Messages and handshakes: cellular interactions in pulmonary fibrosis.

A better understanding of early cellular events following pulmonary injury may permit the identification of those patterns of response which are destined to progress to fibrosis. Interactions between inflammatory, fibroblastic and epithelial cells appear to play crucial roles in fibrogenesis. Intercellular communication may be via "messages" delivered by soluble mediators or "handshakes" at sites of cell-to-cell contact. In this review, we question the validity of some prevailing concepts about the importance of growth factor secretion by alveolar macrophages; examine the possible role of activated T-lymphocytes in regulating macrophage production of mediators; and hypothesise that whereas fibroblast proliferation may primarily be stimulated by macrophage-derived cytokines, accumulation of collagen may be regulated by growth factors expressed by injured alveolar epithelial cells.

An ultrastructural study of hydrogen peroxide production by cultured fetal and neonatal rat lung cells exposed to hyperoxia.

Hydrogen peroxide production in organotypic cultures of fetal and newborn rat lung cells has been investigated using an ultrastructural histochemical method, in which the quantity and location of an electron-dense reaction product derived from the interaction of cerium chloride and hydrogen peroxide was detected. Hydrogen peroxide was present in fetal cell cultures exposed to hyperoxia (50% oxygen) compared to controls maintained in 10% oxygen. This increase could be limited by incubation of cultures with ascorbic acid and preincubation with dexamethasone. On the other hand, in newborn rat lung cell cultures, less hydrogen peroxide was identified in cultures including those exposed to hyperoxia (50% oxygen).

Atypical differentiation of bronchiolar epithelial cells following experimental pneumonia.

In the bronchiolar and alveolar epithelial responses to experimentally induced organizing pneumonia in the rat evoked by Streptococcus pneumoniae type 25, the appearance of lamellar body-containing bronchiolar cells is reported. Such cells, which are interspersed among proliferating type 2 pneumocytes in the form of intraalveolar and bronchiolar buds, also stain immunohistochemically with antisera to alveolyn, a surfactant-associated protein. We believe this phenomenon supports an hypothesis that in response to specific stimuli, proliferation of a common precursor cell of both the bronchiolar Clara cell and the type 2 pneumocyte occurs, with varying expression of a latent or precursor capacity for surfactant secretion.

Abnormal alveolar epithelial repair associated with failure of resolution in experimental streptococcal pneumonia.

We describe an experimental model in Wistar rats of non-resolving bronchopneumonia evoked by Streptococcus pneumoniae type 25. In contrast to a model of resolving streptococcal pneumonia that we have previously described, morphological studies reveal that in this model, there is significant early damage to type 1 pneumocytes which progresses to necrosis, leaving isolated areas of denuded alveolar basement membrane. Furthermore, there is accompanying degeneration and necrosis of a proportion of the type 2 pneumocytes, and alveolar epithelial repair by proliferation and differentiation of these cells appears to be retarded. Isolated, hypertrophic, and hyperplastic foci of type 2 pneumocytes persist as the acute inflammatory response subsides, and organization progresses with proliferation and emigration of fibroblasts into the lumina of alveoli and terminal bronchioles. The resultant lesion is morphologically indistinguishable from bronchiolitis obliterans organizing pneumonia. We hypothesize that the abnormal outcome in this model of pneumonia is a consequence of the failure of proliferating type 2 pneumocytes to transform into type 1 pneumocytes and thus maintain the integrity of the alveolar epithelial surface.

Cloned murine non-malignant, spontaneously transformed and chemical tumour-derived cell lines related to the type 2 pneumocyte.

NAL1A is a murine type 2 pneumocyte-related cell line cultured from normal BALB/c adult mouse lung. In vitro spontaneous transformation of 3 out of 7 clones of NAL1A has led to the isolation and establishment in continuous cell culture of sibling-related non-neoplastic (NAL1A) and spontaneously arising neoplastic (NAL1As) cell strains. NAL1As cells exhibited a similar phenotype to cloned NUL1 cells cultured from urethane-induced mouse lung adenomas. All NAL1As and NUL1 clones grew vigorously in 0.3% agar and formed invasive, poorly differentiated carcinomas following subcutaneous inoculation into immunesuppressed mice. Several subcutaneous nodules metastasised preferentially to the lung. All spontaneous and chemically-derived malignant clones were less differentiated than the non-malignant clones as assessed by staining with a type 2 pneumocyte-specific polyclonal antiserum. The clones described in this report form a useful model in the study of spontaneous and chemically-induced neoplastic transformation in mouse epithelial lung cells.

Effects of hyperoxia on surfactant morphology and cell viability in organotypic cultures of fetal rat lung.

