Human Fibrotic Diseases Research Articles

Biological Effects of Transforming Growth Factor-β1 in Idiopathic Pulmonary Fibrosis May Be Regulated by the Activation of Latent Transforming Growth Factor-β1 and the Differential Expression of Transforming Growth Factor-β Receptors

Transforming growth factor (TGF)-β1 has been demonstrated to be important in the pathogenesis of animal models of pulmonary fibrosis and in human fibrotic lung diseases. 1 Border WA Noble NA Transforming growth factor-β in tissue fibrosis. N Engl J Med. 1994; 331: 1286-1292 Crossref PubMed Scopus (3014) Google Scholar TGF-β1 is usually released in a biologically inactive form because of its noncovalent association with the latency-associated peptide-1. 2 Khalil N TGF-β: from latent to active. Microb Infect. 1999; 1: 1255-1263 Crossref PubMed Scopus (278) Google Scholar Using immunohistochemistry, alveolar macrophages (AMs) and epithelial cells lining honeycomb cysts in lung sections with idiopathic pulmonary fibrosis (IPF) express latent TGF-β1. 3 Khalil N O'Connor R Flanders K et al. TGF-β1 but not TGF-β2 or TGF-β3 is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study. Am J Respir Cell Mol Biol. 1996; 14: 131-138 Crossref PubMed Scopus (282) Google Scholar To be biologically effective, the latency-associated peptide-1 must be removed from its association with TGF-β1. 2 Khalil N TGF-β: from latent to active. Microb Infect. 1999; 1: 1255-1263 Crossref PubMed Scopus (278) Google Scholar Therefore, it was next determined if AMs and epithelial cells lining honeycomb cysts release biologically active TGF-β1. Five patients with the clinical diagnosis of IPF and five patients with no inflammation or fibrosis were used as control subjects. BAL was performed on IPF patients at two sites that included areas with honeycomb cysts and lung that appeared apparently normal as detected by high-resolution computed axial tomography. AMs of IPF patients from both sites but not those from normal control subjects released active TGF-β1. Furthermore, in IPF patients, the BAL fluid from an area of honeycomb lung compared to an area with no apparent changes contained increased quantities of active TGF-β1. BAL fluid from normal control subjects contained only latent TGF-β. For a biological response to occur, the active TGF-β1 must interact with both of its receptors, TGF-β receptor (TβR)-I and TβR-II. 4 Wrana JL TGF-beta receptors and signalling mechanisms. Miner Electrolyte Metab. 1998; 24: 120-130 Crossref PubMed Scopus (82) Google Scholar The distribution of TβR-II by immunohistochemistry was ubiquitous in normal and IPF lungs. However, TβR-I expression was markedly reduced on all cells in areas of honeycomb cysts, especially hyperplastic type 2 alveolar epithelial cells (AECs). In contrast, there was expression of TβR-I on AECs that had a type 1 morphology and interstitial fibroblasts. TGF-β1 is a potent inhibitor of epithelial cell proliferation, 4 Wrana JL TGF-beta receptors and signalling mechanisms. Miner Electrolyte Metab. 1998; 24: 120-130 Crossref PubMed Scopus (82) Google Scholar and the loss of TβR-I on hyperplastic type 2 AECs may be important in rendering these cells resistant to the antiproliferative effects of the TGF-β1 present in honeycomb cysts. This property may be important for regeneration of injured epithelium by proliferation. The presence of TβR-I on AECs would be important for cessation of continued proliferation and induction of differentiation. Since fibroblasts express both TβRs, they are likely to respond to TGF-β1 by proliferation and connective tissue synthesis. In conclusion, these results provide evidence for cell type-specific regulation of TGF-β1 function in vivo and suggest that several mechanisms controlling the effects of TGF-β1 are likely to be involved in the pathogenesis of IPF.

Oxidative Protein Cross-linking Reactions Involvingl-Tyrosine in Transforming Growth Factor-β1-stimulated Fibroblasts

The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.

PDGF-D, a new protease-activated growth factor.

Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.

