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Biomedical subjects

R Paine

Publications and source records attributed to R Paine.

At least 37 records · Page 2Linked to original sources
PubMed1995

Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase.

Products of inducible nitric oxide synthase (iNOS) are known to be involved in lung injury following intrapulmonary deposition of immunoglobulin G immune complexes (IgG-ICx). In the current studies rat alveolar macrophages stimulated in vitro with murine interferon gamma (IFN-gamma), tumor necrosis factor alpha, interleukin 1 alpha, (IL-1 alpha), lipopolysaccharide (LPS), or IgG-ICx immunostained for iNOS and produced nitrite/nitrate- (NO2-/NO3-) in a dose- and time-dependent manner requiring availability of L-arginine. Under the same conditions, IL-4 and IL-10 reduced NO2-/NO3- generation. Type II alveolar epithelial cells, which were obtained from normal rat lungs and stimulated in vitro with IgG-ICx, LPS, or IFN-gamma, also immunostained for iNOS and generated NO2-/NO3-. Special techniques of bronchoalveolar lavage (BAL) were used to retrieve alveolar macrophages and type II alveolar epithelial cells. Under these conditions, intrapulmonary deposition of LPS yielded BAL fluids containing increased amounts of NO2-/NO3- and macrophages that spontaneously released NO2-/NO3- and stained for iNOS. After intrapulmonary deposition of IgG both macrophages as well as type II cells (retrieved by BAL) spontaneously produced NO2-/NO3- and both cell types immunostained for iNOS (approximately 20% of all type II cells and 35% of all alveolar macrophages). Using dual fluorescence staining for cell identification, frozen sections of lung tissue after IgG immune complex deposition revealed iNOS in both alveolar macrophages and type II cells. Finally, in the immune complex model of alveolitis, the appearance of iNOS in macrophages as well as macrophage production in vitro of NO2-/NO3- was dependent on the in vivo availability of tumor necrosis factor alpha, IL-1, and IFN-gamma. These studies suggest a dual cell source for nitric oxide in inflamed lungs and the requirements for iNOS of several cytokines.

Amino Acid Oxidoreductases↗
PubMed1995

Regulation of rat pulmonary dendritic cell immunostimulatory activity by alveolar epithelial cell-derived granulocyte macrophage colony-stimulating factor.

The presentation and recognition of foreign antigen is the critical initial event in the development of local immunity. In the lung, antigen-presenting cell activity is largely attributable to pulmonary dendritic cells (DC) that are distributed along the airways and throughout the pulmonary interstitium in close proximity to overlying alveolar epithelial cells. To test the hypothesis that DC immunostimulatory activity might be locally regulated by overlying alveolar epithelial cells, we evaluated the ability of rat type II alveolar epithelial cells to influence the capacity of purified rat pulmonary DC to stimulate T-cell proliferation in an allogeneic, mixed leukocyte reaction. We found that alveolar epithelial cells greatly enhanced the ability of dendritic cells to induce T-cell proliferation. This effect on DC immunostimulatory activity was mediated by a soluble factor preferentially secreted from the basolateral epithelial cell surface. Alveolar epithelial cultures were found to express mRNA for granulocyte macrophage colony-stimulating factor (GM-CSF), and blocking antibodies against GM-CSF partially neutralized the effect of epithelial cell-conditioned media on DC stimulatory activity, indicating that the effect was due at least in part to alveolar epithelial cell-derived GM-CSF. Through the polar secretion of GM-CSF, alveolar epithelial cells may play an important role in creating distinct immunologic environments within the lung.

Animals↗
PubMed1995

Participation of pulmonary alveolar epithelial cells in lung inflammation.

As our understanding of pulmonary inflammation improves, the complexity of the mechanisms involved becomes more and more apparent. The number of soluble inflammatory mediators that are being recognized and characterized is increasing rapidly. The major purpose of this review is to emphasize that developing a comprehensive understanding of pulmonary inflammation requires investigations that encompass all of the cellular components. More than just a stage on which inflammatory processes are played out, the alveolar epithelium can participate in virtually all aspects of inflammation, including initiation, amplification, down-regulation, and tissue repair. By directing the powerful tools of cellular and molecular biology to study the entire cast of cellular players, a more complete understanding of inflammation will surely result. Such a comprehensive approach should assist in the development of means to prevent and treat inflammatory lung diseases.

Epithelium↗
PubMed1994

Localization of 5-lipoxygenase to the nucleus of unstimulated rat basophilic leukemia cells.

