Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase.

Pulmonary fibrosis is a common and dose-limiting side-effect of ionizing radiation used to treat cancers of the thoracic region. Few effective therapies are available for this disease. Pulmonary fibrosis is characterized by an accumulation of myofibroblasts and excess deposition of extracellular matrix proteins. Although prior studies have reported that ionizing radiation induces fibroblast to myofibroblast differentiation and collagen production, the mechanism remains unclear. Transforming growth factor-ß (TGF-ß) is a key profibrotic cytokine that drives myofibroblast differentiation and extracellular matrix production. However, its activation and precise role in radiation-induced fibrosis are poorly understood. Recently, we reported that lactate activates latent TGF-ß through a pH-dependent mechanism. Here, we wanted to test the hypothesis that ionizing radiation leads to excessive lactate production via expression of the enzyme lactate dehydrogenase-A (LDHA) to promote myofibroblast differentiation. We found that LDHA expression is increased in human and animal lung tissue exposed to ionizing radiation. We demonstrate that ionizing radiation induces LDHA, lactate production, and extracellular acidification in primary human lung fibroblasts in a dose-dependent manner. We also demonstrate that genetic and pharmacologic inhibition of LDHA protects against radiation-induced myofibroblast differentiation. Furthermore, LDHA inhibition protects from radiation-induced activation of TGF-ß. We propose a profibrotic feed forward loop, in which radiation induces LDHA expression and lactate production, which can lead to further activation of TGF-ß to drive the fibrotic process. These studies support the concept of LDHA as an important therapeutic target in radiation-induced pulmonary fibrosis.

Electrophilic PPARγ ligands inhibit corneal fibroblast to myofibroblast differentiation in vitro: a potentially novel therapy for corneal scarring.

A critical component of corneal scarring is the TGFß-induced differentiation of corneal keratocytes into myofibroblasts. Inhibitors of this differentiation are potentially therapeutic for corneal scarring. In this study, we tested the relative effectiveness and mechanisms of action of two electrophilic peroxisome proliferator-activated receptor gamma (PPARγ) ligands: cyano-3,12-dioxolean-1,9-dien-28-oic acid-methyl ester (CDDO-Me) and 15-deoxy-Δ(-12,14)-prostaglandin J(2) (15d-PGJ(2)) for inhibiting TGFß-induced myofibroblast differentiation in vitro. TGFß was used to induce myofibroblast differentiation in cultured, primary human corneal fibroblasts. CDDO-Me and 15d-PGJ(2) were added to cultures to test their ability to inhibit this process. Myofibroblast differentiation was assessed by measuring the expression of myofibroblast-specific proteins (αSMA, collagen I, and fibronectin) and mRNA (αSMA and collagen III). The role of PPARγ in the inhibition of myofibroblast differentiation by these agents was tested in genetically and pharmacologically manipulated cells. Finally, we assayed the importance of electrophilicity in the actions of these agents on TGFß-induced αSMA expression via Western blotting and immunofluorescence. Both electrophilic PPARγ ligands (CDDO-Me and 15d-PGJ(2)) potently inhibited TGFß-induced myofibroblast differentiation, but PPARγ was only partially required for inhibition of myofibroblast differentiation by either agent. Electrophilic PPARγ ligands were able to inhibit myofibroblast differentiation more potently than non-electrophilic PPARγ ligands, suggesting an important role of electrophilicity in this process. CDDO-Me and 15d-PGJ(2) are strong inhibitors of TGFß-induced corneal fibroblast to myofibroblast differentiation in vitro, suggesting this class of agents as potential novel therapies for corneal scarring warranting further study in pre-clinical animal models.

Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung.

Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.

Epithelium-specific adenoviral transfer of a dominant-negative mutant TGF-beta type II receptor stimulates embryonic lung branching morphogenesis in culture and potentiates EGF and PDGF-AA.

