Molecular and cellular mechanisms of pulmonary fibrosis

T lymphocytes

A consistent finding in most fibrotic diseases of the lungs is the presence of T lymphocytes, one of the major inflammatory cell types. Mechanistic studies regarding the role of T cells in pulmonary fibrosis have been performed in animals which lacked T lymphocytes or had selective attraction of T lymphocytes to the lungs. Most of these studies used bleomycin as a model of lung injury, in which fibrosis is preceded by pulmonary inflammation.

In athymic mice lacking T cells, administration of bleomycin resulted in less fibroblast proliferation and less ECM accumulation compared to wild-type mice [123]. Mice whose T lymphocytes lacked CD28, a central co-stimulatory cell surface molecule necessary for full T cell activation, showed markedly attenuated fibrosis following exposure to bleomycin, and transferring CD28-positive T lymphocytes into these CD28-deficient animals restored the fibrotic response to bleomycin [124]. In mice systemically depleted of T lymphocytes, exposure to bleomycin resulted in less collagen accumulation and increased survival compared with controls [125, 126]. In our mouse model in which selective attraction of T lymphocytes to the lung was achieved by over-expressing human CCL18 (PARC), a prolonged infiltration of T cells occurred, moderate collagen accumulation developed, and systemic depletion of T cells prevented the collagen accumulation despite the continuous expression of CCL18, suggesting that T cells were indeed the driving force of fibrosis [113]. It should be noted that although human CCL18 appears to be fully functional in mice in cell culture and in vivo [109, 110, 111, 112, 113,][127], the chemokine CCL18 has not been definitely identified in mice.

There may be a complex interplay among subtypes of T lymphocytes in pulmonary fibrosis, particularly between T effector and T regulatory cells. T regulatory lymphocytes (T regs) are profibrotic and immunosuppressive, and exert their profibrotic actions primarily via transforming growth factor-beta (TGF-β) and platelet-derived growth factor (PDGF) [128]. In an animal model of silica-induced pulmonary fibrosis, T regs were recruited to the lungs, caused fibroblast proliferation, had increased expression of TGF-β1 and PDGF, and caused pulmonary fibrosis upon transfer into silica-unexposed animals [45]. In silica-exposed animals depleted of T regs, pulmonary fibrosis occurred, but increased numbers of CD4+ T effectors were present. These T effectors caused fibroblast proliferation, caused pulmonary fibrosis upon transfer into T reg deficient (but not T reg competent) animals, and neutralizing antibody against these T effectors eliminated collagen accumulation [45]. These results suggest a complex interplay between lymphocyte subsets, with T regulatory cells themselves acting profibrotically, and the role of CD4+ T effector cells depending on the presence or absence of T regulatory cell competence.

Observational studies in humans link T cell infiltration to fibrosis. In patients with IPF, the presence of T cells within lung tissue and BAL of patients has been consistently observed [129–134]. These T cells are activated and antigen experienced [132, 135, 136], and characterization has shown that both CD4+ and CD8+ cells are present, with the suggestion that CD8+ T cells represent the majority [129–132, 137]. Increased numbers of CD8+ T cells in lung tissue from IPF patients was associated with worse pulmonary outcomes [130], and in BAL fluid, higher CD4/CD8 ratios correlated with an improved clinical response to anti-inflammatory therapy [133]. In patients with IPF, well-organized lymph-node-like structures with features of ‘lymphoid neogenesis’ are present, which are composed of T cells expressing CD40L, B cells, and mature dendritic cells [132]. These T and B cells are activated, but not proliferating, and most are CD45RO + indicative of a memory phenotype [132]. These findings support the concept that organized lymphoid tissue can persist in the lung and contribute to chronic inflammation even in the absence of cellular proliferation.

In other forms of interstitial pneumonia, accumulation of T cells has been shown in BAL from patients with scleroderma [138, 139], rheumatoid arthritis [140], and polymyositis/dermatomyositis [141, 142]. In general, it appears that although both CD4+ and CD8+ T cells are associated with pulmonary fibrosis, CD8+ T cells appear to be associated with a worse prognosis. In patients with sarcoidosis [143], beryliosis [144], rheumatoid arthritis [140], and hypersensitivity pneumonitis [145], CD4+ T cells were predominant in the lungs, consistent with the better prognosis of these diseases compared with IPF.

