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.