Fluid and mononuclear cells from healing wounds inhibit thymocyte immune responsiveness

It has been shown previously that fluid obtained from 7-day-old wounds noncytotoxically inhibits normal thymic lymphocyte blastogenesis and that mononuclear cells (MNC) from the same wounds lack mitogenic responsiveness. The present series of experiments studies whether wound MNC are the source of the wound inhibitory factor(s) and the effect of adult thymectomy (ATDX) on their generation. Adult male Sprague-Dawley rats (300-350 g), intact or ATDX (performed at 8-10 weeks of age), underwent dorsal wounding (7 cm) and subcutaneous implantation of sterile Ivalon sponges. Seven days later sponges were harvested, wound fluid was obtained, and the cell pellet was purified to 90% MNC. Normal rat thymocyte blastogenesis (stimulation index) to Con A and PHA evaluated in a microculture system (10 separate experiments) was 169.9 +/- 10.0 and 30.1 +/- 3.7. Addition of 10% wound fluid markedly inhibited thymocyte mitogenesis--6.3 +/- 1.0 and 2.7 +/- 0.6, respectively (P less than 0.001). Heat-inactivated wound fluid (56 degrees C, 30 min) had similar inhibitory activity--3.4 +/- 0.9 and 2.7 +/- 0.6 (P less than 0.001). Normal thymic blastogenesis could also be inhibited by the addition of 5 X 10(4) wound MNC to the microculture system--4.4 +/- 1.1 and 1.9 +/- 0.3 (P less than 0.001). Wound fluid from ATDX rats had much less inhibitory activity (77.1 +/- 22.4 and 7.2 +/- 2.1, P less than 0.01) vs control wound fluid. In addition wound MNC from ADTX animals were also less immune suppressive (30.7 +/- 4.9 and 13.5 +/- 3.7, P less than 0.001) than control MNC. Forty-eight-hour supernatants of wound MNC from intact rats, added in 25% concentration to normal thymocyte cultures, demonstrated inhibition similar to that of the wound fluid from the same animals: 4.4 +/- 0.7 and 3.9 +/- 0.6, while ATDX MNC supernatants had minimal inhibitory activity (110.1 +/- 18.2 and 25.7 +/- 6.5, P less than 0.005). No cytotoxicity could be demonstrated in any of these experiments by trypan blue exclusion. It is concluded that 7-day-old wound fluid noncytotoxically inhibits thymocyte blastogenesis; this effect is also demonstrated by wound MNC and their supernatants, suggesting immune "suppressor" lymphocytes are present in wounds; ATDX, which abrogates suppressor cell induction, leads to marked diminution of wound inhibitory activity. The data suggest that important immune events occur at the wound site; their relation to normal wound healing remains to be elucidated.