Production of collagen type I by mouse peritoneal macrophages.

The origin and identity of the cells that accumulate and produce collagen in wound healing and in granulomatous and fibrotic processes have long remained unsettled. In this study, it was found that pure cultures of peritoneal macrophages produced hydroxy-L-proline not associated with Clq and produced detectable quantities of collagenase-digestible protein. Cultured macrophages synthesized collagen while retaining their phagocytic capacity. Immuno-blot analysis identified the collagen as Type I. This shows that macrophages have collagen-forming capacity and may be productive cells in pathologic collagen deposition.

Localization in the rat spleen of carbon-laden macrophages introduced into the splenic artery: a subpopulation of macrophages entering the white pulp.

Heavily carbon-laden (HC) macrophages, largely derived from the red pulp of the donor spleen, were injected into the splenic artery of recipient rats. Immediately after injection, HC macrophages were found only in the marginal sinus and in the splenic cords. With time after injection, they appeared successively at the periphery of the white pulp, in the deeper white pulp, and finally in and near the germinal centers, suggesting migration of HC macrophages from the marginal sinus towards the germinal centers. The number of HC macrophages in and near the germinal centers reached a peak at 12 h. Most of the HC macrophages in the white pulp were spherical or ovoid in shape with a diameter of 7-11 microns in sections, having an eccentric round or oval nucleus often with a distinct nucleolus and a cap-like or horseshoe-like cytoplasm filled with carbon. When immunostained with monoclonal antibodies against rat macrophage subpopulations, more than 90% of HC macrophages in the white pulp were found to be ED1+2-3-. A population of the same type of macrophages, both in morphology and phenotype, were found in the red pulp of the donor spleen. They were different from the major residents, red pulp scavenger macrophages, which were ED1+2+3- and larger in size and irregular in shape. These results suggest the presence of a distinct subpopulation of macrophages which actively migrate into the splenic white pulp including the germinal centers. A discharge of transferred macrophages from the red pulp to the general circulation is also suggested.

Recognition of N-glycosidic carbohydrates on esophageal carcinoma cells by macrophage cell line THP-1.

Cell-to-cell contact between macrophages and tumor cells is an important initial reaction in a host defense mechanism against tumor cells. The authors have studied cell surface components of human esophageal carcinoma cells recognized by macrophages. Superoxide release from THP-1 cells, a human macrophage cell line, was analyzed in their interaction with a battery of human squamous cell carcinoma cell lines (TE) originated from esophageal cancer patients. The macrophage-triggering ability of TE 1 cell line, a high stimulant, was reduced after treatment with trypsin or tunicamycin, an inhibitor of N-glycosidic glycosylation. Addition of monosaccharides was efficient in competitive inhibition of these cellular interaction. Moreover, con-A-resistant mutation of TE 1 cells was found to reduce their macrophage-triggering ability, associated with increase of L-PHA-binding capacity, suggesting substitution to the GlcNAc beta(1----6)-linked lactosamine antenna in N-glycosidic carbohydrates. These findings suggest that terminal residues of N-glycosidic carbohydrates on some esophageal carcinoma cells may contribute to the recognition sites of macrophages.

Molecular cloning and sequence analysis of cDNA encoding the macrophage lectin specific for galactose and N-acetylgalactosamine.

The primary structure of the macrophage lectin specific for galactose and N-acetylgalactosamine (macrophage asialoglycoprotein-binding protein, M-ASGP-BP) has been deduced from its cDNA sequence. The M-ASGP-BP cDNA encoded a protein consisting of 306 amino acid residues with a molecular mass of 34,242 daltons. The sequence was highly homologous with that of the rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL), particularly that of RHL-1 (the major form of RHL), throughout its whole length, and especially so in its putative membrane-spanning region and carbohydrate recognition domain. There were two N-glycosylation sites in M-ASGP-BP, the location of which were identical to those in RHL-1. However, M-ASGP-BP was characteristic in having a shorter cytoplasmic tail, and an inserted segment of 24 amino acids containing an Arg-Gly-Asp sequence between the membrane-spanning region and carbohydrate recognition domain.

