Differential responses of rat alveolar and peritoneal macrophages to man-made vitreous fibers in vitro.

Different approaches, including inhalation and intraperitoneal injection assays, have been used to assess the potential health effects of man-made vitreous fibers (MMVF). The purpose of this study was to compare the phagocytic activity and the formation of reactive oxygen species by rat alveolar macrophages (AM) and peritoneal macrophages (PM) upon exposure to MMVF10 glass wool and MMVF21 rock wool fibers. Macrophage (Mphi) phagocytosis of mineral fibers was assessed by optical videomicroscopy and computer-aided image analysis. Mphi were classified as cells not associated with fibers, cells with attached fibers, cells with incompletely phagocytized fibers (an appearance known as "frustrated phagocytosis"), and cells with completely phagocytized fibers. The production of superoxide anions by AM and PM upon incubation with MMVF10 and MMVF21 fibers was determined by the superoxide dismutase-inhibitable reduction of ferricytochrome C. PM were found to have a lower phagocytic activity than AM. A significantly higher percentage of AM than of PM underwent frustrated phagocytosis of MMVF10 and MMVF21 fibers. In line with these findings, AM generated higher levels of oxygen radicals than PM upon exposure to MMVF21 fibers. In contrast, MMVF10 fibers failed to induce the generation of reactive oxygen species by both AM and PM. Our in vitro results show that the phagocytic activity, in particular the frustrated phagocytosis of mineral fibers, was significantly lower in PM than in AM. The data support the idea that the durability and biopersistence of mineral fibers are higher in the peritoneal cavity than in the lung.

Phenotypic and functional differences between rat alveolar, pleural, and peritoneal macrophages.

Tissue macrophages (M phi) play a central and essential role in modulating the initiation and perpetuation of the inflammatory response. Phenotypical and functional differences among alveolar M phi (AM) and peritoneal M phi (PM) have been reported, but less is known about pleural M phi (PLM) and their ability and capacity to release biologically active substances. Therefore, the aim of this study was to determine the production of superoxide anion, nitric oxide (NO), and tumor necrosis factor alpha (TNF-alpha) by PLM in comparison to AM and PM in vitro. M phi from rats were isolated by lavage of the respective body compartment and characterized by evaluating the expression of the surface antigens MHC class II molecules, CD11b, and ED2-like antigen. Upon activation, AM produced significantly higher amounts of superoxide anion, NO, and TNF-alpha compared to PM and PLM. Taken together, the findings of this study demonstrate that rat PLM resemble PM more than AM in terms of production of key inflammatory mediators.

Comparison of the phagocytic response of rat and hamster alveolar macrophages to man-made vitreous fibers in vitro.

Rats and hamsters are well known for their disparate response to inhaled mineral fibers/particles. Alveolar macrophages (AM) play an important role in the pulmonary clearance and retention of mineral fibers/particles mainly through the process of phagocytosis. The aim of this study was to investigate whether there exist differences in the phagocytic response and release of reactive oxygen species (ROS) between rat and hamster AM upon exposure to man-made vitreous fibers (MMVF) in vitro. AM were obtained by bronchoalveolar lavage and macrophage-enriched cultures were exposed to MMVF10 and MMVF21 fibers for 20 h. The phagocytic response of macrophages was determined by computer-assisted video-microscopy and the superoxide anion production was evaluated by cytochrome c reduction. A significantly higher percentage of rat AM underwent frustrated phagocytosis of both types of MMVF compared to hamster AM. This was associated with a higher ROS release by rat AM compared to hamster AM. These data may help to explain the cellular mechanisms underlying the disparate pulmonary response of rat and hamster to inhaled particulate matter.

Recombinant human interleukin-10 attenuates TNFalpha production by porcine monocytes.

BACKGROUND: Human recombinant interleukin-10 (rhIL-10) has been found to inhibit endotoxin-induced production of several proinflammatory cytokines including tumor necrosis factor alpha (TNFalpha) from human monocytes. The exogenous therapeutic administration of rhIL-10 in acute and chronic hyperinflammatory conditions has been discussed. For none of the large animal species that have been used to study the role and effects of various mediators during septicemia, crossreactivity of rhIL-10 has been shown so far. Therefore, the aim of the present investigation was to evaluate the crossreactivity of rhIL-10 in a porcine model. METHODS: To determine the effects of rhIL-10 on endotoxin-challenged porcine monocytes, we incubated porcine peripheral blood monocytes from five donors with three different concentrations of rhIL-10 (500 ng/ml, 1000 ng/ml and 2000 ng/ml, respectively) either simultaneously with, or two hours prior to lipopolysaccharide (LPS) administration. RESULTS: As compared to incubation with LPS (1 microg/ml) alone, coincubation with LPS and rhIL-10 (500 ng/ml, 1000 ng/ml and 2000 ng/ml) (n = 5) for four hours resulted in a marked and uniform reduction of immunoreactive TNFalpha. For preincubation (n = 5), only the addition of 500 ng/ml rhIL-10 led to a homogeneous decrease of TNFalpha levels in each sample. There was no consistent reduction in TNFalpha after preincubation with 1000 and 2000 ng/ml rhIL-10. Our results indicate crossreactivity of recombinant human interleukin-10 in porcine peripheral blood monocytes. Further investigations on the potential therapeutical role of exogenously administered rhIL-10 are thus possible in porcine models.

