Isolation of silica-dependent protein from rat lung with special reference to development of fibrosis.

Silicosis was produced experimentally in rats by single intratracheal injections of various doses of SiO2 dust. The weight of the lungs as well as the contents of total nitrogen, collagen, nucleic acids (especially RNA), and lipids increased in accordance with the dose and the time interval. Fibrogenic stimulation in vitro was shown by the supernatant of the homogenized lung in the incorporation of proline into incubated granulation tissue or lung fibroblasts. The fibrogenic factor-activity depended more on the time interval after the injection than on the SiO2 dose. Electrophoresis of the soluble proteins in the silicotic rat lungs showed a protein of 16,000 Da, which was dependent on the time interval following SiO2 administration as well as on the dose itself, and which originated from macrophages. This protein was purified by repeated gel-filtration chromatography. It stimulated collagen synthesis in granulation-tissue cells at a concentration of about 10(-10) M in a dose-dependent way. It was acidic by amino acid composition but differed from calmodulin which also increased collagen synthesis in granulation-tissue cells in vitro. The ability of non-fractionated macrophage preparations to stimulate the incorporation of proline into collagen correlated inversely with the gross alkaline RNase activity.

In vitro assessment of the fibrogenicity of mineral dusts.

Relying on in vitro production of the macrophage fibrogenic factor, an attempt was made to quantitate the fibrogenicity of mineral particles. Having determined the optimal conditions by means of quartz, two series of experiments were conducted with respirable coal mine dusts; the first employed artificial mixtures of a mine dust, having a low natural quartz content, with various proportions of quartz, and the second native dusts from European mines. The fibrogenic responses in both series suggest that dust concentration is more important than its composition. Quartz and ash contents of native dusts bore no evident relation to fibrogenicity, high quartz and ash levels sometimes being associated with low collagen levels and vice versa. Comparison with other information disclosed disparities with epidemiology and different experimental assessments. Factors affecting the disarray may well include the neglected lipid response in vivo and individual susceptibility, aspects which do not appear beyond experimental assay.

Fibroblast RNA and macrophage proteins (including the fibrogenic factor) in experimental silicosis.

A hypothesis is presented for the action of silica-treated macrophages on protein synthesis in fibroblasts and also a method for the isolation of silica-attached materials in lung tissue. The increased protein synthesis in the fibroblasts is due, at least partly, to an increase in mRNA. Silica prevents the suppressing "macrophage effect" of macrophage-originated ribonuclease on fibroblasts. However, under certain conditions, collagen synthesis is stimulated by silica-treated macrophage preparations to such an extent that the effect cannot be explained by the inhibition of macrophage ribonuclease alone. We therefore postulate the existence of a fibrogenic factor, which is released by the macrophages. This factor has been demonstrated and can be purified from lung homogenate of SiO2-treated rats.

Effects of long-term treatment of rats with ethanol, carbon tetrachloride and high fat-low protein diet on the Kupffer cell distribution with reference to chemical composition of the liver.

Rats were treated for 3-4 months with ethanol or carbon tetrachloride or kept on a high fat-low protein diet. The cell distribution of the liver was investigated with special emphasis on the Kupffer cells with reference to lipids and collagen. Lipids were increased both histologically and chemically in all groups treated. All three treatments caused an increase of Kupffer cells, especially in the high fat-low protein group. The number of Kupffer cells and the content of liver lipids were strongly correlated. Collagen rose in the CCl4- and high fat-low protein groups. Cirrhosis was observed in CCl4--treated rats only.

Ethanol and GABA.

A study of the literature showed that the GABA system is an important target of ethanol in the central nervous system, in part as a consequence of damage in membrane-bound enzymes and receptors. A single dose of ethanol initially activates the GABA system which is weakened after chronic administration because of overcompensation. Interest is focused on the GABA receptors because of the involvement of the minor tranquilizer drugs, benzodiazepines. The studies on the GABA system (metabolism, local concentrations, receptors) show great promise in the understanding and treatment of ethanol dependence and withdrawal.

Antifibrogenic effects of antiserum against the macrophage RNase.

