IGSSPOT: an improved method for detection of individual cytokine-secreting cells.
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Animals↗
Biomedical subjects
R K Kumar
Publications and source records attributed to R K Kumar.
Mitogenic activity for fibroblasts induced by silica and titanium dioxide particles in vitro and in vivo.
Experimental studies on particle-induced pulmonary fibrosis have not provided consistent evidence for the specific induction of fibroblast-regulating cytokines by pulmonary macrophages in response to fibrogenic as compared to non-fibrogenic particles. Using an optimized, wholly serum-free bioassay, we assessed mitogenic activity for pulmonary fibroblasts in supernatants of short-term cultures of alveolar macrophages exposed to either fibrogenic silica or non-fibrogenic titanium dioxide ducts. The responses to these supernatants were influenced by the replicative status of the target cells, in that samples which stimulated non-cycling fibroblasts caused inhibition of DNA synthesis by cycling cells when tested at the same concentration. However, both silica and titanium dioxide elicited comparable secretion of growth factor activity by macrophages, following either in-vitro or in-vivo administration of particles. In contrast, bronchoalveolar lavage fluids from animals that received intratracheal injections of silica, but not from those that received titanium dioxide, exhibited a sustained reduction in fibroblast-stimulating activity. We conclude that secretion of growth factor activity by alveolar macrophages in culture is induced by particles in a non-specific manner. However, alterations in mitogenic activity in bronchoalveolar lavage fluid may constitute a biological marker of the pattern of pulmonary injury which progresses to fibrosis.
Animals↗
Role of lymphocytes in silicosis: regulation of secretion of macrophage-derived mitogenic activity for fibroblasts.
We investigated the role of pulmonary lymphocytes in regulating the secretion by alveolar macrophages (AM) of mitogenic activity for lung fibroblasts, in an experimental model of the initial stages of silicotic inflammation and fibrosis. Following intratracheal instillation of silica, pulmonary parenchymal lymphocytes produced a lymphokine(s) that caused modest stimulation of the secretion of mitogenic activity by normal AM. Co-culture of small numbers of lymphocytes from silica-injected animals with AM induced enhanced secretion of fibroblast growth factor activity which was comparable to the maximal response elicited by recombinant interferon-gamma. Lymphocytes from animals given non-fibrogenic titanium dioxide exhibited no such effects. The stimulatory effect of lymphocytes from silica-treated animals in co-culture with macrophages was abrogated when the cells were separated by a microporous membrane. Our findings demonstrate that lymphocytes participating in the response to pulmonary deposition of silica are able to induce the secretion of a growth factor(s) for fibroblasts by pulmonary macrophages, possibly via lymphokines expressed on the cell surface or secreted at sites of cell-to-cell contact.
Animals↗
Prostaglandins enhance DNA synthesis by mouse lung fibroblasts in serum-free culture.
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Alprostadil↗
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.
Animals↗
Lone atrial fibrillation with complete heart block in a child.
An asymptomatic 9-year-old boy presented with a slow heart rate and a structurally normal heart. He was detected to have complete atrioventricular block and fine atrial fibrillation that was not evident on the surface electrocardiogram. This case appears to be an unusual example of a disease of the cardiac conduction system presenting in childhood.
Atrial Fibrillation↗
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.
Animals↗
Mitogen-induced initiation of DNA synthesis by lymphocytes cultured in serum-free media.
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Animals↗
Activation of lymphocytes in the pulmonary inflammatory response to silica.
Lymphocytes were separated from the parenchymal lung tissue of mice administered either silica or titanium dioxide particles by intratracheal injection. There was an approximately 10-fold increase in the number of T-lymphocytes recovered from silica-treated animals at 2 weeks after injection. This was accompanied by an increase in the percentage of separated cells expressing the interleukin-2 receptor and by enhanced spontaneous DNA synthesis in serum-free culture. No such changes were demonstrable in cells obtained from animals administered titanium dioxide. These results confirm that an influx of T-lymphocytes occurs as part of the early pulmonary inflammatory response to silica particles. Furthermore, lymphocytes in the lung are functionally activated by at least two criteria. Activated T-lymphocytes may play a role in the induction of fibroblast proliferation and collagen synthesis that occurs in silicotic lung disease.
Animals↗
Secretion of a platelet-derived growth factor homologue by rat alveolar macrophages exposed to particulates in vitro.