Thus, there are numerous observations in animal models and in patients which describe an association of pulmonary fibrosis with T lymphocytic infiltration. The preponderance of evidence suggests that such association is reflective of T cell profibrotic action, but depending on cell phenotype and the nature of the pulmonary milieu, T cells may promote or diminish the pulmonary fibrotic process. Two broad mechanisms by which T cells may influence the fibrotic process include production of Th-1 or Th-2 cytokines (as seen in Table 1) and cell surface molecule interactions with epithelial or mesenchymal cells, which will be described subsequently below.

Macrophages

Macrophages constitute the majority of cells recovered from BAL of normal individuals, and have essential roles in phagocytosis, innate and adaptive immunity, and surfactant homeostasis [146]. A pathogenic role for macrophages in pulmonary fibrosis has been postulated for many years, and described mechanisms have included overexpression of reactive oxygen species [147–151], proteinase-activated receptors [152–154], Fas ligand [155], and profibrotic cytokines [97, 154, 156–162].

One feature regarding a potential role of macrophages in pulmonary fibrosis relates to the concept of alternative activation. Classical activation of macrophages is induced by interferon-γ, and is characterized by macrophage expression of IL-1β, IL-6, TNF-α, and nitric oxide. Alternative activation of macrophages is induced most notably by the Th-2 cytokines IL-4 and IL-13, and is characterized by macrophage expression of mannose receptor-1 (CD206), arginase-1, the lectin-binding protein Ym1, the resistin-like protein Fizz1, and the chemokine CCL18 [60, 161, 162].

Several animal models of pulmonary fibrosis have demonstrated that alveolar macrophages (AM) display a phenotype consistent with alternative activation. In the silica-induced model, mRNA expression of Ym1 in the lung increased significantly in wild-type mice consistent with alternative activation, but did not increase in silica-exposed IL-4R null mice, indicating that a Th-2 milieu was necessary for alternative activation and fibrosis [162]. In the herpes virus-induced and transgenic TGF-β models of fibrosis, AM accumulated in the lungs and expressed the alternative activation markers Ym1/2, Fizz1 and arginase-1 [163–165].

Likewise, in patients with pulmonary fibrosis, the preponderance of data suggests that AM display an alternatively activated phenotype. In patients with IPF compared to healthy controls, AM showed increased expression of CD206, and spontaneously generated higher levels of the proinflammatory cytokines CCL17, CCL18, and CCL22 [97, 161]. Co-culture of cell supernatants from alternatively activated AM of IPF patients with normal lung fibroblasts generated higher amounts of collagen production than supernatants from AM of healthy controls [161]. In patients with IPF, expression of arginase-1 was increased compared with healthy controls, as demonstrated by immunostaining of AM, immunostaining of lung tissue in areas of interstitial fibrosis, and by higher levels of arginase-1 protein in lung tissue lysates [163].

The above constellation of findings indicate that alternative activation of macrophages may play a mechanistic role in pulmonary fibrosis. Blocking alternative macrophage activation may prove therapeutic, and development of such new approaches to therapies for pulmonary fibrosis is under way. For example, serum amyloid P inhibited bleomycin and TGF-β induced pulmonary fibrosis at least partially through attenuation of alternative macrophage accumulation [164, 165].

B lymphocytes

Though most literature regarding potential lymphocyte involvement in mechanisms of fibrosis has referenced T cells, a role for B cells has been suggested for many years. As mentioned previously, patients with IPF often have well-organized lymph-node-like structures in the lung composed of activated T and B cells [132]. Production of antibody is a major function of B cells, and production of autoantibodies has been demonstrated against alveolar epithelial cell antigens such as vimentin and cytokeratins in patients with IPF [166–171]. Most recently, autoantibodies against periplakin, a component of desmosomes within epithelium, were identified in patients with IPF, and were associated with worse physiologic parameters [172].

A mechanistic role for B cells in pulmonary fibrosis is also supported by animal and patient data regarding CD19, a crucial cell-surface signaling molecule which is expressed on B cells and regulates B cell function. In patients with systemic sclerosis (SSc), an autoimmune disease in which pulmonary fibrosis is a major cause of morbidity and mortality, systemic B cells overexpress CD19 compared to healthy controls, and polymorphisms in CD19 are associated with an increased susceptibility to SSc [173, 174]. In an animal model of SSc, mice deficient in CD19 showed attenuated pulmonary and dermal fibrosis in response to subcutaneous bleomycin compared with wild-type controls [175]. In the intratracheal bleomycin model, overexpression of CD19 correlated with increased histologic lung fibrosis, increased lung hydroxyproline content, and reduced survival compared to wild-type controls, and mice deficient in CD19 had reduced fibrosis and improved survival compared to controls [176]. The number of B cells in BAL correlated with CD19 expression, and CD19 expression was necessary for B cell accumulation [176]. Since it has been demonstrated that the presence of CD19 alters B cell phenotype, the above results suggest that CD19 may have a mechanistic role in fibrosis by skewing B cells towards profibrotic cytokine expression.