Arachidonate metabolism by 5-lipoxygenase (5-LO) coincides with the translocation of the enzyme from a soluble to a pelletable fraction in thoroughly disrupted granulocytic cells. While immunoelectron microscopy has identified the nuclear membrane as the site at which 5-LO, as well as 5-LO activating protein (FLAP), are localized in activated cells, the locale of soluble 5-LO in unstimulated cells could not be established by this technique. We asked whether the nucleus might also be the site for soluble 5-LO in unstimulated cells, and utilized rat basophilic leukemia (RBL) cells as model granulocytic cells to address this question. Using three different techniques to disrupt cells while leaving nuclei intact (mild nitrogen cavitation, Dounce homogenization, and detergent lysis), immunoblot analysis indicated abundant 5-LO in isolated nuclei. Within purified nuclei, 5-LO existed in two pools: a soluble pool that was readily released upon nuclear disruption and a bound pool that was not removed by 300 mM NaCl treatment. In all cases, 5-LO was also found in cytosolic and non-nuclear membrane fractions. Indirect immunofluorescent microscopy confirmed the presence of abundant 5-LO within the nucleus with minimal extranuclear signal in most cells. However, a minority of cells, characterized by condensed chromatin, showed no nuclear-associated staining with increased cytoplasmic staining for 5-LO. This suggested that some of the cytosolic 5-LO found by cell fractionation resulted from these dividing cells. When the contribution from dividing cells was minimized, either by overnight serum deprivation or by isolating cytoplasts of nucleus-containing cells, 5-LO was prominent in the nuclear fraction but negligible in the cytosolic fraction. In contrast to this distribution in RBL cells, 5-LO in unstimulated human neutrophils was predominantly cytosolic, by both immunoblot and immunofluorescence analyses. In both RBL cells and human neutrophils, FLAP was localized at the nuclear membrane and the endoplasmic reticulum. These data provide the first evidence for the localization of 5-LO in unstimulated granulocytic cells. The finding that a substantial proportion of enzyme is localized within the nucleus of unstimulated RBL cells suggests potentially novel roles for 5-LO or its products within the nucleus.

5-Lipoxygenase-Activating Proteins↗
PubMed1994

CD8 cells play a critical role in delayed type hypersensitivity to intact Cryptococcus neoformans.

Although cell-mediated immunity is critical for optimal host defense to C. neoformans, the role of T lymphocyte subsets is complex and poorly understood. CD8 cells are important both for optimal host defense against C. neoformans, and for expression of delayed type hypersensitivity (DTH). Because host defense correlates with the ability to mount a DTH response to C. neoformans, the current studies were performed to determine the mechanism by which CD8 cells participate in DTH. Mice were immunized by the intratracheal route with live C. neoformans, or by the subcutaneous route with heat-killed C. neoformans. Mice were depleted of CD8 cells in vivo by administration of mAb. After challenge with soluble cryptococcal Ag, the DTH response was quantified as footpad swelling. We found that mice depleted of CD8 cells before immunization were unable to express DTH. Mice depleted of CD8 cells after immunization but before challenge also were unable to express DTH. Splenocytes of mice depleted of CD8 cells in vivo, before immunization, failed to transfer DTH to naive, undepleted mice. Immune splenocytes depleted of CD8 cells in vitro also failed to transfer DTH to naive, undepleted mice. These data indicate that CD8 cells were necessary during the challenge and immunizing phases of DTH, and were necessary for expression of DTH. However, CD8 cell depletion did not abrogate DTH in mice immunized with either soluble cryptococcal Ag in complete Freund's adjuvant, or sheep red blood cells, which are mediated by CD4 cells. These data suggest that CD8 cells play a critical role in the cell-mediated immune response to C. neoformans. Based on this information, it may be possible to protect hosts with deficiencies of CD4 cells, such as in AIDS, by designing immunizing strategies for stimulating CD8 cells.

Animals↗
PubMed1994

Regulation of alveolar epithelial cell ICAM-1 expression by cell shape and cell-cell interactions.

