Spleen lymphocytes from Lewis and Buffalo rats and peripheral blood lymphocytes from 10 human donors exhibited high levels of transformation when exposed to Mycoplasma arthritidis supernatants. In contrast, spleen lymphocytes from rabbits and guinea pigs and peripheral blood lymphocytes from sheep and calves failed to transform when exposed to M. arthritidis supernatants. The lymphocytes from all hosts were transformed in the presence of phytohemagglutinin or concanavalin A or both. Serological

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... studies failed to provide evidence that the responding hosts were presensitized against M. arthritidis antigens. We have demonstrated that Mycoplasma arthritidis, an agent of acute or chronic arthritis in rats, mice, and rabbits (8, 10, 11), is mitogenic for normal unsensitized mouse lymphocytes (4, 5) and that the latter develop cytolytic properties for syngeneic and allogeneic target cells (1). Recent studies have shown that lymphocyte activation is dependent upon the mouse strain used and that control of both transformation and lymphocytotoxicity reactions resides within the H2 gene complex (6, 7). Serological and cultural studies have indicated that the lymphocyte transformation observed was not dependent upon a preexisting sensitized lymphocyte subpopulation. Additional work indicated that viable M. arthritidis is predominantly active for the T-cell subpopulation and that the active component of the organisms is a nonsedimentable, heat-labile protein which is present in culture supernatants (Cole et al., J. Immunol., in press). The present studies were undertaken to determine whether this mitogen was specific for murine lymphocytes or whether it was also active for lymphocytes from human and various animal sources. MATERIALS AND METHODS Cultures of mycoplasmas. M. arthritidis 14124P10 (12) was cultured in modified Hayflick medium consisting of PPLO broth (Difco Laboratories, Detroit, Mich.) supplemented to final concentrations of 15% (vol/vol) heat-inactivated horse serum, 5% (vol/vol) fresh yeast extract, 0.5% (wt/vol) L-arginine-hydrochloride, and 1,000 U of penicillin G per ml (3). M. arthritidis supernatants were prepared by centrifuging broth cultures at 27,000 x g for 20 min and passing the supernatant through a 0.45-,um and four successive 0.22-p.m Millex filters (Millipore Corp., Bedford, Mass.). The supernatants were dialyzed for 24 h against phosphate-buffered saline (pH 7.2), refiltered through a 0.22-p.m filter, and cultured on mycoplasma and blood agars to ensure absence of contaminating organisms. Preparation of lymphocyte suspensions. Spleen lymphocyte suspensions from Lewis and Buffalo rats (Microbiological Associates, Bethesda, Md.), Hartley albino guinea pigs, and New Zealand white rabbits were prepared as described previously for mouse lymphocytes (5). Final lymphocyte suspensions were prepared in RPMI 1640 medium containing 5% heatinactivated human serum for rat and rabbit lymphocytes and 5% (vol/vol) heated guinea pig serum for guinea pig lymphocytes. Sheep, bovine, and human lymphocytes were prepared from heparinized blood by Ficoll-Hypaque density gradient centrifugation, using modifications (9) of the procedure of Boyum (2). The bovine lymphocytes received an additional treatment with 0.83% (wt/vol) NH4Cl to lyse contaminating erythrocytes. Final suspensions were prepared in RPMI 1640 medium containing 10% (vol/vol) heat-inactivated fetal calf serum (Hi Clone; Sterile Systems, Logan, Utah), for sheep and bovine lymphocytes and 5% (vol/vol) heat-inactivated human serum for human lymphocytes. Lymphocyte transformation assays. The basic assay was similar to that described previously (6) . In brief, 5 x 105 spleen lymphocytes in RPMI 1640 medium supplemented with 2 mM L-glutamine, 200 U of penicillin G per ml, and appropriate serum were added in 0.2-ml volumes to the wells of microtiter plates. To test for T-cell functions, 0.025 ml of a 1:25 or 1:50 dilution of phytohemagglutinin (PHA) (HA15; Burroughs Wellcome Co., Tuckahoe, N.Y.) was added to triplicate wells to give final concentrations of approximately 45 and 22.5 jig/ml, respectively. Concanavalin A (ConA; Sigma Chemical Co., St. Louis, Mo.) was added in 0.025 ml of medium to give a final concentration of 4.5 ,ug/ml. M. arthritidis supernatants were also added to triplicate wells to give final dilutions of 1:25 to 1:3,125. Wells supplemented with medium alone served as controls for spontaneous uptake of radiolabel (3H control). After 48 h of incubation in 5% CO2 and air, 0.025 ml of medium containing 0.125 p.Ci of 662 on May 5, 2020 by guest http://iai.asm.org/ Downloaded from