Although exogenous transforming growth factor-beta (TGF-beta) is known to inhibit branching morphogenesis in mouse embryonic lungs in culture, whether the principal negative function of endogenous TGF-beta signaling resides in lung epithelium or mesenchyme remains unresolved. A recombinant adenovirus was constructed, containing a mutated human TGF-beta type II receptor with a truncated cytoplasmic kinase domain. We examined whether this dominant-negative receptor could abolish epithelium-specific endogenous TGF-beta signaling. We introduced the recombinant adenovirus into lung explants via intra-tracheal micro-injection. This resulted in over-expression of exogenous truncated TGF-beta type II receptor only in airway epithelium, not in mesenchyme, as assessed by mRNA level and protein localization. Blockade of endogenous TGF-beta receptor signaling in epithelial endoderm by the mutated dominant-negative TGF-beta type II receptor resulted in significant (65%) stimulation of epithelial branching morphogenesis, while exogenous TGF-beta no longer downregulated epithelial PCNA immunoreactivity and surfactant protein C (SP-C) expression. Additionally, the mitogenic responses to epidermal growth factor (EGF) and platelet-derived growth factor, PDGF-AA were potentiated by 33 and 31%, respectively. We conclude that epithelium-specific adenovirus-mediated over-expression of a dominant-negative TGF-beta type II receptor completely and specifically abolished the anti-proliferative effects of both endogenous and exogenous TGF-beta. Therefore, epithelium-specific TGF-beta signaling is sufficient to negatively regulate embryonic lung-branching morphogenesis in culture. We speculate that abrogation of TGF-beta signaling stimulates lung morphogenesis by potentiating the inductive and permissive effects of other endogenous peptide growth factors such as EGF and PDGF-AA.

Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation.

Using adenoviral-mediated gene transfer techniques, the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene is efficiently targeted to and highly expressed by the respiratory epithelium of rat lung. This lung tissue-directed expression of GM-CSF induces accumulation of both eosinophils and macrophages at early stages and an irreversible fibrotic reaction at later stages. These tissue responses to GM-CSF appear to be distinct from those induced by other proinflammatory cytokines, interleukin (IL)-5, IL-6, macrophage inflammatory protein-2 (MIP-2), or RANTES overexpressed in the lung. These findings clearly demonstrate that GM-CSF is more than a hematopoietic cytokine in the lung and may play a pivotal role in the multiple pathological processes underlying numerous respiratory illnesses, including asthma. In this overview, the differences in tissue responses induced by GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF contributes to the development of eosinophilia, macrophage granuloma, and fibrosis are discussed in conjunction with the recent findings from us and others.

Spatial-specific TGF-beta1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung.

The precise spatial-temporal role that expression and activation of transforming growth factor (TGF)-beta plays in mammalian organ morphogenesis remains incompletely understood. Using replication deficient adenoviral vectors containing engineered TGF-beta1 cDNAs, we studied the spatial effects of locally over-expressing either latent or mutated, constitutively active TGF-beta1 protein during embryonic mouse lung branching morphogenesis in culture. Transfer of exogenous genes into lung epithelium was achieved by intra-tracheal micro-injection of recombinant adenovirus, while submerging lungs in virus resulted in gene transfer into the pleura and subjacent mesenchymal cells, as revealed by cytochemical staining for beta-galactosidase. Only lungs transfected with active, but not latent TGF-beta1 gene, showed elevated levels of active TGF-beta. Epithelial over-expression of active, but not latent TGF-beta1, via intra-tracheal micro-injection inhibited lung branching morphogenesis by 36 %. In contrast, lungs submerged with either active or latent TGF-beta1 recombinant virus did not demonstrate an inhibitory effect upon branching. Pulmonary gene regulation was assayed by competitive polymerase chain reaction coupled with reverse transcription. Direct respiratory tract micro-injection of adenovirus over-expressing active TGF-beta1 resulted in a dose-dependent inhibition of epithelial surfactant protein (SP)-C and SP-B mRNA levels by up to 76 % and 70 %, respectively, while in contrast, fibronectin and matrix Gla protein (MGP) mRNA levels remained stable. However, lungs that had been submerged in adenovirus expressing active TGF-beta1 demonstrated a concentration-dependent induction of both fibronectin and MGP mRNA levels up to 4.3- and 4.7-fold respectively in the presence of 1 x 10(11) pfu/ml active TGF-beta1 virus. On the other hand, lungs treated with adenovirus expressing latent TGF-beta1 either by micro-injection or submerging failed to demonstrate any regulatory effect either upon epithelial or mesenchymal gene expression. We conclude that adenovector-mediated over-expression of activated TGF-beta1 in specific spatial compartments results respectively in either inhibition of branching morphogenesis and epithelium-specific gene expression, or in induction of matrix gene expression without affecting morphogenesis or epithelium-specific gene expression, depending on the route of administration. Also, the lack of effect of latent TGF-beta1 over-expression strongly suggests that TGF-beta activation per se provides an important locus of fine regulation of the spatial effects of TGF-beta signaling during embryonic lung branching morphogenesis.

Adenovirus-vector-mediated cytokine gene transfer to lung tissue.