In normal lung, intercellular adhesion molecule 1 (ICAM-1) is expressed at high levels on thin type I alveolar epithelial cells, but is minimally expressed on cuboidal type II cells. ICAM-1 is induced in primary culture on tissue culture-treated plastic as type II cells undergo transition toward a type I cell-like phenotype. We hypothesized that alveolar epithelial cell expression of ICAM-1 might be regulated in part by signals that influence the state of differentiation of these cells. We found that rat type II cells that were cultured as aggregates of cuboidal cells on a hydrated basement membrane gel (Matrigel) or on floating type I collagen gels, expressed markedly less ICAM-1 protein and mRNA compared with cells that had spread on plastic. In contrast, type II cells that had spread as monolayers on dishes coated with basement membrane proteins in planar configuration demonstrated ICAM-1 expression comparable to that of cells on plastic alone. Thus regulation of alveolar epithelial cell expression of this immunologically important adhesion molecule involves complex spatial interactions of the cells with the basement membrane and other epithelial cells.

Animals↗
PubMed1994

Regulation of the immunostimulatory activity of rat pulmonary interstitial dendritic cells by cell-cell interactions and cytokines.

Pulmonary dendritic cells (DC) are potent antigen-presenting cells that are thought to play a critical role in the initiation of immune responses within the lung. Because the lung is both a site of entry into the body for microbial pathogens and the organ of gas exchange, pulmonary immune responses must be meticulously regulated to achieve a balance between host defense and respiration. The initial interaction of DC with T cells in the lung is an excellent point at which to control local immune responses. Studies of the regulation of DC accessory cell function have been greatly hampered by difficulties in obtaining pure populations of pulmonary DC that have not been subjected to prolonged incubations during which the DC may undergo functional alteration. We now describe a method for isolating pulmonary DC from the rat that yields 1 x 10(5) cells/rat with > 90% purity. These cells are potent accessory cells, inducing T cell proliferation in a mixed leukocyte reaction (MLR) at a stimulator-to-responder ratio of 1:1,000. This method, which involves flow cytometric separation of nonphagocytic cells that stain brightly for class II MHC (OX6) from a population of low-density pulmonary interstitial cells, avoids extended incubations at 37 degrees C and thus allows study of a relatively pure population of cells that have functional capacities resembling those of naive cells from the normal lung. With these cells, we demonstrate that the functional capacity of pulmonary DC as stimulator cells in an MLR is significantly increased by exposure to the cytokines interleukin-1 or granulocyte/macrophage colony-stimulating factor (GM-CSF) and by culture with interstitial, but not alveolar, macrophages. Furthermore, DC are heterogeneous with respect to the cell surface expression of receptor for GM-CSF, and this expression is subject to modulation in cell culture. From these studies, we conclude that the immunostimulatory capacity of pulmonary DC is a function of local interactions with cytokines and other parenchymal cells. This suggests that DC function may be an important regulatory point for the local control of pulmonary immune responses.

Animals↗
PubMed1993

MCP-1 expression by rat type II alveolar epithelial cells in primary culture.

Recruitment and activation of mononuclear phagocytes are potentially critical regulatory events for control of pulmonary inflammation. Located at the boundary between the alveolar airspace and the interstitium, alveolar epithelial cells are ideally situated to regulate the recruitment and activation of mononuclear phagocytes through the production of cytokines in response to inflammatory stimulation from the alveolar space. To test this hypothesis, we investigated the production of monocyte chemotactic polypeptide-1 (MCP-1), a protein that is chemotactic for and that activates monocytes, by rat type II alveolar epithelial cells in primary culture. Immunocytochemical staining using anti-murine JE, an antibody recognizing rat MCP-1, demonstrated cell-associated MCP-1 Ag throughout the monolayer. The intensity of staining was increased in response to IL-1 beta. When type II epithelial cells formed a tight monolayer on a filter support, there was polar secretion of MCP-1 Ag into the apical compartment by both control and IL-1-stimulated cells as measured by specific MCP-1 ELISA. Northern blot analysis revealed that IL-1 and TNF-alpha stimulated MCP-1 mRNA expression in a dose-dependent manner, whereas dexamethasone blocked MCP-1 expression by cells stimulated with IL-1. In contrast to previous results using transformed epithelial cell lines, MCP-1 mRNA was induced in these primary cultures directly by stimulation with LPS. These data suggest that alveolar epithelial cells may have an important and previously unrecognized role in the initiation and maintenance of inflammatory processes in the lung by recruiting and activating circulating monocytes through the production of MCP-1.

Animals↗
PubMed1993

Legionella pneumophila replicates within rat alveolar epithelial cells.