Inhibition of bleomycin-induced pulmonary fibrosis in mice by the matrix metalloproteinase inhibitor batimastat

Bleomycin-induced pulmonary fibrosis is known to be associated with the increased activity of two gelatinases, matrix metalloproteinase (MMP)-2 and MMP-9, in bronchoalveolar lavage (BAL). This study has investigated the effect of a synthetic inhibitor of MMP, batimastat, on the development of pulmonary fibrosis induced by bleomycin administration in mice. Animals were intranasally instilled with saline or bleomycin (0.5 mg in 100 microl per mouse). Batimastat (30 mg/kg) or vehicle alone was administered by intraperitoneal injection 24 h and 1 h before saline or bleomycin instillation, and then daily at the same dosage until the end of the study. Fifteen days after bleomycin administration, BAL was performed and the lung was removed. Treatment of mice with batimastat significantly reduced bleomycin-induced lung fibrosis, as shown in the lung by histopathological examination and by a decrease in hydroxyproline levels. Batimastat also prevented the increase in BAL macrophage and lymphocyte numbers, whereas it did not show any effect on the increased expression of active transforming growth factor-beta (TGF-beta) in BAL. Batimastat treatment was effective in reducing MMP-2 and MMP-9 activity as well as the tissue inhibitor of metalloproteinase-1 (TIMP-1) level in BAL. These results suggest that administration of the MMP inhibitor batimastat is useful in preventing experimental pulmonary fibrosis induced by bleomycin and raises the possibility of a therapeutic approach to human pulmonary fibrotic disease.

Connective tissue growth factor expression in pediatric myofibroblastic tumors.

Human connective tissue growth factor (CTGF) is a secreted cysteine-rich peptide and a member of the peptide family that includes serum-induced immediate gene products such as a v-src-induced peptide and a putative proto-oncogene, c-src. CTGF is secreted by endothelial cells, fibroblasts, smooth muscle cells, and myofibroblasts. Its expression is increased in various human and animal fibrotic diseases. We hypothesized that tumors with significant fibrous and vascular components would exhibit increased expression of CTGF. We examined the expression of CTGF mRNA by in situ hybridization in 12 pediatric tumors and tumor-like conditions, including angiofibroma, malignant fibrous histiocytoma, infantile myofibromatosis, and malignant hemangiopericytoma. All the tumors showed moderate to intense CTGF expression in tumor cells and/or endothelial cells of the associated vasculature. Angiofibromas expressed CTGF only in factor VIII-positive endothelial cells and vascular smooth muscle cells. In contrast, infantile myofibromatosis, malignant hemangiopericytomas, and fibrous histiocytomas expressed CTGF in both endothelial cells and in vimentin-positive tumor cells, particularly those around the blood vessels. CTGF mRNA was not detected in the inflammatory cells observed in many of the tumors. The presence of CTGF in the endothelial cells and tumor cells around blood vessels raises the possibility that CTGF is involved in the pathogenesis of these myofibroblastic tumors.

The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases.

Colligin or heat shock protein 47 (HSP47) is a stress protein that resides in the endoplasmic reticulum and is thought to participate in intracellular processing, folding, assembly and secretion of procollagens. Irrespective of the tissue site and organ, induction of colligin/HSP47 expression is always noted during the process of fibrosis, particularly in and around the fibrotic lesions in both humans and experimental models. Its expression is highly tissue- and cell-specific, and restricted to mostly phenotypically altered collagen-producing cells. These observations suggest that upregulation of this collagen-specific chaperone-colligin/HSP47 may play an important role in the subsequent fibrotic process, possibly by regulating increased synthesis/assembly of collagens.

Decorin Attenuates Gliotic Scar Formation in the Rat Cerebral Hemisphere

The transforming growth factor-βs (TGF-βs) are potent fibrogenic factors implicated in numerous CNS pathologies in which fibrosis and neural dysfunction are causally associated. In this study, we aimed to demonstrate significant inhibition of fibrogenesis, glial scarring, and inflammation in penetrating incisional wounds of the rat brain using the proteoglycan decorin, which effectively inhibits TGF-β activity. Adult rats were assigned to two treatment groups each receiving 14 daily intraventricular injections of 10 μl total volume of: (i) saline plus 0.3% autologous rat serum=30 μg protein); or (ii) saline plus 30 μg recombinant human decorin. On day 0 of the experiment, a stereotactically defined unilateral incisional lesion was placed through the cerebral cortex into the lateral ventricle and, after 14 days, brains were processed for immunohistochemical analysis of the lesion site. Specific antibodies were used to visualize the deposition within the wound of matrix molecules and the extent and nature of reactive astrocytosis and inflammation. Quantitative and qualitative image analysis of the fibrous scar was performed in sections from a defined anatomical plane through the wound to detect the antifibrotic effects of decorin treatment. Treatment of wounds with decorin led to a marked attenuation of all aspects of CNS scarring including matrix deposition, formation of an accessory glial limiting membrane, and inflammation. Our findings suggest that decorin is potentially applicable to a number of human CNS fibrotic diseases to arrest the deposition of excessive extracellular matrix components and maintain and/or restore functional integrity.