Lung macrophages secrete a homologue of platelet-derived growth factor (PDGF) which induces the proliferation of fibroblasts in vitro. In previous studies, we showed that such a PDGF homologue is produced by rat alveolar macrophages and that rat lung fibroblasts have specific receptors for the macrophage-derived PDGF. In this study, we demonstrate the biological and physicochemical properties of the growth factor, as well as the time-related production of this factor following macrophage activation in vitro by organic and inorganic particles. Alveolar macrophages (AMs) collected by saline lavage from the lungs of rats were cultured in serum-free Dulbecco's modified Eagle's medium (SF-DMEM) for varying periods of time up to 72 h. The SF-DMEM "conditioned" by the AMs was used to treat early passage rat lung fibroblasts (RLFs), which were rendered quiescent by culturing in 2% platelet-poor plasma (PPP). Alveolar macrophage conditioned media (AMCM) in the presence of PPP caused increases in the number of fibroblasts, the percent of labeled fibroblast nuclei and tritiated [3H]thymidine incorporation. AMCM alone caused no detectable changes in fibroblast growth rate. These results indicate that AMs release a "competence-like" growth factor. The AMs were left untreated or were exposed to opsonized zymosan, carbonyl iron spheres or chrysotile asbestos fibers. Macrophages attached to a plastic substrate spontaneously produced the factor, and subsequent addition of the organic and inorganic particles to the macrophage cultures significantly increased the fibroblast-stimulating activity of the AMCM. The growth factor was stable after concentration (100-fold), lyophilization and reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Immunogold-silver staining by capillary action.
The authors have developed an improved method for immunogold-silver staining of paraffin sections. Using a manual capillary action staining system, they were able to simplify the technical aspects of the procedure, permitting rapid processing of large batches of slides with better reproducibility. Background staining was decreased by use of buffers containing a detergent. The use of a light-stable silver reagent permitted greater control of the enhancement stage. The method yielded a high degree of contrast with negligible nonspecific staining. Sensitivity was comparable to that obtained with conventional enzymatic immunostaining. However, the authors noted that trypsinization of sections was rendered unnecessary for those antigens for which such pretreatment was usually required, and the need for special fixatives could be eliminated. The method was also applicable to immunostaining of frozen sections. Immunogold-silver staining by capillary action deserves consideration as an alternative to existing immunohistochemical methods in diagnostic histopathology.
Capillary Action↗
Immunogold-silver cytochemistry using a capillary action staining system.
An improved method for immunogold-silver staining is described. Rat monoclonal antibodies to T-lymphocyte surface membrane antigens were employed to demonstrate T-cells and their subsets in frozen sections of mouse spleen and in smears of peripheral blood mononuclear cells. A capillary action staining system was used to perform all stages of the procedure, substantially reducing the technical effort involved, saving reagents, and improving reproducibility and control of batch staining. The use of a light-stable silver enhancement reagent simplified the development of the reaction product. Technical modifications were incorporated to obtain good preservation of tissue morphology when employing this reagent on frozen sections and to stabilize the reaction product. The improved immunostaining procedure was rapid, sensitive, and yielded better staining quality, with excellent contrast and minimal nonspecific precipitation of silver.
Animals↗
Quantitative immunohistologic assessment of lymphocyte populations in the pulmonary inflammatory response to intratracheal silica.
Immunogold-silver staining was used to identify T lymphocytes, T lymphocyte subsets, and B lymphocytes in lung tissue from mice injected intratracheally with silica, titanium dioxide, or saline alone. Morphometric quantitation revealed a marked influx of T lymphocytes in the silica-treated animals during the first 3 weeks after injection. The relative numerical density of these cells remained elevated when compared with saline-treated controls throughout the 12 weeks of the experiment. Cells expressing the CD4 and CD8 antigens were both increased in number, with the former accounting for approximately two-thirds of the T lymphocytes. An increased number of B lymphocytes was also apparent from 6 weeks after treatment with silica. The T lymphocyte response preceded the development of significant pulmonary fibrosis by several weeks. No lymphocyte response was observed in the lungs of mice injected with nonfibrogenic titanium dioxide. These observations are consistent with the hypothesis that lymphokines secreted by T lymphocytes play a role in the pathogenesis of silicotic inflammatory lesions and their progression to fibrosis.
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A homologue of platelet-derived growth factor produced by rat alveolar macrophages.
Rat alveolar macrophages secrete a growth factor that renders rat lung fibroblasts competent to initiate DNA synthesis in vitro in the presence of platelet-poor plasma. This biological activity resembles that of platelet-derived growth factor (PDGF). After separation from putative associated binding proteins by chromatography under acidic conditions, the macrophage-derived factor exhibited a relative molecular weight similar to that of highly purified human PDGF. The factor bound to a monospecific antibody to human PDGF and thus could be quantitated in an enzyme immunoassay for PDGF. It competed with radiolabeled human PDGF for receptor sites for PDGF on rat lung fibroblasts, and binding to these receptor sites could be specifically inhibited by anti-PDGF. These data strongly support the view that the factor derived from rat alveolar macrophages is homologous to human PDGF and is similar to human macrophage-derived PDGF-like growth factor. Furthermore, we have demonstrated that the lung contains both an effector cell (pulmonary macrophage) and a potential target cell (interstitial fibroblast) for this cytokine. Therefore the rat appears to be an appropriate animal model in which to study macrophage-derived PDGF-like growth factors as mediators of proliferation in pulmonary fibrogenesis.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Air Pollutants↗