Conversely, the presence of B cells in fibrosis may be protective. In the silica-induced model, overexpression of the anti-inflammatory cytokine IL-9 was accompanied by an expansion of B cells within the lungs and with reduced lung fibrosis [177, 178]. The presence of B cells was necessary for the protective effect of IL-9, demonstrated by lack of reduction in fibrosis in B cell-deficient mice, and by a restoration of the reduction in fibrosis by B cell reconstitution [178]. The concept of conflicting roles for B cells in fibrosis is similar to that postulated for T cells, in which depending on cell phenotype and the nature of the pulmonary milieu, lymphocytes may promote or diminish the pulmonary fibrotic process.

Fibrocytes

Bone-marrow-derived cells that produce collagen and express markers of leukocytes (CD45) and stem cells (CD34) normally circulate at low frequencies, below 1% of circulating cells. These cells were termed fibrocytes in a seminal study [9]. In response to local and systemic injuries, including pulmonary insults, fibrocytes are released from bone marrow in higher numbers, and home to sites of injury, where they differentiate into myofibroblasts and contribute to ECM deposition [8, 9, 120, 121]. Additionally, circulating monocytes may home to sites of injury and differentiate into mesenchymal cells [10, 11]. Recruitment of bone-marrow-derived mesenchymal progenitors is driven by several cytokines that are produced by injured tissues, including CXCL12, CCL2, CCL3, and IL-10 [98, 101, 116, 122]. These observations indicate that anti-fibrotic therapies should be limited not only to the organ affected by the disease, but have systemic mechanisms of action as well.

CD40-CD40L

CD40 ligand (CD40L or CD154) and CD40, members of the TNF family of cytokines and receptors, respectively, are cell surface molecules first identified as co-stimulatory molecules involved in the process of immune cell activation, with activated CD4+ T cells expressing CD40L, and activated antigen presenting cells expressing CD40. It was subsequently demonstrated that CD40 may also be expressed on several non-hematopoietic cell types [179], including human lung fibroblasts [180], which raised the possibility that CD40/CD40L interactions may affect fibroblast function. Subsequent work showed that engagement of CD40 on fibroblasts by CD40L or by agonistic antibody caused cell proliferation [61, 181], mobilization of NF-κB [179, 182], production of IL-6 and IL-8 [179, 181, 182], and expression of ICAM-1 and VCAM-1 [181]. Combined stimulation with IL-4, a potent stimulant for fibroblast proliferation, and ligation of CD40 had synergistic effects on enhancing fibroblast proliferation [61]. In animals, disruption of the engagement of CD40 by antibody against CD40L protected against radiation-induced, oxygen-induced, and autoimmune models of pulmonary injury and fibrosis [183–185].

In patients with IPF, areas of lymphocyte aggregates are often present which contain large numbers of activated T cells expressing CD40L [132]. Additional sources of CD40L include platelets, which are present in areas of lung injury due to the increased procoagulant milieu, and dendritic cells, which are present in lymphocyte aggregates in patients with IPF [132, 186, 187]. Interestingly, CD40L expression was also found in primary human fibroblasts, and found to be expressed and elevated in fibroblasts from patients with IPF compared to normal controls [188].

The constellation of findings suggests that the CD40/CD40L system may be an important pathway by which several cell types rich in CD40L may promote and perpetuate fibrosis through engagement of CD40 on fibroblasts [182, 189]. The finding that lung fibroblasts also express CD40L suggests that fibroblasts themselves may be able to perpetuate fibrosis in an autocrine and paracrine fashion via the CD40/CD40L pathway [188].

