Macrophages in granulomas: histochemical evidence suggesting local control of heterogeneous functions.
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Biomedical subjects
Publications and source records attributed to M Suga.
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Dermal tuberculous lesions were produced in rabbits with Bacille Calmette Guèrin and biopsied on days 4, 14, 21, 35, and 56. Frozen sections prepared from the biopsy specimens were stained by the peroxidase-antiperoxidase immunocytochemical technique with antisera against whole tubercle bacilli and antisera against the protein B, polysaccharide I, and wax D fractions of tubercle bacilli. In the primary lesions, protein B was often undetectable at 21 days, and polysaccharide I was often undetectable at 35 days. Wax D disappeared more slowly than the other bacillary components evaluated, and some wax D was still found at 56 days (if the lesions had not completely healed). Antisera against intact tubercle bacilli produced results similar to those produced by antiserum against wax D. These studies suggest that the chronicity of tuberculous lesions is due, at least in part, to the persistence of the wax D-like component of tubercle bacilli.
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Experimental immunotherapy with a mixture of allogeneic tumor cells and BCG cell-wall skeleton (BCG-CWS) was carried out against established autochthonous fibrosarcomas induced by 3-methylcholanthrene (MCA) in the subcutaneous tissue of rats. The inhibitory effect on growth in the early stage, tumor-size up to 2 cm in diameter, was significantly observed. No significant difference, however, was shown as compared with the effect of treatment with allogeneic tumor cells alone. That is to say, a more effective inhibition of growth due to the addition of BCG-CWS was not demonstrated.
Non-inbred rats of the Gifu strain were intraperitoneally challenged with Hirosaki sarcoma (tetraploid type, 10(5) cells) after repeated immunization with gamma-irradiated (13,000 rads 60Co) allogeneic non-viral tumors of ascites type (tetraploid or diploid type of Hirosaki sarcoma, Usubuchi sarcoma or AH130). In rats immunized not only with the same tumor as the immunizing tumor but also with a different tumor, the growth of the challenge tumor was markedly inhibited as compared with the control in non-immunized rats. It is considered that these tumors retained common antigen(s) by the resistance to irradiation because of their form of ascites tumor. The marked cross-immunity in rats immunized with AH130 may be explained by the fact that gamma-irradiated AH130 cells were alive longer in the peritoneal cavity than other tumors on account of its high resistance to irradiation.
BCG lesions were produced in the skin of rabbits, and biopsies were performed at 7, 21, and 42 days, when they were developing, maximal in size, and almost healed, respectively. Tissue sections were prepared and stained histochemically for several enzymes. The percentage of cells stained for a given enzyme and the distribution of such cells within lesions of various ages were determined. Seven-day BCG lesions contained few esterase- and beta-galactosidase-positive macrophages, but 21-day lesions contained many, especially in the viable and nonviable tuberculous granulation tissue at the edge of the now prominent caseous necrotic center. Both 7-day and 21-day lesions contained many acid phosphatase- and cathepsin-D-positive macrophages, which were numerous in the more peripheral parts of the lesion, where little or no necrosis was present. Enzyme patterns in 42-day lesions resembled those in 21-day lesions. The role of each of these enzymes in the development and regression of the BCG lesion is unknown. Nonetheless, these studies clearly demonstrate that this macrophage population is heterogeneous and that macrophages carry out different functions in different parts of the lesion at different times. Histochemical techniques were developed to stain two enzymes in the same tissue section. The first stain usually contained a naphthol substrate and produced a red color; the second stain contained an indoxyl substrate and produced a blue color. A cell staining with both was colored purple. The peroxidase-antiperoxidase immunocytochemical technique for cathepsin D (producing a red color) was also employed. 1) Red esterase (hydrolyzing naphthol AS-D acetate) and beta-galactosidase, and 2) red esterase and blue esterase (hydrolyzing 5-bromo-4-chloro-indoxyl acetate), probably the same enzyme, were usually present in the same macrophage. In contrast, each of the following enzyme pairs was usually present in a different macrophage: 3) cathepsin D and beta-galactosidase, 4) cathepsin D and blue esterase, 5) acid phosphatase and beta-galactosidase, and 6) acid phosphatase and blue esterase. Roughly 10% of the macrophages stained for one enzyme existed side by side with macrophages stained for a different enzyme. These results suggest that local macrophage activation is under two levels of control. The first, macrolocal control, would determine the overall enzyme distribution in the lesion; whereas the second, microlocal control, would determine enzyme distribution on a cell-by-cell basis, ie, how two neighboring macrophages can each be rich in a different enzyme.