Effects of hyperoxia in an organotypic model consisting of well-differentiated fetal rat type 2 pneumocytes have been studied by light and electron microscopy. In cultures exposed to 50% oxygen for 48 h, hyperoxia caused necrosis of cultured lung cells derived from 18- to 19-day gestation fetal rats, less damage in cells derived from 20- to 21-day gestation fetal rats, and no detectable damage of cells derived from newborn rats. After exposure to hyperoxia in organotypic cultures cocultured with fibroblast monolayers, ultrastructural abnormalities of surfactant (large lamellar bodies with disordered lamellae and abnormal shape) were detected in cells from 18- to 19-day gestation fetuses. These abnormalities were not noted when fibroblast monolayers were absent. Fibroblast conditioned medium from fibroblasts exposed to hyperoxia did not cause significant surfactant abnormalities at the ultrastructural level. These changes were less marked in cultures incubated with glutathione's constituent amino acids and with ascorbic acid during exposure to hyperoxia, and in cultures pretreated with dexamethasone (20 nM) before exposure to hyperoxia.

Alveolar epithelial responses in experimental streptococcal pneumonia.

We describe an experimental model of pneumonia in Wistar rats evoked by Streptococcus sanguis. The lesion developed rapidly as a confluent bronchopneumonia of the single-lobed left lung. Except at the extreme base, where an abscess formed, the pneumonic process thereafter resolved, and most of the lung appeared microscopically and ultrastructurally normal 8 days after infection. Sequential electron microscopic studies revealed that in the areas of lung which subsequently resolved, damage was restricted to type 1 pneumocytes. Within 24 h of infection, the unaffected type 2 pneumocytes were observed to proliferate, transform into elongated pneumocytes of intermediate morphology, and then undermine and strip off the damaged type 1 cells from the subjacent basement membrane. Thereafter, the intermediate type pneumocytes completed their transformation into definitive type 1 cells, thus completing the repair process. We hypothesize that this represents an accelerated form of the normal type 1 replacement mechanism, and that uncomplicated epithelial repair following acute alveolar damage is possible if type 2 pneumocytes escape significant damage, so that they retain their capacity to proliferate and differentiate into type 1 epithelium.

Type 2 pneumocyte responses to cyclophosphamide-induced pulmonary injury: functional and morphological correlation.

We investigated the correlation between the functional and morphological responses of type 2 pneumocytes to experimentally induced subacute lung damage. BALB/c mice were injected with a single dose of 300 mg/kg of cyclophosphamide intraperitoneally to induce alveolar epithelial injury. Groups of six cyclophosphamide-treated animals and three untreated controls were killed at 3 days and 1, 2, 3, 4, 6, 8 and 12 weeks after drug treatment. The net secretory response of type 2 pneumocytes to injury was assessed by an enzyme immunoassay for the surfactant-associated protein alveolyn in bronchoalveolar lavage fluid and the morphological responses of the alveolar epithelial cells were evaluated by light and electron microscopy. Early type I pneumocyte injury occurred without significant endothelial damage and was accompanied by intra-alveolar fibrinous exudation. This was followed by focal hypertrophy and apparent hyperplasia of type 2 pneumocytes, together with the progressive accumulation of large foamy intra-alveolar macrophages and focal pleural fibrosis. In a minority of animals these lesions progressed to intra-alveolar fibrosis with marked epithelial hyperplasia. The type 2 pneumocyte response was initially paralleled by an increase in the concentration of alveolyn in bronchoalveolar lavage fluid, which was significantly greater than control values at 1 and 2 weeks (P less than 0.005) as well as at 3 and 4 weeks (P less than 0.05) after injury induced by cyclophosphamide, but thereafter fell to control levels. This study demonstrates that cyclophosphamide induces morphological alterations of type 2 pneumocytes and altered secretory activity of these cells manifested as an increased net secretion of a surfactant-associated protein.

Pulmonary response to atmospheric pollutants. 3. The role of pulmonary collapse in the induction of aberrations of the type 2 pneumocyte.

Type 2 pneumocyte responses were studied in the rat after experimental right apical lobe collapse induced by surgical occlusion of the apical bronchus. After initial degranulation, type 2 pneumocytes underwent proliferation, and exhibited large numbers of well formed and densely staining intracytoplasmic surfactant lamellar bodies. The proliferative response, which was diffuse in the initial 3 to 25 days, became focal by 50 days and thereafter subsided. These results are discussed in relationship to the responses of the type 2 pneumocyte to toxic injury as previously reported.

