HLA-DR-mediated signals for hematopoiesis and induction of apoptosis involve but are not limited to a nitric oxide pathway

Cross-linking of major histocompatibility complex (MHC) class II antigens by anti-HLA-DR monoclonal antibody (MoAb; H81.9; IgG2a) results in inhibition of hematopoiesis in canine and human models. Inhibition of hematopoiesis is associated with apoptosis in a proportion of marrow cells. Since in murine macrophages class II cross-linking triggers nitric oxide (NO) production, and NO is thought to affect regulation of hematopoiesis, we investigated whether NO was involved in our models. In murine J774 monocytes/macrophages, MoAb H81.9 did induce NO. NO production was blocked by N(G)-monomethyl-L-arginine (NMMA), an inhibitor of NO synthase (NOS), and by the antioxidant N-acetylcysteine (NAC). In human and canine long-term marrow cultures (LTMCs) and in enriched marrow monocytes, however, no measurable increase in NO production was noted after H81.9 exposure. Nevertheless, NAC protected LTMCs against H81.9 induced inhibition of hematopoiesis. Therefore, we determined the effect of an exogenous NO donator, sin-1 (3-morpholinosydnonimine), on canine and human LTMCs and on apoptosis. Sin-1 at concentrations > or =100 microg/mL inhibited LTMCs and induced apoptosis; at low concentrations (1 microg/mL), however, sin-1 stimulated the generation of colony-forming unit granulocyte-macrophage. Combined treatment with sin-1 at 100 microg/mL and MoAb H81.9 resulted in profound inhibition of hematopoiesis in both canine and human LTMCs, and had an additive effect on apoptosis. At 1 microg/mL sin-1 counteracted the effect of H81.9 on hematopoiesis. The effect of sin-1 on apoptosis and hematopoiesis in LTMC was largely prevented by NAC. These results are consistent with the hypothesis that HLA-DR mediated apoptosis and inhibition of hematopoiesis involve oxidative stress. However, the biphasic response of hematopoiesis to sin-1 suggests a complex regulatory network possibly related to differences in NO sensitivity of distinct subpopulations of cells. Signals in addition to NO appear to be involved in the effect of anti-HLA-DR MoAb on hematopoiesis.