition, the IL-13R[alpha]2 chain binds directly to IL-13. (100) Both proteins bind IL-13 but not IL-4. Interleukin 13R[alpha]1 by itself binds IL-13 with low affinity (2-10 nM); the affinity is greatly enhanced in the presence of the IL-4R[alpha] chain. The ability of both cytokines to bind this complex explains the redundancies in function observed between IL-4 and IL-13. In contrast, IL-13R[alpha]2 alone binds IL-13 with high affinity (0.5-1.2 nM). The IL-13R (IL-4R/IL-13R[alpha]1) is expressed on both hematopoietic and nonhematopoietic cells. Although the message for the IL-13Ra1 chain is present in T cells, there appears to be no surface expression of this chain on human or mouse T cells. Consequently, IL-13 is not thought to exert any control over T-cell function.

In order to investigate whether IL-13 regulated its own receptors, we used a variety of in vivo and in vitro methods. (101) In the IL-13 transgenic mice there was a marked increase in IL-13R[alpha]2 and IL-13R[alpha]1 mRNA. This was most prominent in airway epithelial cells and macrophages. The effects of IL-13 on IL-13R[alpha]2 were associated with comparable increases in protein production and were mediated by a blood leukocyte-independent and IL-4R[alpha]-dependent mechanism. Interleukin 13 stimulation of IL-13R[alpha]1 was mediated via a leukocyte-dependent and partially IL-4R[alpha]-dependent pathway. These effects were not specific for IL-13, because transgenic IL-4 stimulated both IL-13R[alpha]2 and IL-13R[alpha]1 mRNA accumulation. These regulatory events were mediated, at least in part, by direct effects of these cytokines, because IL-13, IL-4, and IFN-[gamma] had similar effects on IL-13R[alpha]2 and/or IL-13R[alpha]1 in epithelial cells and macrophages in vitro. (101) Strikingly, IL-13 receptor upregulation was also seen in tissues from patients with IPF. (102-104) It is noteworthy that this upregulation could in theory amplify IL-4/13 signaling even in the presence of modest increases in IL-4/ 13 itself.

In addition to IL-13 itself and its receptors, it is logical to look for evidence for an IL-13 signature in macrophages from IPF lungs. As discussed above, given the data on IL-13 expression, we would expect that macrophages from IPF should show evidence for M2 polarization. The best data on this comes from data on CCL18 in pulmonary fibrosis. CCL18 is a human-specific chemokine previously known as PARC, which is produced predominantly by myeloid cells, especially macrophages under the influence of IL-4, as well as by collagen. (105-110) Strikingly, circulating levels of CCL18 correlated with pulmonary function and outcome better than histology, regardless of disease. Our own work demonstrates that circulating monocytes from patients with scleroderma-related interstitial lung disease display enhanced CCL18 secretion in response to lipopolysaccharide stimulation, suggesting that cells in the circulation may be preprogrammed to adopt a fibrotic phenotype prior to their entry into the diseased lung. (111) These data suggest that alternatively activated macrophages are strongly associated with multiple forms of lung fibrosis and may serve as targets for therapeutic intervention.

It is important to note that even diseases thought to be due largely to IL-13 actually show marked heterogeneity in IL-13 expression. For example, using expression signatures in airway epithelium, only half of asthmatics have an IL-13 signature. Interleukin 13-high patients respond to steroids and have increased eosinophils/IgE, airway remodeling, increased airway hyperresponsiveness, and altered mucin stores. This signature correlates with presence of periostin and CEACAM5 in serum. (112-114) Scleroderma, a disease with more direct application to IPF, also shows multiple signature patterns. Using IL-4, IL-13, and TGF-[beta]1 to create signatures on dermal fibroblasts, the IL-13/4 signature is seen in patients with milder disease, whereas the TGF-[beta]1 signature is expressed in a subset of patients with more severe disease, including those with lung disease. (115,116) There is thus the possibility that patients with pulmonary fibrosis can be equivalently divided into molecularly meaningful subsets with direct therapeutic implications.

What happened to TGF-[beta]1? Transforming growth factor [beta]1 has been shown to be a critical mediator of lung fibrosis in animal models and is probably the most highly studied of any profibrotic mediator. (40) Several studies have shown that antagonizing TGF-[beta]1 prevents the development of tissue fibrosis. (117) Transforming growth factor [beta]1 is clearly upregulated in human pulmonary fibrosis. The data have been sufficiently compelling that a number of therapies directed at TGF-[beta]1 have been developed despite concerns that have been raised about potential consequences of TGF-[beta]1 blockade, including the finding that TGF-[beta]1 knockout mice die of unremitting inflammation. (118) Remarkably, pulmonary fibrosis in the IL-13 mouse was found to be TGF-[beta]1 dependent. (119) Furthermore, this effect was dependent on production and activation of TGF-[beta]1 by those macrophages that are recruited in a CCR2-dependent fashion. (61) Outside of the lung, TGF-[beta]1 independent pathways for IL-13-induced fibrosis have also been established. (120)