In summary, we have been able to demonstrate that adenovirus vectors are valuable tools in examining the roles played by individual cytokines in lung responses and inflammation. These viral vectors have marked trophism for the epithelial cells of the lung and are highly efficient in transferring the genes into these lining cells. This results in significant expression of cytokines both within the lumen and the parenchyma of the lung. As a result of an individual cytokine being overexpressed, there are cytokine-specific changes seen-IL-6 resulting in lymphocytosis, MIP2 resulting in neutrophil accumulation, RANTES resulting in monocyte accumulation, TGF beta resulting in monocytosis but no fibrosis, and GM-CSF most surprisingly resulting in tissue eosinophilia, granuloma formation, and subsequently the onset of fibrosis. These vectors have helped pinpoint the role of a number of the cytokines in inducing chronic inflammatory changes to the lung and imply that a single cytokine may not be the only trigger resulting in chronic changes within the lung parenchyma.

Miliary tuberculosis in Edinburgh--a comparison between 1984-1992 and 1954-1967.

We have reviewed the records of 29 patients notified with miliary tuberculosis in Edinburgh from 1984 to 1992 and compared our findings with those for the 40 patients previously reported in Edinburgh from 1954 to 1967. The incidence of miliary tuberculosis has not changed. Respiratory symptoms of cough and dyspnoea were commoner in 1984-1992 (P < 0.001) perhaps reflecting the increase in mean age at presentation (73.5 vs. 59.4 years; P < 0.001). Mortality was 50% in 1984-1992, significantly higher (P < 0.05) than the 25% recorded in 1954-1967. Forty percent of cases in both time periods were of cryptic miliary disease. The diagnosis of cryptic disease tended to be made more often post-mortem and less often by a trial of anti-tuberculosis chemotherapy in 1984-1992. Our findings emphasize the current poor outcome associated with a diagnosis of miliary tuberculosis. It is important to consider this diagnosis in elderly patients with unexplained pyrexia and implement a trial of specific anti-tuberculosis chemotherapy to confirm it.

Targeting cyclooxygenase-2 in hematological malignancies: rationale and promise.

There is much interest in the potential use of Cox-2 selective inhibitors in combination with other cancer therapeutics. Malignancies of hematopoietic and non-hematopoietic origin often have increased expression of cyclooxygenase-2 (Cox-2), a key modulator of inflammation. For example, hematological malignancies such as chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma often highly express Cox-2, which correlates with poor patient prognosis. Expression of Cox-2 enhances survival and proliferation of malignant cells, while negatively influencing anti-tumor immunity. Hematological malignancies expressing elevated levels of Cox-2 potentially avoid immune responses by producing factors that enhance angiogenesis and metastasis. Cellular immune responses regulated by natural killer cells, cytotoxic T lymphocytes, and T regulatory cells are also influenced by Cox-2 expression. Therefore, Cox-2 selective inhibitors have promising therapeutic potential in patients suffering from certain hematological malignancies.

Severe airflow obstruction and eosinophilic lung disease after Stevens-Johnson syndrome.

Respiratory involvement is a frequent complication of Stevens-Johnson syndrome (SJS). However, there are very few convincing reports of persistent pulmonary sequelae, as demonstrated by spirometry, radiology and pathology. The current study presents a case of a 13-yr-old female with T-cell acute lymphocytic leukaemia who developed persistent, severe, obstructive lung disease following an episode of SJS. A lung biopsy demonstrated bronchiolar submucosal fibrosis consistent with constrictive bronchiolitis, as well as eosinophilic micro-abscesses, which, to the current authors' knowledge, has not been previously described. The present study illustrates specific histopathological features that highlight a possible association between Stevens-Johnson syndrome, constrictive bronchiolitis and eosinophilic micro-abscesses. The eosinophils may be associated with permanent mucosal damage, as seen in the present case, by releasing mediators that have a pro-fibrogenetic role. However, further investigation is warranted.

The aryl hydrocarbon receptor is a regulator of cigarette smoke induction of the cyclooxygenase and prostaglandin pathways in human lung fibroblasts.

Cigarette smoking can lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, predisposes to malignancy. We recently demonstrated that human lung fibroblasts are activated by cigarette smoke to express COX-2 and prostaglandin E(2) (PGE(2)). Little is known about the mechanism whereby smoke activates human lung fibroblasts to produce proinflammatory mediators. Herein, we report the central role of the aryl hydrocarbon receptor (AHR) in cigarette smoke extract (CSE)-induced COX-2, microsomal PGE(2) synthase (mPGES), and PGE(2) production in human lung fibroblasts. Western blot analysis revealed that primary strains of human lung fibroblasts express AHR and aryl hydrocarbon nuclear translocator protein, supporting the possibility that smoke activates lung fibroblasts through this pathway. Experiments were subsequently performed to determine whether the AHR was activated by CSE. Immunocytochemistry and EMSA analysis revealed that CSE induced nuclear translocation of the AHR in human lung fibroblasts. CSE decreased protein levels of the AHR, consistent with AHR ligand-induced proteosome-mediated degradation. CSE also induced mPGES-1 and COX-2 protein and increased PGE(2) production. Treatment of human fibroblasts with AHR antagonists in the presence of CSE inhibited AHR nuclear translocation as well as COX-2, mPGES-1, and PGE(2) production. These data indicate that the AHR pathway plays an important role in cigarette smoke-mediated COX-2 and PG production in human lung fibroblasts and may contribute to tobacco-associated inflammation and lung disease.

