sFasL-The Key to a Riddle: Immune Responses in Aging Lung and Disease.

By dint of the aging population and further deepened with the Covid-19 pandemic, lung disease has turned out to be a major cause of worldwide morbidity and mortality. The condition is exacerbated when the immune system further attacks the healthy, rather than the diseased, tissue within the lung. Governed by unremittingly proliferating mesenchymal cells and increased collagen deposition, if inflammation persists, as frequently occurs in aging lungs, the tissue develops tumors and/or turns into scars (fibrosis), with limited regenerative capacity and organ failure. Fas ligand (FasL, a ligand of the Fas cell death receptor) is a key factor in the regulation of these processes. FasL is primarily found in two forms: full length (membrane, or mFasL) and cleaved (soluble, or sFasL). We and others found that T-cells expressing the mFasL retain autoimmune surveillance that controls mesenchymal, as well as tumor cell accumulation following an inflammatory response. However, mesenchymal cells from fibrotic lungs, tumor cells, or cells from immune-privileged sites, resist FasL+ T-cell-induced cell death. The mechanisms involved are a counterattack of immune cells by FasL, by releasing a soluble form of FasL that competes with the membrane version, and inhibits their cell death, promoting cell survival. This review focuses on understanding the previously unrecognized role of FasL, and in particular its soluble form, sFasL, in the serum of aged subjects, and its association with the evolution of lung disease, paving the way to new methods of diagnosis and treatment.

SIRT1 Deficiency, Specifically in Fibroblasts, Decreases Apoptosis Resistance and Is Associated with Resolution of Lung-Fibrosis.

In contrast to normal regenerating tissue, resistance to Fas- and FasL-positive T cell-induced apoptosis were detected in myofibroblasts from fibrotic-lungs of humans and mice following bleomycin (BLM) exposure. In this study we show, decreased FLIP expression in lung-tissues with resolution of BLM-induced fibrosis and in isolated-lung fibroblasts, with decreased resistance to apoptosis. Using a FLIP-expression vector or a shFLIP-RNA, we further confirmed the critical need for FLIP to regain/lose susceptibility of fibrotic-lung myofibroblast to Fas-induced apoptosis. Our study further show that FLIP is regulated by SIRT1 (Sirtuin 1) deacetylase. Chimeric mice, with SIRT1-deficiency in deacetylase domain (H355Y-Sirt1y/y), specifically in mesenchymal cells, were not only protected from BLM-induced lung fibrosis but, as assessed following Ku70 immunoprecipitation, had also decreased Ku70-deacetylation, decreasedKu70/FLIP complex, and decreased FLIP levels in their lung myofibroblasts. In addition, myofibroblasts isolated from lungs of BLM-treated miR34a-knockout mice, exposed to a miR34a mimic, which we found here to downregulate SIRT1 in the luciferase assay, had a decreased Ku70-deacetylation indicating decrease in SIRT1 activity. Thus, SIRT1 may mediate, miR34a-regulated, persistent FLIP levels by deacetylation of Ku70 in lung myofibroblasts, promoting resistance to cell-death and lung fibrosis.

CMH-Small Molecule Docks into SIRT1, Elicits Human IPF-Lung Fibroblast Cell Death, Inhibits Ku70-deacetylation, FLIP and Experimental Pulmonary Fibrosis.

Regenerative capacity in vital organs is limited by fibrosis propensity. Idiopathic pulmonary fibrosis (IPF), a progressive lung disease linked with aging, is a classic example. In this study, we show that in flow cytometry, immunoblots (IB) and in lung sections, FLIP levels can be regulated, in vivo and in vitro, through SIRT1 activity inhibition by CMH (4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide), a small molecule that, as we determined here by structural biology calculations, docked into its nonhistone substrate Ku70-binding site. Ku70 immunoprecipitations and immunoblots confirmed our theory that Ku70-deacetylation, Ku70/FLIP complex, myofibroblast resistance to apoptosis, cell survival, and lung fibrosis in bleomycin-treated mice, are reduced and regulated by CMH. Thus, small molecules associated with SIRT1-mediated regulation of Ku70 deacetylation, affecting FLIP stabilization in fibrotic-lung myofibroblasts, may be a useful strategy, enabling tissue regeneration.