[FIGURE 2 OMITTED]

Given the importance of TGF-[beta]1 in IL-13-induced lung fibrosis, we decided to create mice that overexpress TGF-[beta]1 in the lung. (121) TGF-[beta]1 overexpression is fetal lethal; thus, it was important to create a transgenic system that was both inducible and extremely tightly regulated. Three "triple-transgenic" lines were established with no significant leak at baseline in which expression of the transgene was regulated by the tetoperon and induced by doxycycline (dox). These mice expressed TGF-[beta]1 in bronchoalveolar lavage fluid ranging from 250 to 16000 pg/mL after 1 week of dox administration. These levels were maintained for the duration (up to 3 months) of dox administration and returned to undetectable levels within 48 hours of the removal of dox from the animals' drinking water. In all cases TGF-[beta]1 was successfully targeted to the lung because TGF-[beta]1 mRNA could not be detected in a variety of other tissues from these animals.

Not surprisingly, the induction of TGF-[beta]1 caused readily apparent airway and parenchymal fibrosis (Figure 2, B, compared with A). On trichrome stains, this response could be seen after as little as 48 hours of transgene expression, predominately in and around airways, especially smaller airways. With longer periods of transgene expression, this fibrotic response could be seen in the alveolar parenchyma. In accord with these findings, lung collagen content continued to increase during the 2-month period of transgene expression.

Remarkably, and consistent with the work concerning cell death in human fibrotic lung disease previously discussed, TGF-[beta]1 expression was associated with a cell death response. After as little as 12 hours of TGF-[beta]1-induction, epithelial cells became positive via terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), a method for detecting DNA fragmentation typically seen in apoptosis. Small numbers of TUNEL-positive macrophages were also seen. Flow cytometry using propidium iodine and annexin V staining confirmed these findings and also showed an increase in so-called secondary necrosis in type II pneumocytes in which apoptotic cells have undergone necrosis. The TUNEL response peaked after 48 hours of dox administration and disappeared despite continuous dox administration. During the apoptotic phase, enhanced levels of mRNA of the apoptosis mediators caspases 3, 7, 8, and 11 were seen. Western blot analysis also revealed increased cleavage of caspase targets also supporting increased apoptosis levels.

In order to determine the significance of this finding, we evaluated multiple interventions that potentially had an impact on apoptosis and/or fibrosis to see if they had a role in fibrosis and/or apoptosis. Backcrossing the TGF-[beta]1 mice onto mice deficient in the mediators sema7A, BAX, Bid, EGR-1, caspase 3, or caspase 7, or treatment with the caspase inhibitor ZVAD, decreased both apoptosis and fibrosis. (121-123) Treatment with serum amyloid protein reduced apoptosis and fibrosis in addition to the change in macrophage polarization mentioned previously. (84) On the other hand, p21 deficiency increased both apoptosis and fibrosis. (124) In contrast to these results, apoptosis in the setting of IFN-[gamma] is not profibrotic. (125) These results are highly consistent with a model in which epithelial injury is a critical step in pulmonary fibrosis but only in the right cytokine environment.

In light of the above observations in the TGF-[beta]1 mice, we reexamined the IL-13 mice for the contribution of apoptosis to this process. Because we had shown that TGF-[beta]1 is required for pulmonary fibrosis in this model and that TGF-[beta]1 requires apoptosis for development of pulmonary fibrosis, it was reassuring that, as expected, apoptosis was dramatically increased in the IL-13 mice. In addition, many of the same interventions that reduce apoptosis and fibrosis in the TGF-[beta]1 mice, as well as some novel ones, also reduce fibrosis in the IL-13 mice. (126-128)

Less expectedly, given the supposed immunosuppressive effect of TGF-[beta]1, expression caused a prominent pulmonary inflammatory infiltrate as soon as 2 days after transgene activation and increased in intensity during the ensuing 10 days. In bronchoalveolar lavage, it manifested as an increase in total cell and macrophage recovery. In lung tissues, it was largely due to an increase in macrophages, with a smaller contribution from eosinophils. Further characterization of these macrophages by gene expression analysis reveals a complex, time-dependent phenotype dominated by M2 activation. (129) In this model macrophage influx peaks at day 5. Interestingly, removal of these macrophages using the specific macrophage toxic agent clodronate ameliorates many aspects of the TGF-[beta]1 phenotype, including collagen accumulation (E.H., unpublished observations). These data suggest that TGF-[beta]1's effects on macrophages may be required for pulmonary fibrosis independent of any direct effects on fibroblasts.