Inhibition of glial scarring in the injured rat brain by a recombinant human monoclonal antibody to transforming growth factor-beta2.

The transforming growth factor-betas (TGF-betas) are potent fibrogenic factors implicated in numerous central nervous system (CNS) pathologies in which fibrosis and neural dysfunction are causally associated. In this study, we aim to limit the fibrogenic process in a model of CNS scarring using a recombinant human monoclonal antibody, derived from phage display libraries and specific to the active form of the TGF-beta2 isoform. The implicit inference of the work was that, as such antibodies are potential pharmacological agents for the treatment of human CNS fibrotic diseases, validation of efficacy in a mammalian animal model is a first step towards this end. Treatment of cerebral wounds with the anti-TGF-beta2 antibody led to a marked attenuation of all aspects of CNS scarring, including matrix deposition, formation of an accessory glial-limiting membrane, inflammation and angiogenesis. For example, in the wound, levels of: (i) the connective tissue components fibronectin, laminin and chondroitin sulphate proteoglycan; and (ii) wound-responsive cells including astrocytes and macrophages/microglia, were markedly reduced. Our findings suggest that such synthetic anti-fibrotic TGF-beta antibodies are potentially applicable to a number of human CNS fibrotic diseases to arrest the deposition of excessive extracellular matrix components, and maintain and/or restore functional integrity.

Mast cells distribution in human liver disease and experimental rat liver fibrosis. Indications for mast cell participation in development of liver fibrosis

The development of liver fibrosis due to chronic liver diseases is thought to be mediated by inflammatory cells releasing fibrogenic mediators that activate fat-storing cells (Ito-cells). Recently, the involvement of mast cells in fibrogenesis has been suggested. We studied the distribution of these cells in normal human liver and human nonfibrotic and fibrotic liver disease as well as in normal rat liver and acutely and chronically injured rat liver (CCl4 model). Mast cells were identified by histochemical and immunohistochemical methods. The immunoreactivity of liver and comparatively of rat peritoneal mast cells to the serpins alpha1-antitrypsin, alpha1-antichymotrypsin and antithrombin III was also studied. In normal human and rat liver, mast cells were rarely found in portal tracts, and there was no change in cell numbers in nonfibrotic human or acutely injured rat livers. In contrast, cirrhotic human and rat livers contained numerous mast cells in the portal tracts and the fibrous septa. They exhibited strong immunoreactivity to the serpins, as did rat peritoneal mast cells. The results indicate that in the late stages of liver fibrogenesis, mast cells may be involved by displaying protease inhibitory activity in the fibrotic septa.

Endothelial cell apoptosis is a primary pathogenetic event underlying skin lesions in avian and human scleroderma.

The mechanism that may cause degenerative fibrotic skin lesions was studied in situ using skin biopsies from patients with systemic sclerosis (SSc), localized scleroderma, or keloids, and at the initial disease stage in the University of California at Davis (UCD) lines 200/206 chickens, which develop a hereditary systemic connective tissue disease resembling human SSc and permit study of disease stages not accessible in humans. Frozen skin sections were analyzed simultaneously for apoptosis by terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick end labeling and indirect immunofluorescence staining of cell markers with tetramethylrhodamine isothiocyanate conjugates. The results showed that endothelial cells are clearly the first cells to undergo apoptosis in the skin of UCD-200/206 chickens, a process that seems to be induced by anti-endothelial cell antibodies. In human fibrotic skin diseases, apoptotic endothelial cells could only be detected in early inflammatory disease stages of SSc and localized scleroderma.

Co-expression of TNF alpha and IL-1 beta in human acute pulmonary fibrotic diseases: an immunohistochemical analysis.

