Macrophage synthesis of nitric oxide in the mouse mixed leucocyte reaction

Abstract

The production of nitric oxide (•NO) in the splenocyte mixed leucocyte reaction (MLR) results in inhibition of allospecific lymphocyte effector function. In order to more clearly define the circumstances which promote •NO synthesis in the MLR, responder accessory cell depleted spleen cells (ACDSC) were co-cultured with allogeneic macrophage cell lines or peritoneal macrophages. •NO synthesis and C57BL/6 (H-2 b) ACDSC proliferation were concurrently monitored in cultures comparing RAW 264.7 (H-2 d, a high •NO producer), P388D1 (H-2 d, a low •NO producer) and BALB/c (H-2 d) peritoneal macrophages as allogeneic antigen presenting cells (APC). A concentration-dependent increase in lymphocyte proliferation was observed in the presence of 1×10 4 to 1×10 5 P388D1. In contrast, addition of N G-monomethyl- L-arginine (NMA), a competitive inhibitor of •NO synthase, was necessary in order to observe lymphocyte proliferation in the presence of increasing numbers of RAW 264.7 and BALB/c peritoneal macrophages. The addition of both anti-IL-2 and anti-IFNγ (interferon-gamma) monoclonal antibodies inhibited •NO synthesis in alloantigen-stimulated cultures. The IFNγ induced expression of class II antigen, as well as the constitutive expression of class I antigen, on RAW 264.7 was similar in the presence or absence of NMA, indicating that induction of •NO synthesis by IFNγ does not inhibit H-2 antigen expression. Thus, cytokines produced as a result of alloimmune interaction initiate macrophage •NO synthesis. However, allogeneic APC function, as assessed by H-2 antigen expression and subsequent stimulatory capacity in MLR, is not affected by initiation of the •NO pathway.