Another interesting aspect of this model is that M2 polarization occurs in the apparent absence of an acquired immune response. Although both the IL-13 mice and the TGF-[beta]1 mice have alternatively activated macrophages, the specific pattern of activation is slightly different. For instance, IL-13 mice macrophages have high expression of the chitinase family members YM1/2, AMCase, BRP39, and chitotriosidase (the former 2 both highly [T.sub.H]2 dependent), whereas the TGF-[beta]1 mice have only high expression of BRP39 and weak chitotriosidase expression. Furthermore, although the TGF-[beta]1 mice have increased expression of IL-4R[alpha] and IL-13R[alpha]2, breeding the TGF-[beta]1 mice onto an IL-4R[alpha] deficient background has no effect on overall fibrosis, indicating that even if IL-13 and/or IL-4 expression occurs, it is not critically important in producing fibrosis (C.G.L. and J.A.E., unpublished observations). Thus, the macrophages in this model show a partial M2 phenotype that is IL-4/13 independent, similar to that described in wound healing. (51)

We have therefore been able to model a system in which both cell death and a polarized macrophage infiltrate are needed for fibrosis to occur. It is possible that these 2 effects are related. Efferocytosis, the process of phagocytosis of apoptotic cells, has been described for many years. It has been established that efferocytosis leads to production of TGF-[beta]1 and IL-10, hence an M2-type response. (130-133) On the other hand, some death responses lead to a [T.sub.H]1, hence M1, response, making the connection more uncertain. (134) Alternatively, it is possible that TGF-[beta]1 signaling directly induces M2 polarization, but this has not yet been formally proven in our system. (135)

Thus we have seen that the so-called inflammatory theory of pulmonary fibrosis, in which inflammation leads to tissue injury and fibrosis, has largely been abandoned in favor of theories that emphasize cell death as a primary driver for the process. However, there is still a major role for inflammatory mediators modifying that response in critically important ways. Interleukin 13 appears to be a potential central mediator of fibrosis in a variety of models and is found upstream of TGF-[beta]1, a well-known mediator of pulmonary fibrosis. Despite the high profile failure of IFN-[gamma] as therapy in IPF, the M2 model of macrophage polarization remains attractive both for targets for intervention and for markers for disease progression. (136) It is noteworthy that one clinical trial has been completed directly targeting IL-13 in pulmonary fibrosis (clinicaltrials.gov identifier NCT00532233), although results have not been published. Direct intervention with macrophage-specific agents is also a highly attractive approach. Future approaches should analyze animal models that more closely replicate human disease based on the concept of epithelial cell death rather than on the highly toxic drug bleomycin. Additional efforts should also be made to correlate specific patterns of inflammatory mediators with human disease (severity, progression, and response to therapy).

This study was supported by grants NIH HL 064242, HL 56389, and HL 081639.

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Robert J. Homer, MD, PhD; Jack A. Elias, MD; Chun Gun Lee, PhD; Erica Herzog, MD, PhD

Accepted for publication July 29, 2010.

From the Departments of Pathology (Dr Homer), Internal Medicine (Dr Elias), and Internal Medicine, Pulmonary and Critical Care (Drs Lee and Herzog), Yale School of Medicine, New Haven, Connecticut.

Financial support was provided by Promedior, Malvern, Pennsylvania. The authors have no relevant financial interest in the products or companies described in this article.

Presented as the keynote address at the Pulmonary Pathology Society meeting;June 24, 2009;Portland, Oregon.

Reprints: Robert J. Homer, MD, PhD, Department of Pathology, Yale School of Medicine, PO Box 208070, New Haven, CT 06520-8070 (e-mail: Robert.homer@yale.edu).
Definitions for Concepts Referenced in This Article

Product Definition

Caspases A family of cysteine proteases (cysteineaspartic
 proteases or cysteine-dependent
 aspartate-directed proteases) that play
 essential roles in apoptosis and
 inflammation.
Chemokines A family of 8-10-kDa proteins that are able
 to induce chemotaxis. Historically,
 chemokines were referred to by descriptive
 names referring to their function, eg,
 monocyte chemotactic protein 1 (MCP-1). The
 modern nomenclature is based on their
 structure. Most chemokines fall into 1 of 2
 families, CC chemokines and CXC chemokines,
 based on a characteristic protein motif
 (cysteine-cysteine versus cysteine-amino
 acid-cysteine). The current nomenclature is
 thus based on these families, eg, CCL1 for
 ligand 1 of the CC family of chemokines and
 CCR1 for its respective receptor.
Fas, Fas ligand A cell surface receptor pathway that
 stimulates apoptosis.
Telomerase An enzyme that adds DNA sequence repeats
 ("TTAGGG" in all vertebrates) to the 3' end
 of DNA strands in the telomere regions, which
 are otherwise shortened when a cell divides.
 Without the presence of telomerase, at some
 point all the cell progeny will reach their
 division limit. A variety of premature aging
 syndromes are associated with short
 telomeres.
Transgenic mice Mice into which an exogenous gene has been
 inserted into the germline. The exogenous
 gene is commonly placed under control of a
 promoter known to drive expression in an
 organ or cell limited fashion.
Unfolded protein A cellular stress response to excess unfolded
 response pathway or improperly folded proteins during protein
 synthesis that if unrelieved can stimulate
 apoptosis.
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Title Annotation:Review Articles
Author:Homer, Robert J.; Elias, Jack A.; Lee, Chun Gun; Herzog, Erica
Publication:Archives of Pathology & Laboratory Medicine
Date:Jun 1, 2011
Words:9462
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