Oestrogen receptors in macrophages.

The presence of receptors for oestradiol-17 beta and 5 alpha-dihydrotestosterone (5 alpha-DHT) in the human monocytic leukaemia cell line J111 and rat peritoneal macrophages was investigated using whole-cell assays. For both cell types, high-affinity binding species for oestrogen were detected, whereas no indication of specific binding was observed for 5 alpha-DHT. Analysis of the data according to Scatchard showed curved lines, indicating the presence of two different oestrogen-binding species. The dissociation constant (Kd) values for the receptors of the rat peritoneal macrophages were calculated to be 1.4 x 10(-10) M and 3.3 x 10(-9) M, while for the J111 cells, the Kd values were 8.7 x 10(-11) M and 2.5 x 10(-9) M. Sucrose-gradient ultracentrifugation identified one oestrogen-binding species of 7.1S. The receptors had a relatively high affinity for diethylstilboestrol (DES) but did not bind to a monoclonal antibody specific for the classical oestrogen receptor, suggesting that oestrogen receptors in macrophages could be of a different type.

Cholera toxin and its B subunit do not change cytosolic free calcium concentration.

Using the fluorescent Ca2+ probe Quin-2 it has been reported that cholera toxin (CT) and its B subunit (B-CT) increase cytosolic free Ca2+ concentration ([Ca2+]i) in entherocytes, thymocytes and fibroblasts. In this work we show, however, that the fluorescence increases of Quin-2-loaded cells (rat thymocytes, mouse splenocytes, P-388 macrophages and 3T3 fibroblasts) observed upon addition of CT or B-CT are not caused by an increase in [Ca2+]i. The observed effect appears to be accounted for by EDTA-2Na admixtures (present as conservation agent in all CT and B-CT preparations) which 'unquenches' the fluorescence of Quin-2 acid leaked out from the cells into the extracellular medium and produces influorescent complexes with contaminating heavy metal ions. Thus the mitogenic effect of B-CT is not obviously connected with the cytosolic free Ca2+ increase but is probably due to ganglioside-mediated protein phosphorylation.

Analysis of the physical state of cholesteryl esters in arterial-smooth-muscle-derived foam cells by differential scanning calorimetry.

The physical state of cholesteryl esters (CE) in the arterial-smooth-muscle-derived foam cells may contribute to the documented reduction in CE hydrolysis. The physical state of CE may also provide a potential enhancing mechanism for increased CE accumulation. To explore these concepts, we therefore examined the influence of alterations in CE and triacylglycerol (TG) content and their fatty acid composition on the thermotropic behaviour of these lipids by differential scanning calorimetry (d.s.c.). After exposure to cationized LDL (cLDL) or after infection with herpes simplex virus type I (HSV), smooth-muscle cells accumulated significant amounts of CE. The CE/TG ratio was significantly higher in cells treated with cLDL compared with HSV infection. TG content was unaffected by either treatment. However, the fatty acid profile of both CE and TG was significantly different between treatment groups, with the polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio being significantly higher in cLDL-treated cells than in HSV-infected cells. The d.s.c.-generated thermograms of intact cells revealed that neutral lipids of both treatment groups were in the isotropic-liquid state, similar to the state of lipids derived from 'fatty streak' types of atherosclerotic lesions. Differences in the thermograms between HSV-infected and cLDL-treated cells can be ascribed to differences in the CE content and the fatty acid composition of CE and TG (PUFA/SFA ratio). Polarizing optical microscopy revealed the presence of isotropic lipids in both groups. Biochemical and physicochemical data confirm the lysosomal localization of engorged CE, and indicate that the cellular isotropic CE in these foam cells are in a physical state which favours enzymic hydrolysis.

[Antimetastatic effect of L-lysine-alpha oxidase]. / Antimetastaticheskii éffekt L-lizin-alfa-oksidazy.