Antiserum against the fibrogenesis controlling macrophage RNase was produced in rabbits. It caused an inhibition of 57% in the RNase activity in vitro. A distinct dose-response relationship was observed in the inhibiting effect of the antiserum on RNase-induced 3H-thymidine incorporation into cultured granulation-tissue fibroblasts. The antifibrogenic properties of the antiserum were also tested in vivo. Rat lungs were made silicotic by intratracheal administration of SiO2. This treatment clearly increased the following parameters: wet weight, DNA, RNA, nitrogen and hydroxyproline content of the lung tissue, and protein concentration, RNase activity, and cell count of the lung lavage fluid. Also, the RNase activity of the lavage fluid cells was increased. Periodical intratracheal administration of the anti-RNase antiserum, optimally at 1:1,000 dilution, decreased the DNA, RNA, and hydroxyproline content of the lung tissue, each by about 30%. The RNase activity of lavage fluid cells was decreased by about 60%. In conclusion the antiserum had no effect on the normal lungs, but it significantly suppressed the development of silicosis.

Qualitative study on the Kupffer cells in the liver of ethanol- and carbon tetrachloride-treated rats.

The treatment of rats with ethanol or CCl4 caused in addition to an increase in numbers, qualitative changes in the Kupffer cells. The rate of phagocytosis of opsonized erythrocytes was greatly increased in the Kupffer cells from CCl4-treated rats, compared with a slightly decreased phagocytosis in similar cells taken from ethanol-treated rats. The number of Fc-receptors per cell was slightly higher in the Kuppffer cells from ethanol- and CCl4-treated rats than in normal rats. Although the nonparenchymal cell fraction from the livers of ethanol- and CCl4-treated rats contained more mononuclear phagocytes than the respective control samples, both the adhering and nonadhering subfractions from the livers of ethanol-treated rats contained proportionally fewer mononuclear phagocytes following an assessment of adhesiveness than the other samples. No difference in esterase staining was observed between the groups.

Penetration of macrophage ribonuclease into fibroblasts and the effects on nucleic acid and collagen metabolism.

RNAase isolated from macrophage culture medium was labelled with 3H-acetic anhydride. The acetylation density of 1.8 acetyl residues/10(4) dalton RNAase was achieved. After just 5 mins' incubation of granulation-tissue slices with 3H-Ac-RNAase, the enzyme had entered fibroblasts, and after 20 min there was only slight increase of the radioactivity. After 1 h incubation 32% of the ingested RNAse was in the nuclei, 23% in the cytosol and about 2% in RER. After the same period of incubation with 3H-Ac-RNAase, 47% of nuclear RNA of the fibroblasts was lost, as compared with the control incubation, and the amount of cytosol RNA increased by about 40%. During a 3-h incubation, in low concentrations, the biologically active macrophage RNAase stimulated. 3H-thymidine incorporation into DNA of proliferative granulation-tissue slices. Thus it was concluded that the fibrogenic effect of the macrophage RNAase on the granulation-tissue fibroblasts takes place through the RNA metabolism and the main target of the enzyme in the fibroblasts is the nuclear RNA.

Neutralization of the fibrogenic silica-released macrophage factor by antiserum.

Antiserum against the fibrogenic factor from the culture medium of silica-treated rat macrophages was produced in rabbits. In dilution 1: 10,000 the antiserum cancelled the stimulating effect of the culture medium of rat macrophages on the 3H-proline incorporation into granulation-tissue fibroblasts. The rabbit antiserum had variable effects on the stimulation by the media of silica-treated human monocytes, but no effect on the media of cultured human SU-DHL-2-cells.

Penetration of various mononuclear ribonucleases into rat experimental granulation-tissue fibroblasts and their intracellular effects.

The penetration of five different mononuclear ribonucleases into the subcellular particles of rat experimental granulation-tissue fibroblasts was compared, along with the effects of the enzymes on the fibroblast RNA fractions. Ribonucleases from normal and silica-treated rat peritoneal macrophages have been shown before to regulate the nucleic acid and protein metabolism of rat experimental granulation-tissue fibroblasts. These biologically active enzymes were taken into the fibroblasts in a greater amount than the corresponding human monocyte enzymes and the biologically inactive rat macrophage ribonuclease. The biologically active macrophage enzymes were incorporated mainly into the nuclear fraction. The other three mononuclear ribonucleases were not found particularly in any subcellular compartment. Both biologically active macrophage enzymes degraded the nuclear RNA of fibroblasts and released it to the soluble fraction in contrast to the biologically inactive macrophage enzyme and ribonuclease from normal human monocytes. Instead the ribonuclease from normal human monocytes seemed to degrade RNA in the soluble fraction. There were no marked differences in the subcellular effects of ribonucleases from normal and silica-treated macrophages. However, the treatment of human monocytes with silica changed their ribonuclease so that it split the nuclear RNA of fibroblast and released it to the soluble fraction in the same way as the biologically active macrophage enzymes did.