Forefront: MiR-34a-Knockout Mice with Wild Type Hematopoietic Cells, Retain Persistent Fibrosis Following Lung Injury.

MicroRNAs (miRs) are known to limit gene expression at the post-transcriptional level and have important roles in the pathogenesis of various conditions, including acute lung injury (ALI) and fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). In this study, we found increased levels of miR-34 at times of fibrosis resolution following injury, in myofibroblasts from Bleomycin-treated mouse lungs, which correlates with susceptibility to cell death induced by immune cells. On the contrary, a substantial downregulation of miR-34 was detected at stages of evolution, when fibroblasts resist cell death. Concomitantly, we found an inverse correlation between miR-34 levels with that of the survival molecule FLICE-like inhibitory protein (FLIP) in lung myofibroblasts from humans with IPF and the experimental model. Forced upregulation of miR-34 with miR-34 mimic in human IPF fibrotic-lung myofibroblasts led to decreased cell survival through downregulation of FLIP. Using chimeric miR-34 knock-out (KO)-C57BL/6 mice with miR34KO myofibroblasts but wild-type (WT) hematopoietic cells, we found, in contrast to WT mice, increased and persistent FLIP levels with a more severe fibrosis and with no signs of resolution as detected in pathology and collagen accumulation. Moreover, a mimic of miR-34a decreased FLIP expression and susceptibility to cell death was regained in miR-34KO fibroblasts. Through this study, we show for the first time an inverse correlation between miR-34a and FLIP expression in myofibroblasts, which affects survival, and accumulation in lung fibrosis. Reprogramming fibrotic-lung myofibroblasts to regain susceptibility to cell-death by specifically increasing their miR34a and downregulating FLIP, may be a useful strategy, enabling tissue regeneration following lung injury.

Matrix Metalloproteinases Retain Soluble FasL-mediated Resistance to Cell Death in Fibrotic-Lung Myofibroblasts.

A prominent feature of obstructed tissue regeneration following injury in general, and fibrotic lung tissue in particular, is fibroblast proliferation and accumulation. The Fas/FasL apoptotic pathway has been shown to be involved in human idiopathic pulmonary fibrosis (IPF) and bleomycin-induced lung fibrosis in rodents. We previously showed that in normal injury repair, myofibroblasts' accumulation is followed by their decline by FasL+ T cell-induced cell death. In pathological lung fibrosis, myofibroblasts resist cell death and accumulate. Like other members of the tumor necrosis factor (TNF) family, membrane-bound FasL can be cleaved from the cell surface to generate a soluble form (sFasL). Metalloproteinases (MMPs) are known to convert the membrane-bound form of FasL to sFasL. MMP-7 knockout (KO) mice were shown to be protected from bleomycin (BLM)-induced lung fibrosis. In this study, we detected increased levels of sFasL in their blood serum, as in the lungs of patients with IPF, and IPF-lung myofibroblast culture medium. In this study, using an MMP-inhibitor, we showed that sFasL is decreased in cultures of IPF-lung myofibroblasts and BLM-treated lung myofibroblasts, and in the blood serum of MMP-7KO mice. Moreover, resistant fibrotic-lung myofibroblasts, from the lungs of humans with IPF and of BLM-treated mice, became susceptible to T-cell induced cell death in a co-culture following MMP-inhibition- vs. control-treatment or BLM-treated MMP-7KO vs. wild-type mice, respectively. sFasL may be an unrecognized mechanism for MMP-7-mediated decreased tissue regeneration following injury and the evolution of lung fibrosis.

Increased Regeneration Following Stress-Induced Lung Injury in Bleomycin-Treated Chimeric Mice with CD44 Knockout Mesenchymal Cells.

