Identification and initial characterization of a nonspecific suppressor factor (macrophage-SF) produced by soluble immune response suppressor (SIRS)-treated macrophages.

Abstract Soluble immune response suppressor (SIRS), a product of concanavalin A-activated murine T cells, nonspecificity suppresses plaque-forming cell (PFC) responses in vitro and acts via macrophages. The mechanism(s) by which macrophages exposed to SIRS suppress PFC responses has been investigated. Macrophage-derived suppressor factor (Mϕ-SF), which also nonspecifically suppresses immune responses in vitro, has been identified in supernatant fluids from cultures of adherent splenic macrophages incubated with SIRS. To reproducibly detect optimal Mϕ-SF activity, conventional SIRS preparations (produced by concanavalin A-activated T cells) are partially purified to remove materials with enhancing activity that interfere with Mϕ-SF; alternatively, supernatant fluids from T cell hybridomas producing SIRS constitutively and that lack enhancing activity are used. Macrophages are exposed to partially purified conventional or hybridioma-derived SIRS for 2 hr, washed, and incubated for 24 hr; the medium is changed, and supernatant fluids collected 48 hr later (72 hr total incubation) contain optimal Mϕ-SF activity. The macrophage-like cell line RAW 264.7, but not several other macrophage-like lines, also produces Mϕ-SF after brief exposure to conventional or hybridoma-derived SIRS. Mϕ-SF has an apparent m.w. of 50,000 to 55,000 daltons; biologic activity is lost after treatment with chymotrypsin, at pH 3 and temperatures greater than 70°C, and after exposure to sulfhydryl reagents 2-mercaptoethanol (2-ME), dithiothreitol (DTT), or cysteine, sodium borohydride, amines (taurine and ethanolamine), and I−, but not SCN− or Br−. When Mϕ-SF is added to spleen cell cultures at initiation, PFC responses are suppressed thoughout the 5-day culture, in contrast to SIRS where suppression is not observed until days 4 and 5. Suppression mediated by Mϕ-SF can be reversed up to 72 hr of culture by high concentrations of 2-ME or DTT (5 × 10−4 M), but not by taurine or cysteine. Collectively, these data suggest that Mϕ-SF may be an oxidizing agent and that a possible mechanism of action may be through oxidation of cellular components.