The influence of a new antitumor enzyme L-lysine alpha-oxidase on Lewis lung carcinoma spreading was studied in mice in which primary tumor had been removed. The enzyme was found to significantly decrease the extent and number of lung metastases as compared to mice which hadn't received L-lysine alpha-oxidase. This was matched by recovery of alveolar macrophages functional activity, as assessed by adenosine deaminase and 5' nucleotidase levels in these cells. Moreover, antimetastatic and cytostatic effect was confirmed by the measuring of polyamine concentration in mice erythrocytes.

Subcellular localization of zirconium in nodular lymphatic cells after administration of soluble salts. Study by electron microprobe.

In the literature two elements, beryllium and zirconium, have been described as inducing identical tissular lesions consisting of granulomas, which some authors have related to an immunological process. Intracellular lesions induced by beryllium sulphate have been studied previously. These lesions were essentially characterized by intranuclear inclusions rich in beryllium. In this work we have studied intracellular concentration sites of zirconium after injection of low doses of zirconium sulphate. Results show that the intracellular concentration sites are very different between both elements, as zirconium is uniquely localized in the lysosomes of the lymph node macrophages where it is associated with phosphorus.

Variation in the composition of gingival inflammatory cell infiltrates.

Biopsy specimens were taken at gingivectomy from 18 adult patients undergoing treatment for chronic marginal periodontitis. They were embedded so that the cut surface of the gingiva was parallel to the top of the block to obtain a comprehensive view in a transversal plane of the inflammatory cell infiltrate near the bottom of the pocket. Sections were stained with HES or with toluidine blue for histological description, and acid alpha-naphthyl acetate esterase (ANAE) was used to differentially stain T lymphocytes, plasma cells and monocytes/macrophages. Sections stained with HES showed that the density and size of the cell infiltrates varied along the circumference of a tooth over very short distances and on various surfaces on neighbouring teeth. Differential counts of cells stained for ANAE demonstrated great variation in the composition of the cell infiltrates, particularly along the pocket epithelium. The predominating ANAE positive cell type in this area was T lymphocytes, while in the central connective tissue, plasma cells predominated. There was no systematic covariation between the localization of the gingiva (i.e. mesial, facial, etc.) and the composition of the cell infiltrates. The local variation in the composition of the cellular infiltrate most likely reflects local variability in the noxious substances (i.e. plaque composition) within the periodontal pocket, and in the resulting local inflammatory response.

[Influence of some antibiotics on eicosanoid production by human macrophages in vitro]. / Influence de quelques antibiotiques sur la production d'Eicosanoïdes par des macrophages humains in vitro.

Non antibacterial effects of antibiotics are presently investigated by several authors. They estimate the effects of different molecules on polynuclear chemotaxis, cytokine production or macrophage activity. In this experiment we have studied the effects of two betalactams, two macrolides, a fluoroquinolone and a tetracycline on eicosanoïd production by stimulated human macrophages in vitro. All evaluated antibiotics are able to modify the prostaglandin and/or leukotriene production. Taking into account the immunomodulative and inflammatory properties of the eicosanoids, the variation of their production could be relevant in clinical practice.

DNA synthesis in alveolar macrophages and other changes in lavaged cells following exposure of CBA/H mice to cigarette smoke.

Traditional methods to determine the proportion of cells in S-phase use radiolabeled precursors of DNA, such as 3H-thymidine, which become incorporated into DNA during its synthesis and are visualized either in tissue sections or in cell preparations by autoradiography. At the Harwell Laboratory the effects of inhaled alpha-emitting actinides on the pulmonary alveolar macrophage population of the rodent lung are being studied. For this research the use of an autoradiographic technique to determine the proportion of cells in S-phase is inappropriate, because of the possible presence of competing sources of radioactivity in the cells under investigation. Consequently, an alternative method has been developed. In this method, 5-bromodeoxyuridine (BrdU), an analogue of thymidine, is incorporated into cells undergoing DNA synthesis. Fluorescein-conjugated monoclonal antibodies, highly specific for BrdU substituted DNA, are available commercially and may be used as a probe for BrdU-labeled cells. This technique for identifying cells in S-phase has been described previously for the flow cytometric analysis of cell suspensions and for cells in tissue sections. An adaptation of this technique for use on cytocentrifuge preparations of cells recovered from mouse lung by bronchoalveolar lavage has been developed and its use is described. Some preliminary results of a short-term experiment with CBA/H mice to determine the effects of exposure to cigarette smoke on the DNA synthesis of alveolar macrophages are also included.