CD44, an adhesion-molecule promoting cell-migration, is shown here to increase in stress conditions following bleomycin-induced apoptosis in alveolar epithelial cells (AECs), a main target of lung injury. In vivo, it inhibits tissue regeneration and leads to fibrosis. We show that some AECs survive by the ataxia-telangiectasia mutated kinase/ATM pathway, and undergo a CD44-mediated epithelial-mesenchymal transdifferentiation (EMT) with migratory capacities in vitro, and in vivo. We assessed apoptosis vs. proliferation of AECs following bleomycin, ATM/P53 signaling pathway in AECs, and CD44 involvement in EMT, cell motility and tissue regeneration in vitro and in vivo. Expression of survival genes, CD44, and ATM/p53 pathway was elevated in AECs surviving bleomycin injury, as were the markers of EMT (downregulation of E-cadherin, upregulation of N-cadherin and vimentin, nuclear translocation of ß-catenin). Inhibition of CD44 decreased AECs transdifferentiation. Bleomycin-treated chimeric CD44KO-mice had decreased EMT markers, ATM, and mesenchymal cells (α-SMA+) accumulation in lung, increased surfactant-b, diminished lung mesenchymal cell motility, and increased lung tissue regenerative capacity following bleomycin injury, as indicated by lung collagen content and semiquantitave morphological index scoring. Thus, AECs surviving lung injury are plastic and undergo ATM-mediated, CD44-dependent transdifferentiation, preventing tissue regeneration and promoting fibrosis. Synthetic or natural compounds that downregulate CD44 may improve tissue regeneration following injury.

A Profibrotic Phenotype in Naïve and in Fibrotic Lung Myofibroblasts Is Governed by Modulations in Thy-1 Expression and Activation.

Lung fibrosis is characterized by abnormal accumulation of Thy-deficient fibroblasts in the interstitium of the alveolar space. We have previously shown in bleomycin-treated chimeric Thy1-deficient mice with wild-type lymphocytes that Thy1-deficient fibroblasts accumulate and promote fibrosis and an "inflammation-free" environment. Here, we aimed to identify the critical effects of Thy1, or the absence of Thy1, in lung myofibroblast profibrotic functions, particularly proliferation and collagen deposition. Using specific Thy1 siRNA in Thy1-positive cells, Thy1 knockout cells, Thy1 cDNA expression vector in Thy1-deficient cells, and Thy1 cross-linking, we evaluated cell proliferation (assessed by cell mass and BrdU uptake), differentiation (using immunofluorescence), and collagen deposition (using Sircol assay). We found that myofibroblast Thy1 cross-linking and genetic manipulation modulate cell proliferation and expression of Fgf (fibroblast growth factor) and Angtl (angiotensin) receptors (using qPCR) that are involved in myofibroblast proliferation, differentiation, and collagen deposition. In conclusion, lung myofibroblast downregulation of Thy1 expression is critical to increase proliferation, differentiation, and collagen deposition.

Bleomycin-Treated Chimeric Thy1-Deficient Mice with Thy1-Deficient Myofibroblasts and Thy-Positive Lymphocytes Resolve Inflammation without Affecting the Fibrotic Response.

Lung fibrosis is characterized by abnormal accumulation of fibroblasts in the interstitium of the alveolar space. Two populations of myofibroblasts, distinguished by Thy1 expression, are detected in human and murine lungs. Accumulation of Thy1-negative (Thy1(-)) myofibroblasts was shown in the lungs of humans with idiopathic pulmonary fibrosis (IPF) and of bleomycin-treated mice. We aimed to identify genetic changes in lung myofibroblasts following Thy1 crosslinking and assess the impact of specific lung myofibroblast Thy1-deficiency, in vivo, in bleomycin-injured mouse lungs. Thy1 increased in mouse lung lymphocytes following bleomycin injury but decreased in myofibroblasts when fibrosis was at the highest point (14 days), as assessed by immunohistochemistry. Using gene chip analysis, we detected that myofibroblast Thy1 crosslinking mediates downregulation of genes promoting cell proliferation, survival, and differentiation, and reduces production of extracellular matrix (ECM) components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions. Chimeric Thy1-deficient mice with Thy1(+) lymphocytes and Thy1(-) myofibroblasts showed fibrosis similar to wild-type mice and an increased number of CD4/CD25 regulatory T cells, with a concomitant decrease in inflammation. Lung myofibroblasts downregulate Thy1 expression to increase their proliferation but to diminish the in vivo inflammatory milieu. Inflammation is not essential for evolution of fibrosis as was previously stated.

Overexpression of Telomerase Protects Human and Murine Lung Epithelial Cells from Fas- and Bleomycin-Induced Apoptosis via FLIP Upregulation.

High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT). Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP). Telomerase in mouse (MLE) and human (A549) lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may be a novel mechanism to confer protection from apoptosis in bleomycin-exposed human lung epithelial cells.