Role of CXCR2 in cigarette smoke-induced lung inflammation.

It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 h, twice per day for 3 days, induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and the neutrophil chemotactic CXC chemokines macrophage inflammatory protein (MIP)-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histological changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. beta-Glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, whereas MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils that either are not present to bind these chemokines or fail to provide a feedback signal to other cells producing these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.

Fibrosis of the lung and other tissues: new concepts in pathogenesis and treatment.

Tissue fibrosis can lead to significant organ dysfunction and resulting patient morbidity and mortality. Unfortunately, the therapeutic repertoire is currently limited, nonspecific, and largely ineffective. While the pathogenesis is incompletely understood, evidence is accumulating that immune and cytokine mediated mechanisms are critical. In this review, data will be provided to support the role of Type 2 cytokines in the pathogenesis of fibrosis. The importance of the role of the pro-fibrogenic cytokine TGF-beta and CD40-CD40 ligand mediated fibroblast activation will also be evaluated. Finally, novel therapeutic options based on inhibiting these pathways will be described.

Immune-inflammatory functions of fibroblasts.

Inflammation is a response that has evolved over millions of years to become an extremely complex process. This complexity reflects the host's need to deal effectively with a wide variety of potentially injurious agents, as well as the need to incorporate an adequate set of checks and balances. An inappropriately checked response, which occurs rarely, results in disease, either acute or chronic. However, in most instances, inflammation is a beneficial response, essential for survival. Inflammation comprises an extensive network of cellular interactions implemented by an overwhelming number of molecules. One category of signal includes soluble products, such as neuropeptide, lipid mediators, cytokines and growth factors, most of which can be produced by inflammatory/haemopoietic cells. However, resident structural cells can also produce many of these products and, on this basis only, fibroblasts, epithelial, endothelial and smooth muscle cells should be considered as active contributors to the regulation of the inflammatory response. Extracellular matrix (ECM) proteins comprise another category of signals. Whilst the most recognized activities of these proteins are those concerned with providing structural tissue integrity, it is clear that they also have powerful inductive effects. Indeed, ECM proteins can influence the shape, movement and state of activation of inflammatory cells in the tissue. Recent evidence indicates that these signals may also play substantial roles in homing of inflammatory cells to certain sites and in the handling of a number of cytokines and growth factors. In so far as fibroblasts are the main producers of ECM proteins, these new data establish an indirect but important role for fibroblasts in the regulation of the inflammatory response.

Differentiation in medulloblastomas and other primitive neuroectodermal tumours.

Fifty-four cases of primitive neuroectodermal tumour (PNET) including 47 medulloblastomas, were examined for evidence of neuronal and glial differentiation, using antibodies to neuron-specific enolase (NSE), neurofilament protein (NF), and glial fibrillary acidic protein (GFAP). In 30 of the cases, antibodies to vimentin, alphafetoprotein, cytokeratin, epithelial membrane antigen and lymphoid markers were also used. Most of the 47 medulloblastomas in the group were NSE positive but NF negative; about half were GFAP positive and three of them were positive for both neuronal markers and for GFAP. Vimentin was demonstrated in four cases and was not always co-expressed with GFAP. Medulloblastomas were negative for all the other markers. Supratentorial PNETs were sometimes positive with neuronal markers but were GFAP negative. The cell specificity of these markers and the interpretation of immunocytochemical findings are discussed in relation to differentiation potential in primitive neuroectodermal tumours.

A case of steroid responsive pulmonary hyalinising granuloma: complicated by deep venous thrombosis.

A case of pulmonary hyalinising granuloma (PHG) complicated by deep venous thrombosis (DVT) is presented. The DVT was associated with the presence of a lupus anticoagulant. In the past PHG has been linked to various auto-antibodies, but to the best of the authors' knowledge, this is the first case reporting PHG in association with a lupus anticoagulant and clinically significant venous thrombosis. Historically, PHG has been regarded as poorly corticosteroid responsive. However, the patient in this case study responded dramatically to prednisone. This case study suggests that in selected patients with pulmonary hyalinising granuloma experiencing disabling symptoms and worsening pulmonary function, a trial of corticosteroids may be warranted.