Legionella pneumophila replicates in the distal pulmonary airspace, causing legionnaires' pneumonia. Legionella organisms replicate within alveolar macrophages and recruited blood monocytes; however, when these cells are activated, they become potent inhibitors of L. pneumophila proliferation. L. pneumophila may replicate in other cells and thereby avoid the host defenses of macrophages. Experiments demonstrated that L. pneumophila replicate within primary cultures of rat pulmonary alveolar epithelial cells. Double-label immunofluorescent and electron microscopy demonstrated L. pneumophila within epithelial cells. Replication of L. pneumophila required similar numbers of alveolar epithelial cells or alveolar macrophages, required viable epithelial cells, and took place intracellularly. While replication of L. pneumophila occurred in both serum-free and serum-containing media, it was enhanced in the presence of serum. Pulmonary alveolar epithelial cells may represent an alternative site for replication of Legionella species in the terminal airspace and thus clarify some previously unexplained aspects of the pathogenesis of legionnaires' disease.

Animals↗
PubMed1993

Differentiation-related expression of ICAM-1 by rat alveolar epithelial cells.

Local regulation of immune and inflammatory responses within the alveolar space is a critical aspect of normal pulmonary host defense. The type I and type II epithelial cells that line the alveolar space are in intimate contact with lymphocytes and macrophages within the alveolar space and are ideally situated to provide regulatory signals to these effector cells. The present studies were undertaken to investigate the expression by rat alveolar epithelial cells in vitro and in vivo of intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule that is involved in migration and activation of T cells and macrophages. An antibody specifically blocking rat ICAM-1 (mAb 1A29) inhibited the adherence of activated T lymphoblasts to monolayers of type II alveolar epithelial cells. The expression of ICAM-1 protein by alveolar epithelial cells in vitro was confirmed both by immunofluorescence microscopy and by Western blot analysis. However, in each instance, ICAM-1 was not detected in type II cells the day of isolation, but appeared at low levels after 1 day and in abundance throughout the monolayer after 2 days, with sustained expression thereafter. This suggested that ICAM-1 expression might be a type I cell feature, which was induced as isolated type II cells underwent transformation towards the type I cell-like phenotype in vitro. Using immunofluorescence microscopy on frozen sections of normal lung, ICAM-1 was found in a linear distribution along the alveolar space, consistent with expression on type I cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Hard metal pneumoconiosis and the association of tumor necrosis factor-alpha.

Hard metal pneumoconiosis is a recently recognized occupational lung disease associated with the exposure to cobalt fumes in the workplace. Chronic exposure in susceptible individuals results in interstitial lung disease histopathologically manifested as interstitial fibrosis with an associated mononuclear cell infiltrate and the presence of "cannibalistic" multinucleated giant cells in the alveolar airspaces. The majority of patients present with symptoms of chronic cough and dyspnea. Interestingly, in addition, patients uniformly report significant weight loss out of proportion to their degree of respiratory impairment. In this case report we demonstrate the association of tumor necrosis factor-alpha (TNF) and hard metal (cobalt) pneumoconiosis and suggest that TNF may have a potential role in the etiology of the constitutional symptoms and the pathogenesis of interstitial lung disease.

Adult↗
PubMed1992

A factor secreted by a human pulmonary alveolar epithelial-like cell line blocks T-cell proliferation between G1 and S phase.

Because the pulmonary alveolar space is both the site of gas exchange for respiration and a portal of entry for foreign antigen, immunologic interactions within that space must be meticulously controlled. Alveolar epithelial cells are ideally situated to play a role in immune regulation within the alveolar space. We have used A549 cells, a cell line that is derived from a human alveolar cell carcinoma and that has been used as a model for alveolar type II epithelial cells, to examine the potential role of alveolar epithelial cells in local pulmonary immune regulation. Medium conditioned by confluent monolayers of A549 cells suppressed proliferation by human peripheral blood mononuclear cells (PBMC) stimulated with lectin, anti-CD3 antibodies, calcium ionophore and phorbol ester, or in a mixed leukocyte reaction. PBMC that had been incubated in and then removed from A549-conditioned medium went on to proliferate normally. Because the suppressive effect was abrogated by heating or acidification and was not blocked by neutralizing antibody to transforming growth factor-beta 1, this effect could not be attributed to transforming growth factor-beta. The factor mediating this effect has an approximate molecular weight of 70,000 D by gel filtration chromatography. Nonalveolar, pulmonary carcinoma cell lines did not exert this immunosuppressive influence nor did the alveolar epithelial cells inhibit proliferation by the transformed, Jurkat, T-cell line. Cell cycle analysis demonstrated that PBMC exposed to A549 cell-conditioned medium failed to enter S phase after mitogen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Factors↗