Cutting edge: FasL(+) immune cells promote resolution of fibrosis.

Immune cells, particularly those expressing the ligand of the Fas-death receptor (FasL), e.g. cytotoxic T cells, induce apoptosis in 'undesirable' self- and non-self-cells, including lung fibroblasts, thus providing a means of immune surveillance. We aimed to validate this mechanism in resolution of lung fibrosis. In particular, we elucidated whether FasL(+) immune cells possess antifibrotic capabilities by induction of FasL-dependent myofibroblast apoptosis and whether antagonists of membrane (m) and soluble (s) FasL can inhibit these capabilities. Myofibroblast interaction with immune cells and its FasL-dependency, were investigated in vitro in coculture with T cells and in vivo, following transplantation into lungs of immune-deficient syngeneic Rag-/- as well as allogeneic SCID mice, and into lungs and air pouches of FasL-deficient (gld) mice, before and after reconstitution of the mice with wild-type (wt), FasL(+) immune cells. We found that myofibroblasts from lungs resolving fibrosis undergo FasL-dependent T cell-induced apoptosis in vitro and demonstrate susceptibility to in vivo immune surveillance in lungs of reconstituted, immune- and FasL-deficient, mice. However, immune-deficient Rag-/- and SCID mice, and gld-mice with FasL-deficiency, endure the accumulation of transplanted myofibroblasts in their lungs with subsequent development of fibrosis. Concomitantly, gld mice, in contrast to chimeric FasL-deficient mice with wt immune cells, accumulated transplanted myofibroblasts in the air pouch model. In humans we found that myofibroblasts from fibrotic lungs secrete sFasL and resist T cell-induced apoptosis, whereas normal lung myofibroblasts are susceptible to apoptosis but acquire resistance upon addition of anti-s/mFasL to the coculture. Immune surveillance, particularly functional FasL(+) immune cells, may represent an important extrinsic component in myofibroblast apoptosis and serve as a barrier to fibrosis. Factors interfering with Fas/FasL-immune cell-myofibroblast interaction such as sFasL secreted by fibrotic-lung myofibroblasts, may abrogate immune surveillance during fibrosis. Annulling these factors may pave a new direction to control human lung fibrosis.

Randomized controlled crossover trial of a new oscillatory device as add-on therapy for COPD.

A new oscillatory device administers predetermined pressure oscillation sequences into the chest cavity over inhaled/exhaled air streams at low positive pressure. We assessed device safety and effect on 6MW performance, pulmonary function, and health-related quality-of-life (HRQOL) in moderate-to-very severe COPD in a randomized, double-blind, controlled, crossover study. Outcomes with an oscillatory device (Pulsehaler(TM), Respinova Ltd, Herzliya, Israel) and a "muted" sham device (control) of identical appearance that delivered continuous positive air pressure were compared in two groups receiving opposite treatment sequences: 2-week oscillatory device/control, 2-week washout, 2-week control/oscillatory device, 2-week washout. The clinical trial was registered ( www.clinicaltrials.gov , NCT00821418) and approved by the Hadassah-Hebrew University Medical Center Institutional Review Board (08-608). All participants signed informed consent; 22 patients completed the study with no marked differences in COPD exacerbations or side effects. A total of 91% of patients treated with the oscillatory device had a clinically significant improvement (increase >40 m) in 6MW performance. The 6MW distance with the oscillatory device increased significantly after 1 week of treatment (51.6 ± 7.6 m, +13.5 ± 2.3%, p < 0.001), and more after 2 weeks (61.8 ± 9.0 m, 16.3 ± 2.7%, p < 0.001). This increase with the oscillatory device was significantly greater (p < 0.001) than the 15.4 ± 10.4 m increase (4.2 ± 2.6%, NS) with control. FVC and inspiratory capacity (IC) improved significantly (p = 0.03 for each) with the oscillatory device but not with control. HRQL improved markedly (≥1 point) for dyspnea and mastery with the oscillatory device (p = 0.02) but not control. Treatment with a new oscillatory device appears to be safe, and to improve 6MW performance, pulmonary function, and HRQL in COPD. Further evaluation is warranted.

Failure of chimerism formation and tolerance induction from Fas ligand mutant bone marrow donors after nonmyeloablative conditioning.