To clarify the involvement of TNF alpha and IL-1 beta in the initiation of fibrotic lung diseases, their localization was examined by immunohistochemistry. Fibrotic lung diseases observed were classified into acute and old pulmonary fibrotic changes. The acute fibrotic changes included adult respiratory distress syndrome, acute interstitial pneumonia and idiopathic pulmonary fibrosis with acute exacerbation. Acute pulmonary fibrotic changes histopathologically corresponded to a mixture of the exudative and proliferative phases of diffuse alveolar damage. Both TNF alpha and IL-1 beta were positive in the alveolar macrophages and proliferating type II pneumocytes in acute fibrotic changes. In contrast, positive cells for TNF alpha and IL-1 beta were sparse in the areas of old fibrotic change and in the normal tissue. These findings suggest that TNF alpha and IL-1 beta play an important role in the initiation of pulmonary fibrotic responses and in the architectural remodeling, irrespective of the etiology of fibrotic lung diseases.

Cytokines in human lung fibrosis.

Fibrosis is a pathological process characterized by the replacement of normal tissue by mesenchymal cells and the extracellular matrix produced by these cells. The sequence of events leading to fibrosis of an organ involves the subsequent processes of injury with inflammation and disruption of the normal tissue architecture, followed by tissue repair with accumulation of mesenchymal cells in the area of derangement. The same sequence of events occurs in wound healing with normal granulation tissue and scar formation, but, while normal scar formation is very localized and transient, in contrast, in fibrosis, the repair process is exaggerated and usually widespread and can be chronic. Inflammatory cells (mainly mononuclear phagocytes), platelets, endothelial cells, and type II pneumocytes play a direct and indirect role in tissue injury and repair. The evaluation of three human fibrotic lung diseases, two diffuse [idiopathic pulmonary fibrosis (IPF), and the adult respiratory distress syndrome (ARDS)], and one focal (tumor stroma in lung cancer), has shown that several cytokines participate to the local injury and inflammatory reaction [interleukin-1 (IL-1), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-alpha)], while other cytokines are involved in tissue repair and fibrosis [platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-beta), and basic-fibroblast growth factor (b-FGF)]. A better understanding of the cytokines and cytokine networks involved in lung fibrosis leads to the possibility of new therapeutic approaches.

Bronchiolarized Metaplasia and Interstitial Fibrosis in Rat Lungs Chronically Exposed to High Ambient Levels of Ozone

The cellular and tissue changes in the lungs of rats were studied using electron microscopy following 20 months exposure to a range of ozone levels from 0.12 to 1.0 ppm. Male and female Fischer 344 rats were exposed and morphometric methods were used to determine the volume, surface area, and cellular changes observed in bronchiole-alveolar duct regions following chronic ozone exposure. No major gender-related effects were observed in response to chronic inhalation of ozone nor were significant effects of ozone exposure found in either terminal bronchioles or the proximal alveolar regions in animals chronically exposed to 0.12 ppm ozone. The proximal alveolar regions of animals exposed for 20 months to 0.5 and 1.0 ppm ozone were significantly altered with exposure. The high-dose, long-term exposure to ozone resulted in a pronounced increase in volume of both the interstitium and epithelium in the proximal alveolar regions. The thickening of the epithelium was due to a change in tissue type from the normal squamous epithelium to a cuboidal epithelium similar, but not identical, to that found in terminal bronchioles. This bronchiolar epithelial metaplasia of proximal alveolar ducts, which was dose related, was composed of differentiated ciliated and Clara cells similar to those found in terminal bronchioles. In addition, unique cells which contained morphologic features of many different cell types were observed. These cells, which may represent stem cells or differentiated but transformed cells, were found associated with the bronchiolar metaplasia of alveolar ducts. In conjunction with the epithelial changes, cellular and matrix components in the interstitium were increased with chronic exposure to 0.5 and 1.0 ppm ozone. All matrix components were increased including collagen, elastin, and basement membrane, as well as other acellular spaces which did not contain identifiable structures. The total volume of interstitial fibroblasts was also increased in the high-dose exposure group. Alveolar macrophages were increased only in the 1.0 ppm exposed animals. The cell and tissue changes in the terminal bronchioles were less pronounced indicating a relative resistance of this tissue to ozone and mainly consisted of a change in cell type from ciliated to Clara cells in the 1.0 ppm exposed animals. The relative resistance of bronchiolar tissue to high concentration ozone exposure and the extensive bronchiolar epithelial metaplasia seen in the alveolar ducts suggest that bronchiolar metaplasia may be an adaptive mechanism following chronic ozone exposure. The bronchiolar epithelial metaplasia and interstitial fibrosis seen with high-dose chronic ozone exposure are similar to, and may be a model for, the early steps in human fibrotic diseases such as idiopathic pulmonary fibrosis.