Fas-FasL

Fas (Fas antigen, Fas receptor, or CD95) is a member of the TNF family of cell surface receptors, is expressed in numerous cell types, and induces cellular apoptosis once engaged by FasL. FasL (Fas ligand or CD178) is a transmembrane protein belonging to the TNF family, is predominantly expressed on activated T lymphocytes and natural killer cells, and induces apoptosis in Fas-bearing cells. The role of the Fas-FasL pathway in pulmonary fibrosis has been examined in both epithelial and mesenchymal cells.

Bronchiolar and alveolar epithelial cell apoptosis has been a consistent finding in the bleomycin model, along with up-regulation of Fas mRNA and Fas pathway genes in epithelial cells, and up-regulation of FasL in lung tissue and infiltrating lymphocytes [190, 191]. In mice, inhalation of agonistic anti-Fas antibody alone caused apoptosis of bronchiolar and alveolar epithelial cells, increased inflammation in BAL and lung tissue, increased collagen content, and increased lung tissue TGF-β mRNA similar to that observed with bleomycin [192, 193]. Inhalation or injection of soluble Fas (aimed at binding and neutralizing inherent FasL) along with bleomycin reduced epithelial cell apoptosis, tissue inflammatory cell infiltration, and collagen accumulation [194]. Mice deficient in Fas (lpr) or FasL (gld) had substantially reduced tissue inflammatory cells, epithelial cell apoptosis, and collagen accumulation compared to controls in response to bleomycin challenge [191, 194]. Conversely, selective inactivation of Fas in T lymphocytes (via Cre-mediated recombination) led to up-regulation of T cell FasL, massive infiltration of inflammatory cells in the lungs, and development of pulmonary fibrosis [195]. Treatment with neutralizing anti-FasL antibody completely prevented the accumulation of lymphocytes in the lung [195].

In mesenchymal cells, the preponderance of mechanistic data suggests inherent resistance to Fas-mediated apoptosis. In primary lung fibroblasts, ligation of cell surface Fas by agonistic antibody was unable to induce apoptosis, and resulted in increased levels of the anti-apoptotic proteins X-linked inhibitor of apoptosis (ILP) and FLICE-like inhibitor protein (FLIP_L) [27]. Similarly, fibroblasts from patients with pulmonary fibrosis demonstrated resistance to apoptosis when exposed to recombinant FasL, and demonstrated prominent signals for ILP and FLIP_L in lung tissue [27, 196]. Despite inherent resistance to Fas-mediated apoptosis, several studies have shown that TNF-α and IFN-γ, particularly in combination, increase Fas expression in fibroblasts and increase susceptibility to apoptosis [27, 69, 196, 197]. Attenuation of Fas expression in fibroblasts by small interfering RNA (siRNA) inhibited the ability of TNF-α and IFN-γ to increase susceptibility to apoptosis, whereas transduction of fibroblasts with Fas-expressing adenovirus-enhanced apoptosis when engaged with agonistic antibody [69]. The ability of TNF-α and IFN-γ to sensitize fibroblasts to apoptosis suggests that altered pro-inflammatory cytokine milieus may contribute to the development of pulmonary fibrosis.

It appears that enhanced apoptosis in epithelial cells coupled with resistance to apoptosis in mesenchymal cells affects the cross-talk between these two cell types. This altered cross-talk is often mediated by TGF-β. In the bleomycin model, myofibroblasts demonstrated overexpression of FasL and induced epithelial cell apoptosis in vitro [191]. In epithelial cells, TGF-β induced apoptosis in vitro, and low concentrations of TGF-β, which were unable to induce epithelial cell apoptosis alone, increased apoptosis in epithelial cells stimulated with agonistic anti-Fas antibody or soluble FasL [28]. In vivo administration of TGF-β along with agonistic anti-Fas antibody increased epithelial cell apoptosis to a degree greater than with either agent alone, and the induction of epithelial cell apoptosis by soluble FasL was inhibited by antibodies against TGF-β [28].

Observational studies in patients with IPF have demonstrated up-regulation of Fas and Fas-signaling molecules in epithelial cells compared to normal controls [69, 198, 199]. Mesenchymal cells within fibroblastic foci demonstrated minimal or absent expression of Fas, and fibroblasts from patients with pulmonary fibrosis had lower expression of surface bound Fas, but higher levels of soluble Fas in the supernatant [196]. In regards to FasL, there was increased expression of soluble FasL in BAL and serum in patients with active IPF and connective tissue disease-interstitial pneumonia [198, 200, 201]. In lung tissue from patients with IPF, increased FasL protein was demonstrated in infiltrating granulocytes and T cells, and strong expression of FasL was seen in myofibroblasts [191, 198]. In non-smoking patients with IPF, BAL showed increased percentages of alveolar macrophages and CD8+ cells expressing FasL along with increased levels of soluble FasL, and these findings were inversely correlated with vital capacity [155]. The summative observations in cell culture, animal models, and patients support a mechanistic role for disturbances in Fas and FasL in the development of pulmonary fibrosis.