Formation of donor-recipient mixed chimerism after nonmyeloablative conditioning allows co-existence of donor and recipient hematopoietic stem cells, with solid organ allograft tolerance and less likeliness of graft versus host development. Using a post-transplant bronchiolitis obliterans murine model, we aimed to test the hypothesis that allograft preservation after mixed chimerism formation is dependent on the presence of a functional Fas ligand (FasL) on donor hematopoietic cells. To form mixed chimerism, two aliquots of 30 × 10(6) whole bone marrow cells (BMC) from either wild-type C57BL/6 in one group, or transgenic gld mice with mutant FasL (d = 0 and 2+) in the other were used, with both groups receiving intravenous busulfan (10mg/kg) on d-1 and intraperitoneal cyclophosphamide (200mg/kg) on d+1. Tracheal allografts obtained from C57BL/6 mice were implanted into recipient BALB/c mice subcutaneously on d = 0. Tracheal allografts were harvested at d+28 post-transplant and were evaluated by histopathology. Mixed chimerism formation was detected in wild type C57BL/6 whole BMC recipients, which was accompanied by tracheal allograft acceptance with near normal structure at d+28 post implantation. However, in recipients of FasL mutant whole BMC, neither mixed chimerism formation nor tracheal allograft acceptance was obtained. We thus conclude that bone marrow cells lacking functional FasL molecules could not be engrafted in allogeneic recipients to form stable mixed chimerism after nonmyeloablative conditioning, thus not allowing tracheal allograft acceptance.

Cellular FLICE-like inhibitory protein deviates myofibroblast fas-induced apoptosis toward proliferation during lung fibrosis.

A prominent feature of fibrotic tissue in general and of lungs in particular is fibroblast proliferation and accumulation. In patients overcoming fibrosis, apoptosis limits this excessive cell growth. We have previously shown resistance to Fas-induced apoptosis of primary lung fibroblasts from mice with bleomycin-induced lung fibrosis, their escape from immune surveillance, and continued accumulation in spite of overexpression of the Fas death receptor. Cellular FLICE-like inhibitory protein (c-FLIP) is a regulator of cell death receptor-induced apoptosis in many cell types. We aimed to determine c-FLIP levels in myofibroblasts from fibrotic lungs and to directly assess c-FLIP's role in apoptosis and proliferation of primary lung myofibroblasts. c-FLIP levels were determined by apoptosis gene array, flow cytometry, Western blot, and immunofluorescence before and after down-regulation with a specific small interfering RNA. Apoptosis was assessed by caspase cleavage in Western blot and by Annexin V affinity labeling after FACS and tissue immunofluorescence. Proliferation was assessed by BrdU uptake, also using FACS and immunofluorescence. We show that myofibroblasts from lungs of humans with idiopathic pulmonary fibrosis and from bleomycin-treated versus normal saline-treated mice up-regulate c-FLIP levels. Using the animal model, we show that fibrotic lung myofibroblasts divert Fas signaling from apoptosis to proliferation and that this requires signaling by TNF receptor-associated factor (TRAF) and NF-κB. c-FLIP down-regulation reverses the effect of Fas activation, causing increased apoptosis, decreased proliferation, and diminished recruitment of TRAF to the DISC complex. This indicates that c-FLIP is essential for myofibroblast accumulation and may serve as a potential target to manipulate tissue fibrosis.

Osteopontin induces airway remodeling and lung fibroblast activation in a murine model of asthma.

Airway remodeling is a central feature of asthma; however, the mechanisms underlying its development have not been fully elucidated. We have demonstrated that osteopontin, an inflammatory cytokine and an extracellular matrix glycoprotein with profibrotic properties, is up-regulated in a murine model of allergen-induced airway remodeling. In the present study, we determined whether osteopontin plays a functional role in airway remodeling. Osteopontin (OPN)-deficient (OPN(-/-)) and wild-type mice were sensitized and exposed to inhaled ovalbumin (OVA) or saline for 5 weeks. Collagen production, peribronchial smooth muscle area, mucus-producing cell number, and bronchoalveolar cell counts were assessed. The functional behavior and phenotype of lung fibroblasts from OVA-treated OPN(-/-) and from wild-type mice were studied using ex vivo cultures. OVA-treated OPN(-/-) mice exhibited reduced lung collagen content, smooth muscle area, mucus-producing cells, and inflammatory cell accumulation as compared with wild-type mice. Reduced matrix metalloproteinase-2 activity and expression of transforming growth factor-beta1 and vascular endothelial growth factor were observed in OVA-treated OPN(-/-) mice. Lung fibroblasts from OVA-treated OPN(-/-) mice showed reduced proliferation, migration, collagen deposition, and alpha-smooth muscle actin expression in comparison with OVA-treated wild-type lung fibroblasts. Thus, OPN is key for the development of allergen-induced airway remodeling in mice. In response to allergen, OPN induces the switching of lung fibroblasts to a pro-fibrogenic myofibroblast phenotype.

