Stimulation of [3H]thymidine incorporation of thymocytes and splenocytes from guinea pigs by various bacterial cell walls and their peptidoglycans, by enzymatic digests, and by synthetic muramyl dipeptides was studied as an indication of mitogenic activity. Cell wall and peptidoglycan preparations, isolated from 19 strains belonging to 18 different species, definitely increased [3H]thymidine incorporation of thymocytes as well as splenocytes, regardless of mycolic acid contents as a

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... lycan component. Both the cell walls from Nocardia corynebacteriodes (containing mycolic acids) and those from Streptomyces gardneri (lacking mycolic acids) showed far stronger mitogenic activities on splenocytes than other cell walls (stimulation index, 25 to 30). Furthermore, water-soluble enzymatic digests, notably the endopeptidase digests, which generally were greater in degree of polymerization of peptidoglycan subunits than the glycosidase digests obtained from representative cell walls, were found to have as distinct a stimulating activity on splenocytes as the original cell walls. In contrast, solubilization of the cell walls by enzymes, irrespective of endopeptidases or glycosidases, was accompanied by disappearance of the mitogenic activity on thymocytes. On the other hand, studies with synthetic 6-0-acyl-MurNAc-L-Ala-D-isoGln preparations (6-0-acyl-MDPs) revealed that 6-0-stearoyl-MDP and 6-0-(2-tetradecylhexadecanoyl)-MDP, unlike MDP, had distinct mitogenic activity on thymocytes, whereas their activity on splenocytes was rather weaker than MDP itself. The findings presented here suggest that MDP is the minimal structure for the mitogenic activities of bacterial cell walls on guinea pig splenocytes, but that MDP, though distinctively active by itself, requires a polymerized form to exert effectively its inherent stimulating activities on splenocytes. On the other hand, on thymocytes, MDP, unless it takes a particular form or has appropriate additive groups, cannot exert its mitogenic activities. Damais et al. reported that cell wall peptidoglycans obtained from Bacillus megaterium and Escherichia coli exerted mitogenic effects on spleen lymphocytes of nude mice and rabbit splenocytes, but those from Micrococcus lysodeikticus and the monomeric subunit of E. coli peptidoglycan lacked these activities (8). This study was followed by that of Ciorbaru et al., which demonstrated that a water-soluble polymer of peptidoglycan subunits prepared from Nocardia rubra cell walls by use of Streptomyces albus G (endo)peptidase exhibited mitogenic activity on murine B cells, but that a 645 peptidoglycan monomer obtained from the same cell walls digested by lysozyme could not (7) . Then we revealed that adjuvant-active synthetic MurNAc-L-Ala-D-isoGln (MDP) (1, 11, 21) and MurNAc-L-Ala-D-Glu (21) had weak but significant mitogenic activity on splenocytes of guinea pigs and ICR mice, but none of the adjuvantinactive analogs, MurNAc-L-Ala-L-isoGln, MurNAc-L-Ala-D-Gln, MurNAc-L-Ala-L-Gln, MurNAc-L-Ala-L-Glu, and MurNAc-L-Ala-D-isoAsn, had such activity (38). Our findings were confirmed by Damais et al., who demonstrated the mitogenic activity of synthetic MDP by cul-on May 8, 2020 by guest http://iai.asm.org/ Downloaded from