[Histochemical study of proliferating cells in the spleen in mice with acute hepatic injury].

When heat-killed Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) were injected into mice, liver cell necrosis with infiltrating neutrophils and macrophages were induced. Proliferating cells in the spleen were histochemically investigated. P. acnes injection rapidly produced hyperplasia of neutrophils and macrophages in the red pulp of the spleen. The proportion of Bromodeoxyuridine positive cells reached a peak at 5 days after injection of P. acnes. These results indicate that proliferating cells in the spleen after injection of P. acnes are mainly neutrophils and macrophages, which are the same kinds of infiltrating cells into the liver after injection of P. acnes and LPS.

Comportamiento de los receptores de insulina de macrofagos inducidos con tioglicolato y en condiciones de cultivo / Behaviour of insulin receptors in macrophages induced with thioglycate and under culture conditions

Se estudian los receptores de insulina en macrófagos peritoneales de ratón, inducidos con medio tioglicolato y en macrófagos residentes cultivados in vitro durante distintos tiempos. Los macrófagos inducidos presentan una disminución del número de receptores de insulina (1,5 x 10 4/célula con respecto a las residencias (5,2 x 10 4/célula) El número de receptores aumenta en los macrófagos peritoneales de 3,7 x 10 4/célula hasta 2,1 x 10 5/célula durante 72 horas de cultivo in vitro

Microquantification of cholesterol and cholesteryl esters in rat peritoneal macrophages by reverse-phase high-performance liquid chromatography.

A simple and rapid method for the microquantification of cholesterol and cholesteryl esters by reverse-phase high performance liquid chromatography has been established. Comparison of elution patterns of authentic cholesterol and cholesteryl esters revealed that a mu Bondasphere reverse-phase C8 (300-A) column was more suitable than a corresponding reverse-phase C4 or C18 column in terms of rapidity and sensitivity. Recovery of cholesterol and cholesteryl esters from a C8 column was greater than 98% when determined either by radioactive cholesterol and cholesteryl oleate or by cholesteryl heptadecanoate. The sensitivity of the quantification ranged from 5 ng to 50 micrograms for both cholesterol and cholesteryl esters. This method was applied to determination of cellular cholesterol and cholesteryl esters of rat peritoneal macrophages. Lipid extracts of these cells were found to contain 38.01 +/- 2.60 micrograms of cholesterol and 3.18 +/- 0.36 micrograms of cholesteryl esters per milligram of cell protein. When the cells were loaded with cholesteryl esters by incubation for 24 h with various concentrations of acetylated low-density lipoprotein, a cellular level of cholesteryl esters showed a dose-dependent increase and reached a maximal level of 106.60 +/- 3.05 micrograms/mg cell protein. Thus, the present method is useful for the microquantification of cholesterol and cholesteryl esters from lipid extracts of biological samples.

Increases in HLA-DQ, DP, DR, and transferrin receptors on alveolar macrophages in sarcoidosis and allergic alveolitis compared with fibrosing alveolitis.

We have used flow cytometric methods to detect and quantify HLA-DR, DQ, and DP antigens and transferrin receptors on alveolar macrophages in lavage samples from 36 patients with granulomatous lung diseases (extrinsic allergic alveolitis [EAA], n = 13; sarcoidosis, n = 23), and 12 patients having fibrosing alveolitis (FA) (cryptogenic fibrosing alveolitis, n = 3; FA and scleroderma, n = 8; FA and primary biliary cirrhosis, n = 1). HLA-DR, DQ, and DP antigens were expressed on the majority of alveolar macrophages in all the patients, and the percentages of positive cells were similar to those in control subjects without lung disease. However, the amounts expressed were higher in those with EAA and sarcoidosis than in the FA group or control subjects, the most significant differences being in HLA-DQ and HLA-DP expression. Transferrin receptor expression was also higher in the granulomatous lung diseases. In sarcoidosis, higher levels of HLA-DQ correlated with lower lung function measurements (Dco p less than 0.025, FVC p less than 0.025, FEV1 p less than 0.005), suggesting this may be a marker of disease activity. HLA-DP levels also showed a trend (p less than 0.1) of inverse correlation with lung function. Levels of HLA-DQ (p less than 0.005) and HLA-DP (p less than 0.001) correlated more closely than HLA-DR with numbers of lymphocytes in the lavage fluids, and HLA-DQ levels correlated with increasing proportions of lymphocytes in proliferation (p less than 0.05). We suggest that high levels of HLA-DQ and DP on alveolar macrophages may be more relevant than HLA-DR to the enhanced antigen-presenting function of these cells in sarcoidosis, and possibly also in EAA.