Cell size of alveolar macrophages: an interspecies comparison.

Alveolar macrophages (AM) play a critical role in the removal of inhaled particles or fibers from the lung. Species differences in AM size may affect the number and size range of particles/fibers that can be actually phagocytized and cleared by AM. The purpose of this study was to compare the cell size of rat, hamster, monkey, and human AM by selective flow cytometric analysis of cell volume. Resident AM from CD rats, Syrian golden hamsters, cynomolgus monkeys, and nonsmoking, healthy human volunteers were harvested by standard bronchoalveolar lavage procedures. Morphometric analysis of AM was performed using a flow cytometer that generates volume signals based on the Coulter-type measurement of electrical resistance. We found that hamster and rat AM had diameters of 13.6 +/- 0.4 microns (n = 8) and 13.1 +/- 0.2 microns (n = 12), respectively. Comparatively, the AM from monkeys (15.3 +/- 0.5 microns, n = 7) and human volunteers (21.2 +/- 0.3 microns, n = 10) were larger than those from rats and hamsters. The AM from humans were significantly larger (p < 0.05) than those from all other species studied, corresponding to a 4-fold larger cell volume of human AM (4990 +/- 174 microns 3) compared to hamster (1328 +/- 123 microns 3) and rat (1166 +/- 42 microns 3) AM. In summary, we have found marked species differences in the cell size of AM. We suggest that the number and size range of particles/fibers that can be phagocytized and cleared by AM may differ among species due to inherent or acquired species differences in AM cell size.

Interspecies comparison of rat and hamster alveolar macrophage antioxidative and oxidative capacity.

Generation of oxidants has been implicated in lung injury and disease caused by a variety of inhaled agents such as ozone, particles, and mineral fibers. Antioxidants in the pulmonary system presumably provide the initial defense against such oxidants. We designed the present study to assess the oxidative and antioxidative capacity of alveolar macrophages (AM) from rats and hamsters. These two laboratory animal species commonly used in biomedical research are well known for their disparate response to pulmonary irritants/toxicants. AM from CD rats and Syrian golden hamsters were obtained by bronchoalveolar lavage. We assessed AM antioxidant levels by measuring the catalase and superoxide dismutase (SOD) activity and the intracellular concentrations of total glutathione, ascorbic acid, and alpha-tocopherol. We determined the AM oxidative capacity by assessing the ability of AM to oxidize extracellular glutathione (GSH) and to release superoxide anions. There were no significant differences in the intracellular antioxidant levels, except for catalase activity that was significantly (p < 0.05) higher in hamster AM than in rat AM. However, AM oxidative capacity was markedly different between the two species studied. The amount of spontaneous and phorbol myristate acetate (PMA)-induced GSH oxidation was about 5-fold higher in rat AM than in hamster AM, whereas the PMA-induced superoxide anion release did not differ significantly between the two rodents. In summary, our data suggest that species variation exists between the oxidative capacity of rat and that of hamster AM. Whereas the oxidative capacity of hamster AM appears to be based mainly on the formation of reactive oxygen species, it is suggested that rat AM possess an additional oxidative system.

Immunophenotyping of lymphocyte subsets in bronchoalveolar lavage fluid. Comparison of flow cytometric and immunocytochemical techniques.

In order to compare flow cytometry with the conventional peroxidase anti-peroxidase method for the immunophenotyping of bronchoalveolar lavage fluid (BALF) lymphocytes, we studied BALF samples from 27 patients with various interstitial lung diseases. The results achieved with both methods were consistent concerning CD3+ pan T cells, CD4+ T helper/inducer, CD8+ T suppressor/cytotoxic and CD57+ natural killer cells. In contrast, a statistically significant lower anti-HLA-DR positive subset was obtained with flow cytometry than with the immunoperoxidase method (p less than 0.005). Since regression analyses and reliability counts showed further agreement between the methods, we conclude that flow cytometric immunophenotyping of BALF lymphocytes leads to similar, if not better, subset analyses than the immunoperoxidase method.