Integrins

Integrins are heterodimeric, transmembrane cell surface molecules which primarily mediate cell-cell and cell-ECM adhesion, but also play major roles in cell migration, growth, and survival [202–205]. Currently, eighteen α-subunits and eight β-subunits have been identified which associate to form 24 known integrins. Mechanistic roles for integrins in the pathogenesis of pulmonary fibrosis have been described in epithelial, inflammatory, and mesenchymal cells.

The integrin αvβ6, which is expressed principally in epithelial cells, is one of the αv-integrins which has the ability to activate latent TGF-β by binding to the tripeptide RGD sequence on TGF-β latency-associated peptide (LAP). In a landmark study, αvβ6 activated latent TGF-β by binding to the RGD sequence, and in the bleomycin model, mice lacking β6 developed increased pulmonary inflammation, but were protected from pulmonary fibrosis [206]. In the animal model of radiation-induced fibrosis, β6 was up-regulated following injury, and lack of β6 or mutation of integrin binding site on TGF-β LAP significantly reduced pulmonary fibrosis [207]. In the radiation-induced or bleomycin model, antibody against αvβ6 reduced histologic evidence of fibrosis, hydroxyproline content, and phosphorylation of nuclear Smad 2/3 [207, 208]. In patients with IPF, αvβ6 was overexpressed within alveolar epithelial cells compared with normal controls, and in patients with systemic sclerosis, was overexpressed to a greater extent in patients with a UIP vs. NSIP pathologic pattern [208].

The integrin αvβ8 is also expressed in epithelial cells and fibroblasts. In fibroblasts, αvβ8 contributes to TGF-β activation, fibrosis, and regulation of immune processes including dendritic cell function [209]. In airway epithelial cells, the β8 subunit was highly expressed, active TGF-β was produced, and airway proliferation was minimal [210]. Antibody against β8 or TGF-β reduced active TGF-β production and resulted in enhanced airway proliferation, indicating that β8 activation of latent TGF-β was regulating epithelial cell proliferation. In an epithelial wounding model, administration of TGF-β delayed wound closure, and antibody against αvβ8 reduced activation of latent TGF-β and enhanced epithelial wound closure [211]. Both of these studies suggest that activation of latent TGF-β by αvβ8 may contribute to the broad mechanism of impaired epithelial cell regeneration coupled with mesenchymal cell proliferation in patients with pulmonary fibrosis.

The integrin α3β1 is an epithelial cell integrin and laminin receptor. Specific loss of α3 expression in lung epithelial cells of mice exposed to bleomycin resulted in typical findings of acute inflammation and lung injury, but had reduced accumulation of myofibroblasts and type I collagen [212]. Specific loss of α3 expression resulted in an inability to form β-catenin/Smad2 complexes, a process implicated in the development of epithelial-mesenchymal transition (EMT), suggesting that the α3β1 integrin may play a central role in EMT [212, 213].

Several integrins have been examined in inflammatory cells in pulmonary fibrosis. First, in our animal model of CCL18 overexpression, T lymphocytes accumulated in the lungs, expressed αvβ3 and αvβ5 integrins, and administration of neutralizing antibody against αv or genetic deficiency of β3 significantly reduced pulmonary T cell infiltration and collagen accumulation [214]. Transformed T cells that overexpressed αvβ3 and αvβ5 stimulated collagen accumulation in co-cultured fibroblasts, which was mediated by TGF-β, and pulmonary T cells from patients with systemic sclerosis expressed αvβ3 and αvβ5 integrins [214]. Second, many T cells express αEβ7, which is up-regulated by TGF-β, and binds to E-cadherin on epithelial cells [215]. In the bleomycin model, the majority of CD8+ and γδ T cells in BAL expressed αEβ7 [216], and in patients with IPF, a significantly higher percentage of CD4+ and CD8+ T cells in BAL expressed αEβ7 when compared to peripheral blood [217]. Third, lymphocytes and eosinophils may express α4 integrin, which binds to vascular cell adhesion molecule-1 (VCAM-1) on endothelium [218]. In the bleomycin model, treatment with neutralizing antibody against α4 resulted in reduced cellular inflammation, lipid peroxidation, hydroxyproline content, histologic fibrosis, and α-SMA compared with controls [219]. Though the precise role of T cells in the pathogenesis of pulmonary fibrosis remains incompletely understood, T cell expression of several integrins which bind epithelium or ECM skew the pulmonary milieu towards a pro- or anti-phenotype depending on the T cell phenotype.

