Unscheduled apoptosis during acute inflammatory lung injury.

Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.

The clearance of heterologous antibodies in experimental antibasement membrane antibody mediated glomerulonephritis.

A study of the clearance of anti GBM antibodies is important for an understanding of the pathogenesis of Goodpasture's syndrome. This paper reports a study of the sequential clearance of heterologous anti-glomerular basement membrane antibodies in the neonatal rat. A single intraperitoneal injection was followed by rapid and linear binding of injected antibodies to glomerular basement membranes, particularly to those glomeruli in the corticomedullary region. GBM bound antibodies were cleared gradually through the mesangium and significant amounts of antibodies still remained bound after 6 months. Subsequent injections of antibodies failed to provoke morphological abnormalities. These experiments have shown that the glomerular basement membrane of neonatal and adult rats possesses similar antigenic sites. The mesangium plays a major role in the clearance of injected heterologous antibodies. The slow clearance of antibodies from the GBM indicates a strong affinity of antibodies to antigenic sites and that the removal of antibodies is intimately related to the slow metabolic turnover of the glomerular basement membrane. The findings help to explain some of the observations in human antibasement membrane antibody mediated disease of the Goodpasture's type.

Cardiovascular lesions in experimental acute and chronic renal failure in the rat.

Adult Wistar rats were subjected to partial or total nephrectomy or injected with mercuric chloride to induce a state of acute or chronic renal failure. They developed sequential pathological changes in the cardiovascular system characterized by fibrin deposition in the pericardium and fibrinoid changes in many systemic arteries and arterioles. Focal myocardial necrosis and interstitial fibrosis were a feature of long term chronic renal failure. Increased vascular permeability was evident in the pericardium of rats with chronic renal failure especially when autologous blood was also injected into the pericardial sac of previously nephrectomized rats.

Enhanced malignant phenotype of urethane-induced lung adenoma associated with sialoadenectomy in BALB/c mice.

Mouse lung adenoma were induced in adult female BALB/c mice by chronic i.p. urethane injection. Sialoadenectomy of the mice prior to carcinogen treatment did not alter the frequency or size of lung adenoma. However, sialoadenectomized mice exhibited a decrease in the proportion (18 versus 52%) of tumours which exhibited a low nuclear to cytoplasmic ratio and which grew along alveolar septa. Sialoadenectomy was also related to a complementary increase in the proportion (82 versus 48%) of tumours with large vesicular nuclei, prominent nucleoli and generally increased phenotypic features of malignancy. Replacement of epidermal growth factor (EGF) in sialoadenectomized mice restored the tumour-type ratio observed in non-sialoadenectomized mice. These data are discussed with respect to the possible roles of EGF in mouse lung alveologenic carcinoma formation and the cell type of origin for the tumours observed.

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.

Primary culture of adult mouse lung fibroblasts in serum-free medium: responses to growth factors.

We report a completely serum-free system for primary culture of fibroblasts from explants of adult mouse lung tissue which permits bioassays for cytokine activity to be performed using unselected populations of cells at low passage number, without interference by serum binding proteins or interacting growth factors. Cultures were established on collagen-coated surfaces in medium MCDB 201 containing albumin, transferrin, epidermal growth factor, lipids, prostaglandin E1, vitamin E, and reducing agents. The cells were morphologically and ultrastructurally typical of fibroblasts in culture and demonstrated expression of vimentin and induction of expression of desmin in culture. Proliferation of the cells was reproducible between different primary cultures and was growth factor dependent. Both cycling and growth-arrested cells exhibited increased DNA synthesis when stimulated with epidermal growth factor, platelet-derived growth factor, or basic fibroblast growth factor, which functioned as complete mitogens, but did not respond to insulin, tumor necrosis factor or interleukin-1 beta. Maximal induction of DNA synthesis by epidermal growth factor required the continued presence of the mitogen in the culture medium. These results cannot be satisfactorily explained by the competence-progression model of responses to mitogenic stimuli but support and extend the findings of other studies using diploid fibroblasts.

EGF-receptor and extracellular matrix changes in mouse pulmonary carcinogenesis.