Lymphokine-induced phenotypic changes in cells of a type 2 pneumocyte-related strain: characterization of activity in mitogen-stimulated spleen cell supernatants.

Supernatants from concanavalin A-stimulated spleen cells contain a factor which induces morphological alterations, inhibition of replication, and altered phospholipid biosynthesis in cells of a type 2 pneumocyte-related strain designated NAL 1A. This study has demonstrated that the active factor is generated by nonadherent spleen cells in the presence of supernatants from cultures of stimulated adherent cells. The active factor inhibits proliferation of NAL 1A cells in a dose-dependent fashion, exerts an irreversible effect following overnight exposure of these cells to stimulated supernatants, and appears to be continuously synthesized by stimulated spleen cells in culture. It is stable at pH 2 for 4 hours, does not bind to Blue Sepharose CL-6B, and exhibits molecular heterogeneity on gel filtration chromatography, with maximal activity recovered in fractions of molecular size 34,000 and 43,000. Comparable inhibition of growth was induced by exposure of NAL 1A cells to recombinant murine and human interleukin 2, as well as by recombinant murine interferon-gamma. These findings provide evidence for an interaction between type 2 pneumocytes and lymphokines such as interleukin-2 and interferon-gamma. The possible implications of a similar interaction occurring in vivo and its effects on type 2 pneumocyte morphology and function are discussed in the context of chronic interstitial inflammatory lung disease.

Characterization of an alveolyn-like protein associated with mouse type 2 pneumocytes using immunochemical methods.

Delipidated proteins from a fraction of mouse lung homogenate enriched for lamellar bodies of type 2 pneumocytes were characterized by a combination of gel filtration and enzyme immunoassay for the presence of a surfactant-associated antigen. Immunoreactivity was associated with a protein of Mr 250-270,000 as well as its multimers and proteolytic fragments. The characteristics of the antigenic protein closely resembled those of a surfactant-associated protein termed alveolyn which is reported to be secreted by type 2 pneumocytes of various other species. Surfactant-associated proteins of M 32-38,000 in bronchoalveolar lavage fluid may be derived from this molecule by proteolysis.

An enzyme immunoassay for quantitation of a type 2 pneumocyte-specific surfactant-associated antigen in bronchoalveolar lavage fluid.

A sensitive, specific and reproducible assay for quantitation of a surfactant-associated antigen in mouse bronchoalveolar lavage fluid is described. The procedure employed was a non-equilibrium competitive enzyme immunoassay in which detection of unreacted antibody was significantly enhanced by the use of a second antibody and peroxidase-antiperoxidase as the reporter label. The sensitivity of the assay was such that bronchoalveolar lavage fluid had to be diluted 15 to 30-fold prior to assay. No interference by serum proteins or soluble tissue proteins was observed. This assay may provide in vitro quantitative assessment of the functional status of type 2 pneumocytes.

Altered morphology, growth, and biosynthetic activity of a type 2 pneumocyte-related cell strain induced by lymphokine-enriched supernatants of mitogen-stimulated spleen cells.

Hypertrophy and hyperplasia of type 2 pneumocytes during the evolution of subacute and chronic pulmonary injury cannot always be satisfactorily explained in terms of reparative responses to type 1 pneumocyte injury. We hypothesized that immunocompetent cells, evoked as part of the interstitial inflammatory response, may be secreting factors which affect proliferation and surfactant biosynthesis by type 2 pneumocytes. To evaluate this hypothesis in vitro, we tested the effects of lymphokine-enriched supernatants, from serum-free cultures of concanavalin A-stimulated spleen cells, upon the type 2 pneumocyte-related cell strain NAL 1A. Addition of these supernatants in culture induced irreversible morphologic and ultrastructural alterations in the NAL 1A cells, inhibited cell replication, and evoked increased and apparently abnormal surfactant phospholipid biosynthesis. Supernatants from unstimulated spleen cells had no effect. There was no evidence of toxic injury to the cells in culture, and an immunologically specific cytoplasmic protein continued to be expressed. The active factor(s) appeared to be a protein or peptide of greater than 10,000 molecular weight. A specific soluble factor such as is present in the mitogen-stimulated lymphoid cell supernatants may be capable of mediating a similar interaction with type 2 pneumocytes in vivo.

Expression of a type 2 pneumocyte-specific antigen by a cell strain from normal adult mouse lung.

