Evidence that Ia-Antigens Are not Secreted by Antigen or Mitogen Stimulated Mouse or Guinea Pig Lymphocytes

Lymph node cells of tuberculin sensitive C3H mice (H-2Mk) and strain 13 guinea pigs were labeled in culture with 3H-leucine and simultaneously stimulated either with PPD or Con A. Unstimulated cultures were labeled in parallel. After incubation for 24 hours the cells were separated from the supernatants by centrifugation and the membranes solubilized by the non-ionic detergent NP 40. Cell lysates were reacted with murine or guinea pig anti-Ia alloantisera in order to test the efficiency of the labeling technique and the specificity of the antisera. The immunocomplexes were analysed on SDS-polyacrylamide gels without reduction. Highly labeled murine (Mw 30,000 and 60,000) and guinea pig Ia-antigens (Mw 25,000; 33,000; 58,000) could readily be detected. Stimulated or unstimulated supernatants of murine lymphocytes, however, when analysed with the same sera, gave no indication for secreted Ia-antigens even when the supernatants were examined either directly or after preprecipitation with normal mouse serum. Furthermore, supernatants of stimulated 3H-labeled and non-stimulated 14C-labeled cultures were combined, chromatographed on Sephadex G 75, and individual fractions were reacted with anti-Iak-serum and control sera. No specific binding or labeled material was observed in any fraction. When double-labeled supernatants from stimulated versus non-stimulated guinea pig lymphocyte supernatants were analysed with an anti-Ia-serum, again no Ia-antigens were detectable in either stimulated or control culture supernatants. On the other hand when 2 heterologous antisera, 1 prepared against guinea pig migration inhibitory factor, the other against guinea pig lymphotoxin were reacted against the same aliquots of double-labeled supernatants, both sera clearly recognized products of activated lymphocytes which were not present in the control culture supernatants. This experiment indicated that the methodology used is sufficiently sensitive to detect some early lymphocyte activation products in culture supernatants. With respect to the consistent failure to detect both murine and guinea pig Ia-antigens in the culture supernatants of control or activated lymphocytes, it is concluded that Ia-antigens are not secreted by resting or activated lymphocytes or perhaps secreted to a much lesser extent than lymphokines such as MIF and lymphotoxin.