Increased expression of mRNA for gelatinase A and TIMP-2 in human fibrotic liver disease

Increased expression of mRNA for gelatinase A and TIMP-2 in human fibrotic liver disease

The Tight Skin (Tsk) Mutation in the Mouse, a Model for Human Fibrotic Diseases, Is Tightly Linked to the β2-Microglobulin (B2m) Gene on Chromosome 2

The Tsk mutation in the mouse is characterized by the excessive accumulation of collagen in skin and various internal organs, including the heart and lungs. These connective tissue abnormalities are similar to those present in human systemic sclerosis or scleroderma. The Tsk mutation provides an opportunity to investigate, at the molecular level, the pathogenesis of tissue fibrosis. As a first step to cloning the Tsk gene, we report the localization of the Tsk mutation with respect to known molecular markers on mouse chromosome 2. N2 progeny carrying the Tsk mutation were obtained from an intersubspecific backcross of [(C57BL/6-pa +/+ Tsk × Mus castaneus)F1 × M. castaneus ] mice. Genomic DNA from each N2 mouse was subjected to Southern and PCR analyses to identify restriction fragment length polymorphisms and simple sequence length polymorphisms, respectively. Our results refine the location of Tsk to a 3-cM region, eliminate several genes from consideration as the Tsk mutation, identify molecular probes tightly linked with Tsk, and suggest candidate genes responsible for the Tsk phenotype.

Increased Production and Immunohistochemical Localization of Transforming Growth Factor-α in Idiopathic Pulmonary Fibrosis

Transforming growth factor-beta (TGF-beta) can regulate cell growth and differentiation as well as production of extracellular matrix proteins. Elevated production of TGF-beta has been associated with human and rodent chronic inflammatory and fibrotic diseases. Using immunohistochemical staining, we have examined lung sections of patients with advanced idiopathic pulmonary fibrosis (IPF), a disease characterized by chronic inflammation and fibrosis and demonstrated a marked and consistent increase in TGF-beta production in epithelial cells and macrophages when compared to patients with nonspecific inflammation and those with no inflammation or fibrosis. In patients with advanced IPF, intracellular staining with anti-LC (1-30) TGF-beta antibody was seen prominently in bronchiolar epithelial cells. In addition, epithelial cells of honeycomb cysts and hyperplastic type II pneumocytes stained intensely. Anti-CC (1-30) TGF-beta antibody, which reacts with extracellular TGF-beta, was localized in the lamina propria of bronchioles and in subepithelial regions of honeycomb cysts in areas of dense fibroconnective tissue deposition. The close association of subepithelial TGF-beta to the intracellular form in advanced IPF suggests that TGF-beta was produced and secreted primarily by epithelial cells. Because of the well-known effects of TGF-beta on extracellular matrix formation and on epithelial cell differentiation, the increased production of TGF-beta in advanced IPF may be pathogenic to the pulmonary fibrotic and regenerative responses seen in this disease.

A molecular marker for fibrotic collagen in lungs of infants with respiratory distress syndrome.

Lung samples from four infants who died of respiratory complications of prematurity (Infant Respiratory Distress Syndrome, IRDS) were analyzed for their content of various collagen crosslink amino acids by newly developed techniques of high-performance liquid chromatography. Comparable analyses were performed with tissue from stillborn infants with apparently normal lungs (control group) and from adults without apparent lung disease. We observed increased amounts of the difunctional crosslink dihydroxylysinonorleucine (DHLNL) in the IRDS lungs. Gestational age seemed to be the most important determinant of total lung content of the trifunctional crosslink hydroxypyridinium (OHP). Term infants had about one-third of the OHP content in their lung collagen as was found in the adult lungs. These observations suggest that there are important changes in the molecular structure of collagen in human fibrotic lung disease, changes that are paralleled in various animal models of experimental pulmonary fibrosis, and in various human diseases involving abnormalities of skin or bone collagen metabolism.

