Inflammatory mediators applied to in vitro toxicology: Studies on mediator release and two-cell systems

Release of mediators is a manifestation of tissue stimulation or damage applicable to in vitro toxicological investigations. Intercell mediators, or cytokines, are released on tissue stimulation or as a result of mild damage. Intracell mediators, or enzymes of respiration and metabolism, released from damaged cells, participate in inflammatory change and are the usual mediator determinants in in vitro tests. Cells examined for in vitro mediator release are mast cells, which are applicable to anaphylactic allergy and acute inflammation, macrophages, which are particularly applicable to toxicity of inhalable dusts, and fibroblasts/fibrocytes, which are widely used as a general model for cytotoxicity. In tests on organs, lungs may be perfused to identify cytokines released by test substances, which may be used to distinguish between effects due to allergy and those resulting from chemically-induced inflammation. Skin, both human and animal, as full depth or keratome slices of epidermis, has an application in the detection of irritant and corrosive substances. Weak irritants are identified by release of enzymes, for example acid phosphatase, neutral protease and lactate dehydrogenase. Strong irritants and corrosive substances inhibit release or destroy the enzymes. There are inconsistencies in the identification of moderate irritants, both chemicals and bacterial toxins, in which the rank order of severity of effect does not correspond to other in vitro features or in vivo potency. Detection of cytokines (prostaglandins, leukotrienes and interleukin-1) is possible with appropriate conditions of culture, but has been little applied to toxicology. Studies on two-organ/cell systems are briefly reported in which biological mediator release is detectable without specific identification. Cultured skin preparations were treated in vitro with irritants. The culture media were transferred to cultures of normal skin (examined for stimulus to DNA synthesis), to fibroblasts (examined for DNA and polysaccharide/heparan sulphate synthesis) and to suspensions of neutrophils in Boyden chambers (for chemotaxis). The media were also examined for histamine (none found), acid protease cathepsins, acid phosphatase and lactate dehydrogenase. As before, weak chemical irritants were detectable by stimulation of normal skin and fibroblasts to DNA synthesis, fibroblasts to increased heparan sulphate-like synthesis, and analytically by release of enzymes. Moderate irritants and Clostridium perfringens type C toxins depressed these features. Reference is made to the possibility to identify substances likely to induce progressive pulmonary fibrosis, by transfer to fibrocyte cultures of media from macrophages ingesting the dusts or co-culturing macrophages and fibrocytes, to detect a sustained stimulus to fibrogenesis.