Work undertaken at the IOM on the production of macrophage fibrogenic factors in vitro

It has been accepted for many years that, during the pathogenesis of fibrosis, reactions occur between macrophages and fibroblasts. One such reaction is thought to be the release, from macrophages, of a fibroblast-stimulating factor which induces increased collagen production by fibroblasts.This work has attempted to define an in vitro model of a macrophage-derived, fibrogenic factor using DQ12 -treated rat peritoneal macrophages and chick embryo fibroblasts as the target cells.Our initial experiments were based on those described by HEPPLESTON and STYLES (1967) and under these conditions poor cultures resulted and no fibrogenic factor was observed. Subsequent modifications to the culture conditions resulted in healthy preparations of both cell types, but there was still no evidence of any fibrogenic factor.The macrophage extracts prepared by this technique proved to be markedly cytotoxic towards the fibroblast cultures. Diluting these extracts 1 in 2 with fresh medium failed to reduce the cytotoxicity and still no evidence of any factor was found. The cytotoxic effect of the extracts was thought to be due to the release of intracellular enzymes from macrophages which were damaged during the incubation period. In order to reduce this damage, further variations in the technique were introduced:- the macrophages were incubated without shaking, the quartz dose was lowered and the cells were incubated for shorter periods. Although these measures did result in reduced cytotoxicity, the levels of collagen production in the test fibroblast cultures were still lower than those found in the controls. Thus, it appeared that there was still some inhibitory compoment in the maorophage extracts, which may have been released during their preparation. To try to overcome this problem, monolayer cultures (as opposed to the normal suspension culture) were incubated with a low quartz dose. During this experiment there was no deliberate disruption of the macrophages and only the supernatant medium from the cultures was tested for its potential fibroblast-stimulating content. The only effect of this different type of preparation was a marked cytotoxicity towards the fibroblast cultures and once again there was no evidence of any fibrogenic factor.At no time could any fibrogenic factor be demonstrated despite several alterations in the preparation and assay techniques.