Collagen deposition in autoimmune diseases: the expanding role of the fibroblast in human fibrotic disease.

Fibrosis, usually considered an extension of the normal process of wound healing, follows to a variable degree host responses of an inflammatory nature, both immune and non-immune. The immediate mesenchymal effector cell of fibrosis, the fibroblast, has more or less been taken for granted in this process, due largely to its monotonous morphological uniformity. An emerging realization that the fibroblast can react in a variety of ways, both as an amplifying population of cells and in selective ways as the final arbiter of the extracellular matrix, is the subject of this report. This heterogeneity of fibroblast behaviour is characterized and, since many types of cells (endothelial, smooth muscle, epithelial) have now been shown to respond to matrix-derived signals, the potential for mesenchymal cell selection as a mechanism for understanding human fibrotic disease is probed. Signals which selectively alter fibroblast proliferation and fibroblast phenotypic expression are emphasized.

Immunohistochemical study of collagen types in human foetal lung and fibrotic lung disease.

Highly purified type-specific anti-collagen antibodies (prepared in animals to types I, II, III, and IV bovine collagen) were used in an indirect immunofluorescence method for the study of human lung collagen. The tissue localisation of each collagen type, and the apparent type I:III collagen ratio was assessed in normal foetal and adult lung and in fibrotic lung lesions. In the latter, the relationship of the findings to the natural history of the lesion was considered. This method was compared with routine connective tissue stains. The following observations were made. (1) Foetal lung in the canalicular phase of development proved a useful substrate for validating and standardizing the procedure. (2) Collagen fluorescence was more sensitive than connective tissue stains in detecting collagen in foetal tissues and sites of early fibrosis. (3) On the basis of collagen-type fluorescence, two distinctive patterns of fibrosis were recognised. Areas of mature collagen surrounding vessels and bronchi and in established scar tissue, for example in asbestotic pleural plaques, were virtually exclusively type I collagen. By contrast, areas of early active fibrosis like sarcoid nodules and organising pneumonia, which usually contained variable numbers of fibroblasts and chronic inflammatory cells, were characterised by an increased proportion of type III collagen and a greater intensity of both types I and III collagen fluorescence. The possible significance of this change in type III:I collagen ratio is discussed. Determination of the stage of fibrotic lesions by this method might have applications in the prediction of disease progression, and influence management of some conditions.

Pathogenic mechanisms in pulmonary fibrosis: collagen-induced migration inhibition factor production and cytotoxicity mediated by lymphocytes.

The universal features of the histopathology of fibrotic lung disease are derangement of parenchymal collagen and infiltration of the parenchyma with chronic inflammatory cells. To determine if this cellular reaction might be associated with autoimmunity to a consitituent of the alveolar interstitium, peripheral blood lymphocytes were exposed to human type I collagen in vitro and evaluated for the production of migration inhibition factor and cytotoxicity. Data from 18 patients with idiopathic pulmonary fibrosis, 8 patients with pulmonary fibrosis other than idiopathic pulmonary fibrosis, 12 patients with nonfibrotic lung disease, and 9 normals demonstrated that circulating lymphocytes from more than 94% of patients with fibrotic lung disease take part in processes where the recognition of collagen results in migration inhibition factor production and lysis of collagen-coated sheep red blood cells. These collagen-induced cell-mediated phenomena are obviated with human T-lymphocyte antiserum. Collagen-induced migration inhibition factor production and cytotoxicity were found in less than 20% of patients with nonfibrotic disease and were not found in normals. Qualitatively, there was no organ (lung, skin) or species (human, rabbit) collagen specificity in these assays, but human lung alpha 2 chains were recognized more often than alpha 1(I) chains. Circulating lymphocytes from patients with fibrotic disease are present in a normal T to B ratio. These lymphocytes did not incorporate [3H]thymidine when exposed to collagen but did when exposed to T-cell mitogens. These in vitro observations suggest that circulating T-lymphocytes and lung collagen may be intimately associated in the pathogenesis of human fibrotic lung disease.