INDUCTION OF EARLY-RESPONSE GENES KC AND JE BY MYCOBACTERIAL LIPOARABINOMANNANS - REGULATION OF KC EXPRESSION IN MURINE MACROPHAGES BY LSH ITY/BCG (CANDIDATE NRAMP)/

The murine chromosome 1 gene Lsh/Ity/Bcg (candidate Nramp) regulates m acrophage activation for antimicrobial activity against Salmonella typ himurium, Leishmania donovani, and Mycobacterium spp. To determine ear ly events in the activation pathway, the ability of mycobacterial lipo arabinomannan (LAM) to induce early gene (KC and JE) expression in mac rophages from susceptible (S) C57BL/10ScSn (Lsh(s)) and congenic resis tant (R) B10.L-Lsh(r) mice was investigated. Stimulation with 1.8 mu g of arabinofuranosyl-terminated LAM (AraLAM) per ml resulted in simila r kinetics for KC or JE expression in S and R macrophages. However, wh ereas JE/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA ratios remained equivalent, R macrophages consistently showed enhanced KC/GAP DH ratios within 30 to 40 min of stimulation compared with S macrophag es. Significant differences in KC/GAPDH ratios were observed throughou t the peak period (0.5 to 6 h) of the KC response and with doses of Ar aLAM ranging from 0.01 to 2.5 mu g/ml. Heavily mannosylated LAM from v irulent Mycobacterium tuberculosis Erdman, in doses of up to 2.5 mu g/ ml, failed to stimulate KC or JE in S or R macrophages. Gamma interfer on alone (25 U/ml) stimulated equivalent JE expression in S and R macr ophages and synergized with AraLAM to enhance JE in both. In contrast, AraLAM-induced KC expression was inhibited in the presence of gamma i nterferon. Agonist/ inhibitor studies were undertaken to determine the signal transduction pathways mediating KC expression. The protein kin ase C (PKC) inhibitor Calphostin C (200 nM) inhibited AraLAM-induced K C by 34% +/- 4% in S macrophages and 43% +/- 5% in R macrophages; the cyclic AMP-dependent PKA inhibitor KT5720 (2 mu M) inhibited AraLAM-in duced KC by 33% +/- 4% (S) and 25% +/- 5% (R). A role for Ca2+ was ind icated because ionophore alone stimulated KC expression and synergized with AraLAM to give a dramatically enhanced response. Induction of KC was also inhibited by (i) blocking constitutive nitric oxide (NO) pro duction by preincubation of macrophages with N-G-monomethyl-L-arginine (400 mu M) (48% +/- 8% [S] and 40% +/- 11% [RI) and (ii) incubation o f macrophages with the cyclic GMP-dependent kinase inhibitor KT5823 (4 mu M) (65% +/- 4% [S] and 72% +/- 6% [R]). The manner in which these PKC-, PKA-, and Ca2+-dependent, NO-mediated cyclic GMP-dependent kinas e signal transduction pathways may relate to function of the candidate Lsh/Ity/Bcg gene Nramp is discussed.