Integrins expressed on fibroblasts have been shown to activate latent TGF-β and promote fibrosis. The α2β1 integrin is a major type I collagen receptor [220]. In normal fibroblasts, exposure to polymerized collagen inhibited proliferation, whereas fibroblasts from patients with IPF demonstrated abnormal proliferation due to activation of the PI3K-Akt-S6K1 pathway and low activity of the tumor suppressor phosphatase and tensin homologue (PTEN) [221]. Protein expression of PTEN was preserved, but there was defective regulation of PTEN function by the α2β1-polymerized collagen interaction. Consistent with these in-vitro experiments, mice haploinsufficient for PTEN showed an exaggerated fibroproliferative response and increased collagen deposition in a cutaneous and bleomycin injury model, and immunohistochemistry in patients with IPF showed activation of Akt in fibroblastic foci [221].

Myofibroblast contraction has been shown to activate TGF-β by inducing conformational changes in LAP mediated by integrin binding. Contraction of myofibroblasts along with mechanically-resistant ECM activated latent TGF-β via αvβ5 binding, and blocking antibodies against αvβ5 prevented this TGF-β activation [222]. Thy-1, a cell surface glycoprotein which inhibits a fibrogenic phenotype in fibroblasts and is associated with decreased fibrosis in the bleomycin model [223, 224], may have a role in cell contraction-mediated TGF-β activation. Several studies have shown that Thy-1 can bind to integrins, and specifically, Thy-1 bound αvβ5 in a cell-free system and on the surface of lung fibroblasts [225]. Upon exposure to cell contraction agonists, fibroblasts either lacking Thy-1 or in which Thy-1/αvβ5 binding was prevented were able to activate latent TGF-β and promote myofibroblast differentiation, whereas these effects were absent in fibroblasts expressing Thy-1 [225]. Thesis data suggested that Thy-1 is able to bind αvβ5 on the cell surface, and prevent myofibroblast contraction-induced integrin-dependent activation of latent TGF-β.

In patients with localized or diffuse scleroderma, fibroblasts demonstrated increased expression of αvβ5, and exposure to exogenous latent TGF-β increased collagen production [226, 227]. Overexpression of αvβ5 on normal fibroblasts recruited latent TGF-β on the cell surface, increased collagen promoter activity, and resulted in differentiation to a myofibroblastic phenotype, with each of these reduced or reversed with antibody against αvβ5 [226–228]. Thesis data in aggregate from patients with scleroderma showed that up-regulation of αvβ5 expression in fibroblasts increased ECM production and promoted myofibroblastic differentiation through enhanced autocrine TGF-β signaling.

Absence of cellular infiltration does not equate with lack of inflammation

As mentioned previously, recent pathologic descriptions of patients with IPF have emphasized the epithelial, mesenchymal, and ECM abnormalities, while de-emphasizing the possible contributions of inflammation to mechanisms of the disease. Most patients with IPF will manifest a mild to moderate degree of chronic cellular inflammation in the lung pathologically, but it is true that some patients with end-stage lung disease and advanced UIP on biopsy will have minimal evidence of chronic inflammatory cells in the lung parenchyma. Although infiltration with inflammatory cells is an overall common feature of inflammation, the degree of cellular infiltration may vary significantly depending on the particular tissue which is involved. In disease of tendons (tendinopathies), the presence of inflammatory cells is rather limited, due most likely to tissue architecture constraints, but an active inflammatory process is present based on levels of cytokines, growth factors, prostaglandins, and neuropeptides [229, 230]. In the spatially confined brain, often considered an “immune-privileged” organ, cellular infiltration following injuries or autoimmune processes is minor, but the brain is fully capable of an active inflammatory response, with cytokines playing key roles [231–233]. Molecular rather than cellular inflammation appears to drive the response to injury in tendons and brain, and the lack of inflammatory cells does not equate with lack of inflammation.