Thy1 up-regulates FasL expression in lung myofibroblasts via Src family kinases.

We have previously demonstrated that myofibroblasts from lungs with bleomycin-induced fibrosis overexpress FasL molecules. Two subpopulations of fibroblasts, distinguished by their expression of Thy1 molecules, have been shown in the lungs of both mice and humans. Thy1-mediated FasL induction has been reported in T cells through the use of anti-Thy1 (G7). We therefore sought to determine whether FasL expression in lung myofibroblasts is associated with and/or dependent on Thy1 expression, and to examine the underlying mechanism of regulation. We show that myofibroblast Thy1 expression is associated with increased FasL expression. Moreover, we directly show that Thy1 activation induces FasL up-regulation. Initially, Thy1 activation causes translocation of FasL to the membrane surface, and later induces de novo synthesis of FasL at the mRNA and protein levels. In contrast to Thy1 activation, TNF-alpha and IFN-gamma do not induce FasL myofibroblast up-regulation. Using Src family kinase (SFKs) inhibitor (PP2), we show the general involvement of SFKs in Thy1 signal transduction leading to FasL up-regulation; and, using specific siRNA, we show the particular involvement of Fyn, one protein in the SFK family. These results demonstrate that Thy1 in myofibroblasts is not just a marker, but is a functional protein that transmits signals into the cell, up-regulating its FasL expression.

Airway eosinophil accumulation and eotaxin-2/CCL24 expression following allergen challenge in BALB/c mice.

Eotaxin-1/CCL11 is important for early eosinophil recruitment to the airways of asthmatics. In order to clarify whether eotaxin-2/CCL24 accounts for prolonged airway eosinophilia, the authors determined the expression of CCL11 and CCL24 in lung tissue and bronchoalveolar lavage (BAL) as well as eosinophil infiltration over 14 days in BALB/c mice sensitised (intraperitonealy) and challenged (inhalations) with ovalbumin (OVA). Allergen exposure induced perivascular, peribronchial, and BAL eosinophilia for up to 7 days. CCL11 and CCL24 were highly expressed in lung tissue from 6 and up to 72 hours. Peak expression of CCL11 protein was 1557 +/- 109 pg/mL for OVA (mean +/- SEM) versus 404 +/- 73 pg/mL in controls (SAL) (P < .001) and 1690 +/- 54 versus 455 +/- 165 pg/mL for CCL24 (P < .01). In BAL, only eotaxin-2/CCL24 was significantly increased (1623 +/- 85 pg/mL for OVA versus 157 +/- 22 pg/mL for SAL, P < .01). Peak eosinophilia and CCL24 expression occurred later in BAL than in lung tissue. These data suggest that both CCL11 and CCL24 are important for recruitment of eosinophils to perivascular and peribronchial tissue seen up to 72 hours. This finding implies redundancy between these chemokines rather than differentially regulated expression over time. In contrast, only CCL24 seems important for recruitment of eosinophils into BAL. Specific inhibition of CCL11 alone is therefore unlikely to inhibit eosinophil recruitment to the airways.

DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis.