The use of quantitative two-dimensional gel electrophoresis to analyze changes in alveolar macrophage proteins in humans exposed to ozone.

Acute exposure of humans to 0.4 ppm ozone is known to cause production of components that mediate inflammation and damage in the lung. However, ozone may cause even more extensive changes in the lung than those currently measured by traditional enzymatic or immunologic methods. The contribution of alveolar macrophages to these processes is not well understood. Therefore we have used molecular techniques to measure changes in the total spectrum of alveolar macrophage proteins in humans exposed to ozone. In this study, 8 human volunteers were each exposed to 0.4 ppm ozone and to filtered air for 2 h with intermittent exercise. Eighteen hours later, bronchoalveolar lavage was performed and alveolar macrophages were isolated. Changes in proteins made by these cells after air or ozone exposure were analyzed by high-resolution two-dimensional gel electrophoresis, using computerized densitometry to quantify changes in individual proteins. Of the nearly 900 proteins analyzed, 45 (5.1%) were synthesized at a significantly increased rate following ozone exposure, while 78 (8.8%) were synthesized at a significantly reduced rate. These results indicate that exposure of humans to ozone causes extensive changes in the spectrum of macrophage proteins being produced. Quantitative two-dimensional gel electrophoresis is a highly sensitive technique that may reveal much more information about the in vivo effects of a pollutant than has previously been available. Furthermore, the ability to survey large numbers of macrophage proteins after exposure to various inhaled pollutants may allow a better understanding of the mechanisms of action of these agents, as well as provide new biomarkers of pollutant exposure.

[The expression of Fc gamma receptors on the surface of macrophages activated by a vaccinal strain of Yersinia pestis]. / Ekspressiia Fc gamma-retseptorov na poverkhnosti makrofagov, aktivirovannykh vaktsinnym shtammom chumnogo mikroba.

The expression of Fc gamma R on the surface of macrophages in the process of antiplague immunity formation is analyzed. The stud is performed on the alveolar and peritoneal macrophages obtained from intact and immunized guinea pigs in different periods after vaccination (the 1st, 7th, 14th and 21st day). It is established that during the formation of the antiplague immunity there occurs activation of macrophages which is accompanied by an increase of the Fc gamma R expression on the outer surface of the membrane both of peritoneal and alveolar macrophages and the pattern of response of these cells to the interaction with the vaccine strain of the plague microbe changes. The Fc gamma R expression heterogeneity of certain macrophage populations is revealed both in an intact and in immune organism as well as different pattern of the intact alveolar and peritoneal macrophage response during the interaction with the vaccine strain of the antiplague microbe. These differences are levelled in the process of the antiplague immunity formation.

HIV-1 in trophoblastic and villous Hofbauer cells, and haematological precursors in eight-week fetuses.

AIDS in children is usually caused by vertical transmission of human immunodeficiency virus type 1 (HIV-1). Aborted eight-week fetal and placental tissue from HIV-1 positive and negative (enzyme-linked immunosorbent assay and Western blot) women was analysed by immunocytochemistry and in-situ hybridisation. Maternal decidual leucocytes, villous trophoblastic derivatives, villous mesenchymal cells, and embryonic blood cell precursors in tissues from seropositive patients all stained for HIV-1 antigen and hybridised for nucleic acids. These observations suggest that a cytological pathway for vertical transmission of HIV-1 is established by eight weeks gestational age.