The mouse lung epithelial cell strain NAL 1A has been confirmed as type 2 pneumocyte-related by immunostaining with a type 2 pneumocyte-specific antiserum. Cytoplasmic immunoreactivity with the antiserum paralleled the appearance of phospholipid-containing osmiophilic cytoplasmic inclusions, which were much more abundant in confluent cell cultures at low passage number than in exponentially growing cultures. However, phospholipid analysis indicated that NAL 1A cells were impoverished in phosphatidylglycerol as compared to lung type 2 pneumocytes. Confluent cultures of the neoplastic cell lines NAL 1AM and NUL 1 did not reveal any reactivity with the specific antiserum.

Characterization of a neoplastic epithelial cell strain derived by dexamethasone treatment of cultured normal mouse type 2 pneumocytes.

The epithelial cell strain NAL1A cultured from normal adult mouse lung has been transformed by culturing in dexamethasone into an invasive neoplastic cell strain. The criteria for neoplastic transformation include the capacity for anchorage independent growth in soft agar as well as the formation of invasive neoplastic nodules after subcutaneous transplantation in thymectomized irradiated newborn mice. The cells of the invasive neoplastic nodules induced by dexamethasone culturing of NAL1A were indistinguishable histopathologically and by electron microscopy from invasive nodules evoked by the subcutaneous inoculation of CMT64, a cell line cultured from a metastasizing mouse lung tumour and cell strain NUL1 derived from mouse pulmonary adenomata induced by urethane. Cells of the nodules derived from all three cultured strains possessed desmosomes, surface microvilli and phospholipid lamellar bodies characteristic of the type 2 pneumocyte. It is concluded that cultured cell strains NAL1A, cultured in dexamethasone, NUL1 and CMT64 evoke invasive subcutaneous neoplasms derived from a common ancestor, presumably a type 2 pneumocyte related stem cell.

Pulmonary responses to bleomycin-induced injury: an immunomorphologic and electron microscopic study.

Intratracheal injection of a small dose of bleomycin in rats induced early alveolar epithelial cell injury and a pneumonitis which subsequently evolved to pulmonary fibrosis. Hydropic degeneration of type I pneumocytes was apparent at 3 days after treatment. Marked interstitial and intra-alveolar pneumonitis developed at 7 days after treatment and was accompanied by hypertrophy and hyperplasia of type II pneumocytes. The inflammatory cell population consisted predominantly of cells with the morphology of large lymphocytes together with a number of eosinophils. Examination by immunoperoxidase and histochemical staining of frozen sections revealed that the lymphoid cells stained positively with the monoclonal antibodies W3/13 and W3/25 but not with other markers. Thus these cells appeared to be helper T lymphocytes. The later development of interstitial fibrosis was accompanied by alveolar microcollapse which contributed to the thickening of alveolar septa observed by light microscopy. The possible role of immunologic and other mechanisms in the pathogenesis of bleomycin-induced pulmonary fibrosis is discussed.

Development and characterization of type 2 pneumocyte-related cell lines from normal adult mouse lung.

Cell lines which exhibit epithelial morphology with surface microvilli and inclusion bodies characteristic of type 2 pneumocytes have been derived from normal adult mouse lung by a simple procedure involving enzymatic dispersal and mechanical elimination of other cell types. One of these cell lines designated NAL 1A, examined in detail, shows features consistent with its being related to type 2 pneumocytes of mouse lung. These features include desmosomes, dense lamellar bodies as well as phospholipid profiles related to immature surface active material, the inhibition of cell growth rate by dexamethasone, and the close similarity of the cytoskeletal protein patterns of this cell line to those of a metastatic type 2 pneumocyte-related cell line of mouse lung. The cell line from normal lung demonstrated near diploid chromosome number at low passage number with some evidence of karyotype instability at high passage number.

Establishment of epithelial cell strains from normal adult mouse lung resembling a urethane-induced lung adenoma cell strain and a metastasizing mouse lung carcinoma cell line.

Epithelial cell strains, designated NAL, have been established from normal lungs of adult female Balb/c mice. The ultrastructural characteristics, effect of dexamethasone on cellular morphology and proliferation rate, and the cytoskeletal protein pattern of NAL suggests that these cell strains may be related to a urethane-induced mouse lung adenoma cell strain NUL and to a metastasizing mouse lung tumour cell line CMT. NAL exhibited no anchorage-independent growth under normal conditions, however extensive passaging in the presence of 5 X 10(-6)M dexamethasone resulted in a colony forming efficiency in soft agar of 8.4% at passage number 30.

Cell separation: a review.

This review summarizes currently available techniques for cell separation. Techniques that exploit differences in physical properties of cells are widely used but have a number of limitations. Those that are based on differences in surface properties may more readily permit reproducible separation of a functionally homogeneous population of cells. Unfortunately very few techniques achieve separation of cells on the basis of differences in their functional characteristics.