Malignant Balb/c mouse lung cell clones related to alveologenic carcinoma exhibited low levels of epidermal growth factor (EGF) receptor activity compared to nonmalignant cell clones. Immunoprecipitation of cell homogenates and immunohistochemistry on urethane-induced lung tumors suggest that the absence of activity reflects decreased amounts of EGF receptor protein. Low levels of EGF receptor alone cannot cause neoplastic transformation, since a nonneoplastic cell cone, B5D3, exhibited low levels of EGF receptor despite its nontransformed phenotype. The reduced levels of EGF receptor in malignant clones have been mimicked by long-term (12 h) treatment of a nontransformed cell clone with 200 nM phorbol dibutyrate. The detection of mutated ras oncogene in the transformed cell lines, taken together with the EGF receptor findings, suggests that more than one alteration in the signal transduction pathway may be necessary for transformation in alveologenic adenoma and carcinoma cell systems. A further phenotypic feature of transformation, reduced expression of the extracellular matrix proteins fibronectin and laminin, may be mediated at the transcriptional level.

Enhanced invasiveness and metastatic potential of epithelial cell lines cultured in the presence of dimethyl sulphoxide.

Several passage cycles of poorly metastatic malignant epithelial cells through immunosuppressed mice failed to induce enhanced metastasis-forming ability of cells derived from either the primary subcutaneous tumours or the resultant lung metastases. In vitro treatment of cultured malignant cells with dimethyl sulphoxide (DMSO) induced a reversible change in phenotype towards increased invasiveness but did not significantly increase metastasis formation. A cloned-cell line from a spontaneous in vitro transformant in the presence of DMSO was highly invasive and highly metastatic. In vitro treatment of cultured cells with 2% (v/v) DMSO produced alterations in morphology with decreased growth rate of all cell lines and decreased anchorage-independent colony formation in several malignant cell lines. All in vitro and in vivo effects were reversible following both short- and long-term (1 year) culture of cells in the presence of DMSO, suggesting epigenetic effects. These data support the concept of independent genetic controls for the invasiveness of tumours and the ability to form metastases.

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.

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.

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.

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.

The effect of oxygen toxicity and cigarette smoking on the binding of heterologous antibodies to alveolar basement membrane in the rat.

It has been suspected that pulmonary lesions of the Goodpasture type may be the result of circulating antibodies to alveolar basement membrane and that environmental factors such as cigarette smoke may influence antibody binding. Carefully designed experiments in this study have shown that exposure to cigarette smoke for 3 weeks or 100% oxygen for 65 h did not influence the binding of heterologous antibodies to alveolar basement membrane in Wistar rats, nor did these regimes increase any pathological changes associated with the antibody binding as detected by light microscopy. The results which are at variance with other reported findings are discussed with reference to reported animal models of antibody mediated pneumonitis and Goodpasture's syndrome in man.

Differential patterns of apoptosis in resolving and nonresolving bacterial pneumonia.

Infection with either Streptococcus sanguis or Streptococcus pneumoniae type 25 causes acute pneumonitis in rats. Pneumonia caused by S. sanguis resolves over the course of 8 d, whereas pneumonia caused by S. pneumoniae type 25 progresses to fibrosis. To examine the role of apoptosis in these models, we performed assays with the terminal deoxynucleotidyltransferase-uridine nucleotide end-labeling technique on tissue sections from rat lungs at various times, and quantified the results with image analysis. Apoptosis was a feature of both the acute and resolving stages of pneumonia. The pattern and extent of apoptosis were similar in both models during the acute stage, and the number of apoptotic nuclei increased in both models through 4 d after infection. Although there were differences in the cellular pattern of apoptosis after 2 d and 4 d of infection, the extent of apoptosis was the same in both models. After 8 d, major differences were observed. In the resolving model, apoptosis was limited primarily to an abscess in the base of the lung. In the nonresolving model, apoptosis was persistent. We also found that cyclin-dependent kinase-5 expression is upregulated during apoptosis induced by bacterial infection. These data indicate that the location and timing of apoptosis may determine whether pneumonia resolves or progresses to fibrosis.