Resistance to corticosteroids does not equate with lack of inflammation

An additional concept that should not be equated with lack of inflammation is resistance to corticosteroids. There are several well-established diseases in which inflammation is clearly accepted to be the predominant underlying mechanism, but traditional anti-inflammatory therapy has been poorly or completely ineffective in groups or subgroups of patients [23]. Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the lungs, with high levels of pro-inflammatory cells and cytokines, but most studies have shown that inhaled or systemic corticosteroids do not significantly reduce cellular or molecular markers of inflammation, and do not improve long-term lung function or survival in patients [234–241]. Asthma is also a prototypical inflammatory lung disease of the airways, and although most patients with asthma respond well to corticosteroids, a subset of patients have steroid-resistant asthma, manifested clinically by a poor response to treatment, and mechanistically by continued expression of pro-inflammatory and profibrotic cytokines in alveolar macrophages and T cells despite exposure to corticosteroids [242–247]. In the autoimmune inflammatory diseases rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE), a subset of patients will respond poorly to corticosteroids, and mononuclear and T cells from these patients showed less inhibition of proliferation and less apoptosis when exposed to corticosteroids [248, 249]. Similarly, a proportion of patients with inflammatory bowel disease (IBD) will have a poor or absent response to corticosteroids, and peripheral blood T cells from these patients showed less inhibition of proliferation when exposed to corticosteroids [250, 251].

The normal mechanisms by which corticosteroids reduce inflammation have been reviewed in detail [235]. Several mechanisms have been postulated by which patients with inflammatory diseases may manifest resistance to corticosteroid therapy, and these have included abnormalities in bone morphogenetic protein receptor [252], increased phosphorylation and subsequent reduced nuclear translocation of the activated glucocorticoid receptor (GR) [253], reduced expression of the anti-inflammatory protein MKP-1 resulting from increased p38 mitogen-activated protein kinase activity [254], reduced binding affinity of GR for corticosteroids due to nitrosylation of GR by inducible nitric oxide synthase [255], or excessive activation of the transcription factor AP-1 which prevents GR interaction with DNA glucocorticoid response elements [256]. One other particularly noteworthy cause of corticosteroid-resistant inflammation in chronic lung disease results from decreased histone deacetylase activity. Corticosteroids normally reduce inflammation by decreasing transcription of activated inflammatory genes, which occurs at least in part by recruitment of histone deacetylase-2 (HDAC2) [235]. In epithelial cells, inhibition of histone deacetylase activity promoted corticosteroid resistance, and this resistance was reversed by HDAC2 overexpression [257]. Reduced levels of histone deacetylase-2 (HDAC2) have been observed in patients with COPD and refractory asthma [258, 259], and one mechanism of reduced deacetylase activity is inactivation of HDAC2 by oxidative stress, which has been demonstrated in epithelial cells, animal models, and patients with COPD [260–262].

Based on all the available mechanistic, animal model, and observational data regarding inflammation and immune mechanisms, it may be a reasonable conclusion that IPF is a chronic inflammatory disease of the lung, but cellular infiltration is minor compared to the substantial changes in pulmonary cytokines, and the absence of clinical improvement in treated patients may be a manifestation of resistance to conventional anti-inflammatory therapies. The fact that oxidative stress has been found capable of promoting steroid-resistant inflammation is certainly a thought provoking concept, given the large amount of data demonstrating high levels of oxidative stress in patients with pulmonary fibrosis, which will be discussed below.

Oxidative stress and oxidative signaling

Molecular oxygen is central to aerobic metabolism, is critically required for vertebrates, and must be constantly supplied to ensure survival. However, oxygen is a strong oxidant, and has damaging effects on cells and biologic macromolecules due to formation of reactive oxygen species (ROS). Oxidative stress occurs when there is an imbalance between the generation of ROS and the capacity to detoxify these intermediates, which occurs when generation of ROS is excessive, antioxidant defenses are reduced, or both disturbances occur together. Additionally, extensive evidence suggests that ROS may be also involved with post-translational processing of proteins and intracellular signaling mechanisms in health and disease, including activation or deactivation of signaling factors, regulation of gene expression, and cell differentiation. Over many years, there has been accumulating evidence that oxidative stress and/or oxidative signaling may play a major role in the pathogenesis of pulmonary fibrosis [263–266].