We have shown recently that cDNA vaccination, using a virtual lymph node, ameliorates experimental allergic encephalomyelitis. Successful cure from mammary tumor requires resolution of local tumor growth and metastases. We have examined whether targeting of CD44 cell surface adhesion molecule by cDNA vaccination plays a role in resolving mammary tumor development. We show here that CD44 cDNA vaccination decreases the tumor mass and metastatic potential in experimental mammary tumor of BALB/c mice. Vaccination of mice, inoculated with the mammary tumors, by cDNA of CD44 variant (CD44v) but not by cDNA of standard CD44, markedly reduced local tumor development and lung metastasis. Concomitantly, transfection of CD44 antisense into a highly metastatic mammary tumor cell line disrupted the CD44 expression of the cells and reduced their ability to establish local tumors as well as metastatic colonies in the lung. Moreover, when CD44v, but not standard CD44 sense cDNA, was transfected into the poorly metastatic cell line, tumor development was markedly enhanced. It is possible therefore that DNA vaccination with a specific CD44v construct could induce an immune resistance to mammary tumor progression.

Evasion of myofibroblasts from immune surveillance: a mechanism for tissue fibrosis.

Tissue fibrosis evolving from impaired tissue remodeling after injury is characterized by myofibroblast accumulation. We propose that during the development of fibrosis myofibroblasts acquire an immune-privileged cell phenotype, allowing their uninterrupted accumulation. Using the murine model of bleomycin-induced lung fibrosis in mice, we show that myofibroblasts that accumulate in lungs with fibrosis, but not in normal lungs, kill Fas(+) lymphocytes, resist Fas-induced apoptosis, and survive longer when grafted into allogeneic mice. In contrast, bleomycin-treated FasLigand (FasL)-deficient (gld) chimeric mice did not accumulate myofibroblasts or collagen in their lungs, and their FasL(-) myofibroblasts did not survive after alloengraftment. This finding indicates that myofibroblasts possess Fas/FasL-pathway-dependent characteristics that allow them to escape from immune surveillance and resulting organ fibrosis.

Positron emission tomography in interstitial lung disease.

BACKGROUND AND OBJECTIVES: A high rate of glycolysis may reflect the inflammatory activity of interstitial lung disease (ILD). This prospective study investigated whether PET can distinguish IPF, a primarily fibrotic process, from other entities of ILD that have a marked inflammatory component. METHODS: Twenty-one patients referred for surgical lung biopsy because of diffuse ILD underwent PET at the time of the lung biopsy. PET transmission scans were obtained after injecting (18)FDG intravenously. Regions of interest (ROI) were drawn on the lung images, and the standardized uptake value (SUV) was calculated for these ROI. RESULTS: Of the 21 patients studied, 14 had IPF, four had non-specific interstitial pneumonia, and the remaining three patients each had respiratory bronchiolitis-ILD, sarcoidosis and Langerhan's cell histiocytosis. There was no statistically significant difference in the SUV between IPF patients and non-IPF patients (P = 0.26), although patients with IPF tended to have higher SUV. Radiographic changes tended to be more prominent in the lung bases in patients with IPF compared with non-IPF patients; however, the median ratio of SUV measured in the upper fields to the whole lungs in IPF patients was not significantly different compared with the median ratio in non-IPF patients (P = 0.31). CONCLUSION: PET does not allow differentiation of IPF from a non-IPF diffuse interstitial pulmonary process.

Epithelial cell apoptosis by fas ligand-positive myofibroblasts in lung fibrosis.

The Fas/Fas ligand (FasL) apoptotic pathway has been shown to be involved in bleomycin-induced lung fibrosis. We examined the hypothesis that myofibroblasts from fibrotic lungs possess a cytotoxic phenotype that causes apoptosis of epithelial cells via the Fas/FasL pathway. We show in vivo epithelial cell apoptosis and associated upregulation of Fas and apoptotic Fas pathway genes in epithelial cells of lungs with bleomycin-induced fibrosis. In addition, we show that FasL surface molecules are overexpressed on alpha-SMA-positive cells in mice with bleomycin-induced fibrosis, and in humans with idiopathic pulmonary fibrosis. This enables the molecules to kill Fas-positive epithelial cells. In contrast, FasL-deficient myofibroblasts lose this myofibroblast cytotoxic phenotype, both in vivo and in vitro. In vivo, there was no bleomycin-induced epithelial cell apoptosis, as assessed by specific M30 staining in chimeric FasL-deficient mice that lacked FasL-positive myofibroblasts. In vitro, FasL-positive, but not FasL-negative myofibroblasts, induce mouse lung epithelial cell apoptosis. Thus myofibroblast cytotoxicity may underlie the absence of re-epithelialization, resulting in persistent lung fibrosis.