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. ImagesFIGURE 2.FIGURE 5.FIGURE 6. (a)FIGURE 6. (b)


Introduction
Macrophage preparations have been shown to suppress protein synthesis in adjacent fibroblasts (1,2) but this effect can be canceled out by pretreatment with silica ( Fig. 1). We have now demonstrated the effect of SiO2 on protein synthesis in vitro, thus imitating the effect of silica in vivo, both in disease and under experimental conditions (3,4). This model has provided the opportunity for studying in detail several problems relevant to silicotic fibrosis (5,6) and to fibroses in general (7,8).
The problems can be divided into two groups: changes in protein synthesis in the fibroblasts and changes in macrophage proteins or secretion products caused by the silica treatment, which may have subsequent effects on the fibroblasts.

Materials and Methods
We used SiO2-treated rat peritoneal macrophages and fibroblasts from experimental granulation tissue in vitro. The experimental conditions have already been described (3,4,(9)(10)(11), but we would like to stress two important details: only intact, non-*Department of Medical Chemistry, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku 52, Finland. elicited macrophages can be used, and the fibroblasts should be "active," (e.g., from experimental granulation tissue or synovial tissue). Established cell lines or "committed" fibroblasts cultured from, e.g., skin, are not suitable. Human monocytes and related malignant cell lines can be used instead of peritoneal macrophages (12). We checked our in vitro results with experiments using rats treated intratracheally with silica dust (DQ 12) (6, 7). The in vivo procedure gave better yields of biologically active materials for purification than did analogous preparations from rat peritoneal macrophages.
In the nucleic acid experiment, (3H)cytidine was used as a precursor. It was incorporated in both DNA and RNA, which were separated by conventional KOH treatment according to Schmidt and Thannhauser (13).
The RNA was purified as described by Rowe et al. (16) and translated with commercial rabbit reticulocyte lysate (The Radiochemical Centre, Amersham, U.K., No 50495) according to the manufacturer's instructions. The gradient gel electrophoresis was performed as described by O'Farrel (17).
The SiO2-bound material was isolated by density KULONENETAL.  gradient centrifugation. The separation of the subcellular particles in a stepwise sucrose gradient has been described earlier (18). The silica particles with the attached material sediment with the debris and nuclei to the bottom of the tube. The DNA and collagen are degraded with specific enzymes and the remnants washed. The materials adhering to silica were solubilized, the lipids by chloroform:methanol extraction, the proteins with sodium dodecyl sulfate-urea solution, and were then studied by conventional methods.

RNA and Other Tissue Components
The essential feature in the silicosis was the increased protein synthesis which was due, at least partially, to an increase of mRNA. In silicotic rat lung, DNA and nitrogen remained relatively constant with regard to the increasing wet weight (19), but whole RNA and hydroxyproline (indicating collagen) increased. A most remarkable 12-fold increase was seen in purified, translatable RNA. Surprisingly, the in vitro translation products mainly contained proteins other than collagen (Fig. 2). This was confirmed to a certain extent by experiments with whole cultured fibroblasts. The specific trans- Translation products of the RNA purified from silicotic rat lung as seen in gel electrophoretic pattern. The reticulocyte lysate was used with ("S)-methionine as isotope marker; 4-42 days indicates the time interval after intratracheal injection of silica, C control lung. The numbers in the right-hand margin give the molecular weights in 10g.
The procollagen band (135 x 10') was very weak.  (2). We tried to elaborate on this by incorporating .\ ' (3H)-cytidine in incubated fibroblasts and occasion-E 30 _ \ " oO ally following it with a "chase." In nonconfluent granulation-tissue fibroblasts, the incorporated activity of DNA and RNA was highest in the presz \e zence of extracts from SiO2-treated macrophages in comparison with the respective controls from un-O \ E treated macrophages. In the degradation of DNA from confluent fibroblasts, we observed that the SiO2-macrophage preparation had a retarding effect 20 lduring the "chase" (Fig. 3  homogenate we found changes in the activities of several RNase fractions following intratracheal silica administration (Fig. 4). The aggregation state of one RNase moiety increases during the silicotic process. The migration of inflammatory cells from the blood stream may contribute to the initial increase in RNase activity. The RNase activity that was precipitated at 40% saturation of ammonium sulfate and which by immunochemical evidence was supposed to originate from macrophages decreased. Rbrogenic Factor and Other Macrophage Protein The isolation of a fibrogenic factor, although only in small yield, from the peritoneal macrophage medium, has been described previously (6,10), as has the preparation of antiserum in rabbits (22). We found that the whole supernatant from silicotic rat lung homogenate contained an activity which stimulated the incorporation of proline to collagen in cultured granulation-tissue fibroblasts (in preparation). The fractionation of this supernatant with ammonium sulfate precipitation combined with gel-filtration chromatography revealed many differences in the protein pattern between the silicotic and control rats (7). Of special interest was a protein with a molecular weight of about 16,000. It was related to the action of silica (6) and originated from macrophages. It could be isolated by gel filtration in almost pure state (Fig. 5). It stimulated the incorporation of proline to collagen by 35-40% at a concentration of 1.5 x 10'M, in the same way as a fibrogenic factor (in preparation). In its molecular weight and acidity, it resembled the light chain of macrophage myosin, calmodulin or troponin C, but differed in the characteristic details in its amino acid composition. The fibrogenic activity seemed to be released, not synthesized, during the disintegration of the macrophages by silica.

Si(2-Binding Components of Macrophages The
SiO2 particles were attached to vesicular subcellular structures of the macrophages (18). The isolation of the attaching subcellular material has been summarized in the experimental section. The resulting simple patterns (Fig. 6) provide a tool for the preparation and analysis of the "attachment sites" between SiO2 particles and cell structures.

Discussion
Both direct analyses and translation experiments show that messenger RNA is increased in experimental silicosis. Although general gene expression is enhanced, the minor role of collagen is surprising. This may be due to increased DNA, increased transcription to hnRNA, increased conversion of hnRNA to cytoplasmic mRNA or a decreased degradation of mRNA for which there is some evidence. DNA is also a target, even in isolated nuclei, for SiOg-macrophage preparations (20) which stimulated the incorporation of cytidine (5). Tolstoshev et al. (23) discuss at length how collagen synthesis in the lung is regulated by the level and utilization of pro-collagen mRNA, and by procollagen degradation. The amount and quality of mRNA are key factors (24) for understanding the imiolecular biology of silicosis. The role of the fibrogenic factors is as yet unclear, as is the nature of the other proteins whose mRNA's show simultaneous increase.
There are many pharmacological means known for supressing collagen synthesis in a developing fibrosis but they are all nonspecific and produce harmful side effects. When the mechanism of silicotic fibrosis can be understood in terms of molecular biology and the role of macrophages defined, silicosis and other fibroses can then be treated by methods designed to have local and specific effects.
We are grateful to the Medical Research Council of Finland for financial support.