Gregory C. Rhodes

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.

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.

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.

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 Goodpastures syndrome in man.

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

Summary 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. In order to test this hypothesis, we studied the reactivity of the type 2 pneumocyte in the rat to surgically induced limited pulmonary collapse, amounting to approximately 10% of the total pulmonary parenchyma.

Atypical differentiation of bronchiolar epithelial cells following experimental pneumonia

SummaryIn the bronchiolar and alveolar epithelial responses to experimentally induced organizing pneumonia in the rat evoked byStreptococcus 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.

The relationship between pneumocyte reactivity and the outcome of experimental streptococcal pneumonia

Two models of bacterial pneumonia in SPF Australian Albino Wistar rats have been developed and characterised in order that alveolar epithelial (pneumocyte) reactivity may be studied during the evolution of this disease. In the first model, in which pneumonia is induced by a strain of Streptococcus sanguis, resolution of the pneumonic lesion is associated with rapid repair of damaged alveolar epithelium mediated by hyperplasia of type 2 pneumocytes and transformation of a proportion of daughter cells into type 1 pneumocytes 1 . In the second model, in which pneumonia is induced by a strain of Streptococcus pneumoniae type 25, failure of resolution is associated with damage to transforming type 2 pneumocytes and subsequent inhibition of the normal alveolar epithelial repair process 2 . Using immunohistochemistry to identify type 2 pneumocytes and a comparative morphometric analysis, we have confirmed significant differences between the two models in the pattern of this cell’s response. These findings have suggested that the type 2 pneumocyte’s response to an injurious stimulus may be of fundamental importance in determining the ultimate outcome of the acute injury.

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 Goodpastures 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 Goodpastures type.