Collagen synthesis in cultured mesothelial cells. Response to silica.

Mesothelial cells were isolated from the peritoneal surface of rats using trypsinization. The cells were polygonal-shaped and proliferated rapidly forming confluent cultures. Application of colloidal silica to mesothelial cell cultures in doses known to induce peritoneal adhesion disease was injurious to the cells and reduced their synthesis of total proteins and collagen. This effect was dose dependent. Media from silica-treated mesothelial cells and granulation tissue fibroblasts were applied to similar but non-treated mesothelial cell and granulation tissue fibroblast cultures. Media from SiO2-treated mesothelial cells increased collagen synthesis markedly both in the mesothelial cell and fibroblast cultures compared with the control cultures as estimated from the incorporation of radioactive proline into hydroxy-proline-containing proteins. However, no stimulation of collagen synthesis was induced by media from the SiO2-treated granulation tissue fibroblasts. Similar effects are known to be induced in fibroblast cultured by media from silica-treated peritoneal macrophage cultures. The alkaline ribonuclease activities of the media were decreased in silica-treated mesothelial cell and macrophage cultures but not in the media of fibroblast cultures. These results suggest that mesothelial cells, like macrophages, can interfere with the protein synthesis of fibroblasts and thus contribute to the regeneration of mesothelium after injury and to the formation of adhesions.

Effects of purified macrophage RNase on granuloma fibroblasts with reference to silicosis.

Two alkaline RNases, designated RNase 1 and RNase 2, were isolated from the culture media of silica-treated and non-treated macrophages. The yield of RNase from the medium of silica-treated macrophages was 30% of that from the non-treated control. The effects of these RNases on cultured granuloma fibroblasts and on granulation-tissue nuclei were studied. RNase 1 inhibited thymidine incorporation into fibroblasts except at low concentrations, where it was observed to be stimulatory. RNase 1 also inhibited the protein synthesis of fibroblasts. The incorporation of cytidine into RNA in cultured fibroblasts was not affected by RNase 1, but the incorporation into isolated nuclei was decreased. In pulse chase experiments RNase 1 increased the release of cytidine, but not that of thymidine, from the cells. RNase 2 had no effect on the protein or nucleic acid metabolism of the fibroblasts or on the RNA metabolism of isolated nuclei, perhaps because of impermeability. These experiments confirm that macrophage RNase activity is able to regulate the metabolism of granulation-tissue fibroblasts by increasing RNA degradation. Through this action it also regulates DNA and protein synthesis and other metabolic functions of those cells.

A rapid assay to measure collagen synthesis in cell cultures.

Limited pepsin digestion and precipitation of resistant parts of proteins with perchloric acid on glass-fiber filters has been used as a rapid way to determine the radioactive collagen secreted into fibroblast culture media. The specificity of the pepsin cleavage was tested by digesting [14C]- or [3H]proline- and [3H]tyrosine-labeled procollagens. Radioactivities obtained with this method were comparable with those obtained with collagenase digestions or hydroxyproline determinations. Dialysis of the samples is avoided and the radioactive collagen can thus be determined from the small medium samples obtained from microtest plates. The method was used to localize a collagen synthesis-increasing factor in preparative isoelectric focusing of microphage culture media.

Liberation of a fibrogenic factor from human blood monocytes, ascites cells, cultured histiocytes and transformed mouse macrophages by treatment with SiO2.

Human monocytes and ascites macrophages from cirrhotic patients were isolated in Percoll-gradient and cultured with and without silica. Similar experiments were carried out also with cultured malignant human histiocytes and transformed mouse macrophages. The fibrogenic activity of the culture media was tested by measuring the incorporation of [3H]proline and [3H]thymidine into cultured rat granuloma and human synovial cells. Media from silica-treated monocytes, ascites macrophages and certain histiocyte and mouse macrophage lines caused an increase in the incorporation of both [3H]proline and [3H]thymidine into collagen and DNA, respectively, in both cell systems. Alkaline RNase activities were decreased markedly in the media from silica-treated ascites macrophages but not in the media of the monocytes or histiocytes.