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The superoxide production of BCG-infected and noninfected alveolar macrophages was measured by superoxide dismutase-inhibitable nitro blue tetrazolium reduction. The cells were incubated with or without cell-free bronchial lavage fluid (pulmonary washings). When control alveolar macrophages were infected by BCG, superoxide production was decreased markedly, probably due to bacterial cytotoxic factors. In contrast, the production of superoxide in alveolar macrophages exposed to pulmonary washings was increased and not appreciably influenced by BCG infection. Superoxide production by alveolar macrophages was dependent on time and on the protein concentration in the pulmonary washings. In controls, it was inversely proportional to the infecting dose of BCG. We observed previously that alveolar macrophages activated by pulmonary washings inhibited intracellular growth of BCG. We now present evidence that enhanced production of superoxide contributes to such inhibition, especially in the presence of catalase at acid pH. These findings are pertinent to the defense of inflamed lungs, where serum and serum immunoglobulin G transuded from blood into alveolar spaces probably induce such activation on alveolar macrophages.
The tumor-inhibitory effect of injections of allogeneic tumor cells was observed. Established autochthonous sarcomas induced in the subcutaneous tissue of rats by 3-methylcholanthrene (MCA) were treated with immunization by using allogeneic Hirosaki sarcoma cells. When MCA-induced sarcomas grew to approximately 1 cm in mean diameter, Hirosaki sarcoma cells were inoculated into various tissues of primary tumor-bearing rats. Immunizing procedures consisted of intraperitoneal and subcutaneous injections in one experimental group, and of intradermal and intraperitoneal injections in another. Significantly inhibitory effect on the growth of autochthonous sarcomas was observed in the initial stage up to 2 cm in diameter as compared with that of control sarcomas. No significant inhibition was seen in the course of the growth of sarcomas larger than 2 cm in diameter. This results may indicate that immunotherapy by using allogeneic tumor cells should be considered to be valuable for the treatment of human cancer.
Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, beta-galactosidase, and cytochrome oxidase in plastic embedded and ruotine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 micrometers, were far superior to frozen sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.
Chickens were exposed to SO2 in relatively low concentrations (3.4 to 18.5 parts per million (ppm)) for 1 to 14 days. A portion of their tracheas was embedded in water-soluble methacrylate, cut at 2 micrometer and stained with hematoxylin and eosin, Wright's stain, methyl green-pyronin, Alcian blue - periodic and Schiff, and for acid phosphatase. An increase was found in (a) the mucosa to wall ratio; (b) the number of mucosal cells in mitosis; (c) the number of macrophages, lymphocytes, plasma cells, and neutrophils in the epithelium and lamina propria; and (d) the number of these infiltrating cells which contained acid phosphatase. The number of mucus- and seromucus- secreting cells and vasoamine-containing cells were sometimes increased, but not consistently. The percentage of cells containing sialidase-sensitive sialomucins was elevated, and percentage of cells containing neutral mucins was reduced. These changes were only partly related to the SO2 concentration and the duration of SO2 exposure, in that increasing amounts of SO2 did not always cause increasing changes in the mucin composition. Evidently, the altered mucins sometimes protected against further mucin modification.
Pulmonary washings from rabbits were freed of cells and added to the monolayers of homologous alveolar macrophages (AM). At 1 h after incubation with the pulmonary washings, many more cells adhered to glass, spread out, and showed enhanced Nitro Blue Tetrazolium reduction. The maximal effect of the pulmonary washings on AM activation was obtained 12 h after incubation. The AM activated by the pulmonary washings showed a higher capacity to inhibit the growth of intracellular BCG, and that capacity was correlated with the intensity of Nitro Blue Tetrazolium reduction by the AM. Gel filtration of the pulmonary washings through Sepharose 4B yielded five fractions. The factor that activated the AM functions was in fraction 4. When the immunoglobulin G in the fraction was removed by an immunoadsorbent column, AM activity was abolished. The effect of the immunoglobulin G was dose dependent, and minimal responses to 10(6) cells per ml were obtained at a protein concentration of 20 mug/ml. Lymphokines had no effect on AM activation with respect to the morphological alterations and Nitro Blue Tetrazolium reduction during the 24-h observation time. In summary, AM from normal rabbits were soon activated markedly by lavage-procured immunoglobulin G, but not by lymphokines.
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