Procoagulant milieu in the lung

Essential in the process of wound healing in response to tissue injury are procoagulant signaling mechanisms. Activation of the coagulation cascade by tissue injury contributes to both cessation of bleeding as well as tissue repair. Over the past 15 years, there has been an accumulation of evidence in humans and animal models that imbalances in procoagulant or antifibrinolytic activity are present in the lungs of patients with pulmonary fibrosis. Most of the descriptions of these imbalances have centered on abnormalities of tissue factor, factor Xa, thrombin, or proteinase-activated receptors.

Epidemiologic clues

Epidemiologic observations in humans have suggested a relationship between disorders of the coagulation system and the development of pulmonary fibrosis. In a large cohort of patients in Denmark, patients who were homozygous for a mutation in factor V (factor V Leiden) had abnormalities of several respiratory indices when compared to non-carriers, which included an increase in severe dyspnea, lower forced expiratory volume in one second (FEV₁) and forced vital capacity (FVC), and increased rate of annual decline in the FEV₁ and FVC [307]. In another large cohort of patients from Denmark, the relationship between a history of deep venous thrombosis or pulmonary embolism was correlated with the presence of interstitial lung disease or idiopathic interstitial pneumonia [308]. The incidence rates of idiopathic interstitial pneumonia and interstitial lung disease were higher in patients with a history of venous thromboembolism or pulmonary embolism than control patients.

Tissue factor (TF)

Elevated levels of TF in BAL fluid have been found in patients with IPF compared with normal controls, and in patients with an acute exacerbation of IPF, BAL levels of TF were markedly elevated [309]. Immunohistochemical analyses revealed large amounts of TF in cuboidal epithelial cells in patients with IPF [309], and in type II pneumocytes of patients with IPF, systemic sclerosis, and cryptogenic organizing pneumonia [310]. Increased TF mRNA and protein were seen in fibroblasts from IPF patients compared with normal lung fibroblasts, and expression of TF assessed by qRT-PCR was up-regulated in fibroblastic foci of these IPF patients [311]. Procoagulant activity was increased in patients with IPF and other forms of diffuse parenchymal lung disease, and this increase in procoagulant activity was TF-dependent [312]. Interestingly, this TF-dependent increase in procoagulant activity correlated with a reduction in lung compliance, which is well-known to occur in patients with IPF.

Factor VII and Factor X

Increases in factor VII antigen in BAL fluid and mRNA in alveolar type II epithelial cells were detected in the lungs of patients with IPF compared with normal lung tissue [311]. Similarly, increased expression of factor X mRNA was seen in alveolar septa in the lungs of patients with IPF, whereas there was no significant expression of factor X in normal healthy lung tissue [152].

Anti-fibrinolytic activity

Contributing to the balance between procoagulant and anticoagulant mechanisms in the lung are the fibrinolytic plasminogen activators (urokinase-type and tissue-type) and the anti-fibrinolytic plasminogen activator inhibitors (plasminogen activator inhibitor 1 [PAI-1] and plasminogen activator inhibitor 2 [PAI-2]). BAL levels of PAI-1 and PAI-2 were elevated in patients with IPF vs control patients, accompanied by no difference in urokinase-type plasminogen activator levels, suggesting an imbalance shifted towards anti-fibrinolytic (or procoagulant) activity in the lungs of patients with IPF [309].

PAR-1 and PAR-2

The major thrombin receptor PAR-1 was highly expressed in macrophages and fibroblasts within fibroproliferative foci in patients with IPF, whereas there was only weak staining by immunohistochemistry for PAR-1 in resident alveolar macrophages from normal lung tissue [152, 153]. Similarly, in patients with systemic sclerosis, PAR-1 expression by immunohistochemistry was elevated in areas of inflammation and fibroproliferation, but showed only minimal expression in normal control patients [313]. Recently, PAR-1 expression was shown to be up-regulated on activated epithelium within the fibrotic areas of IPF patients, and additionally, was co-expressed in these epithelial cells with chemokine CCL2, also known as monocyte chemotactic protein-1 (MCP-1) [314].

In addition to PAR-1, increased expression of mRNA and protein of PAR-2, which is activated by tissue factor, factor VIIa, and factor Xa, but not by thrombin, was found in lung homogenates and fibroblasts of patients with IPF compared with controls [305, 311]. Increased expression of PAR-2 by immunohistochemistry was also seen in type II alveolar epithelial cells and in fibroblastic foci, and tissue factor (TF) co-localized with PAR-2 in these fibroblastic foci [311].