Macrophage synthesis of nitric oxide in the mouse mixed leucocyte reaction.

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-2b) ACDSC proliferation were concurrently monitored in cultures comparing RAW 264.7 (H-2d, a high .N = O producer), P388D1 (H-2d, a low .N = O and BALB/c (H-2d) peritoneal macrophages as allogeneic antigen presenting cells (APC). A concentration-dependent increase in lymphocyte proliferation was observed in the presence of 1 x 10(4) to 1 x 10(5) P388D1. In contrast, addition of NG-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-IFN gamma (interferon-gamma) monoclonal antibodies inhibited .N = O synthesis in alloantigen-stimulated cultures. The IFN gamma 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 IFN gamma 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 of MLR, is not affected by initiation of the .N = O pathway.

Characterization of the immunosuppressive effects of nitric oxide in graft vs host disease.

The generation of nitric oxide (.N = O) during in vitro assays involving lymphocyte-macrophage interaction can result in profound inhibition of lymphocyte proliferation. The present study examined whether .N = O synthesis plays a role in the suppression observed in immune function assays during graft vs host disease (GvHD). By using a parent to F1 model to induce GvHD (C57BL/6J to C57BL/6J x DBA 2J F1), a mild but transient increase in serum NO2- plus NO3- levels was observed on day 12 after inoculation. Resident peritoneal macrophages obtained from mice with GvHD demonstrated enhanced .N = O synthesis in response to LPS, compared with control F1 peritoneal macrophages. Similarly, when splenocytes from GvHD mice were cultured with Con A or LPS enhanced supernatant NO2- levels were observed, compared with control F1 mice. Addition of NG-monomethyl-L-arginine (NMA), a competitive inhibitor of .N = O synthesis, resulted in decreased NO2- levels and greatly enhanced proliferation in response to Con A. Addition of NMA to LPS-stimulated cultures did not enhance proliferation, perhaps as the result of the paucity of B cells in the GvHD population. LPS-induced .N = O synthesis by GvHD splenocytes was blocked by anti-IFN-gamma mAb, whereas Con A-induced .N = O synthesis was relatively unaffected by similar concentrations of anti-IFN-gamma mAb, suggesting different mechanisms of induction of .N = O synthesis. A proliferative response of splenocytes from mice with GvHD to third-party alloantigen was not detectable, even in the presence of NMA. The suppression observed when splenocytes from GvHD animals were added to control TNP-modified self cultures was partially reversed in the presence of NMA. These results demonstrate that .N = O synthesis in both splenocyte and peritoneal macrophage populations from GvHD mice is enhanced, revealing that in vivo priming of macrophages for .N = O synthesis occurs during GvHD. Some, but not all, in vitro tests of immune function by using GvHD splenocytes are suppressed by the generation of .N = O.

Nitric oxide production by mouse sponge matrix allograft-infiltrating cells. Comparison with the rat species.

We have recently demonstrated in the rat species that sponge matrix allograft infiltrating cells spontaneously produce nitric oxide (.N = 0) and this .N = 0 production precedes the development of CTL. Compared with our experience in the mouse species, the CTL activity recovered from rat sponge grafts is of shorter duration and less intense. Assessment of the spontaneous .N = 0 production by mouse allograft infiltrating cells reveals a more delayed time course of production, paralleling the recovery of CTL activity from the graft. The in vitro spontaneous .N = 0 production by mouse allograft-infiltrating cells was greater than the production by syngeneic graft-infiltrating cells on all days tested. Exposure of allogeneic but not syngeneic graft infiltrating cells to the sensitizing alloantigen in vitro resulted in enhanced .N = 0 synthesis. In contrast, LPS stimulated .N = 0 production by both syngeneic and allogeneic graft cells on all days postgrafting. Culture of day-14 allograft infiltrating cells with alloantigen in the absence of NG-monomethyl-L-arginine (NMA), the competitive inhibitor of .N = 0 synthesis, resulted in elevated supernatant NO2- levels and decreased 3H-TdR uptake and CTL activity compared with cultures carried out in the presence of NMA. The supernatant NO2- levels, as well as the CTL activity and 3H-TdR incorporation of the cultured cells, was dependent on the concentration of NMA present, and these effects could be reversed by excess L-arginine. Thus, the species difference in .N = 0 synthesis (rat > mouse), observed by others, is evident in the sponge allograft model and may explain why CTL activity recovered from rat allografts is of shorter duration and less intense than that from the mouse allografts.

Evidence that nitric oxide production by in vivo allosensitized cells inhibits the development of allospecific CTL.

The oxidative metabolism of L-arginine to its bioactive product, nitric oxide (.N = O) has been shown to inhibit rat splenocyte mixed lymphocyte reactions. To determine if alloantigen-induced .N = O production might be operative in vivo, cells that had infiltrated a rat sponge matrix allograft were tested for de novo .N = O production as well as .N = O production upon restimulation with the sensitizing alloantigen. When graft-infiltrating cells were placed in culture, a peak in de novo .N = O production was observed by day 6 graft-infiltrating cells, the time when donor-specific CTL activity by the graft-infiltrating cells was first observed. Upon restimulation with alloantigen, allograft-infiltrating cells produced greatly increased levels of .N = O, and this production was associated with inhibition of lymphocyte cytolytic function. The addition of NG-monomethyl-L-arginine (NMA), the competitive inhibitor of oxidative L-arginine metabolism, inhibited .N = O production and promoted allospecific CTL development. Both observed effects of NMA were reversed by addition of excess L-arginine. Cytokine(s) able to induce proliferation of the IL-2-dependent T cell line CTLL-2 could be detected in alloantigen-stimulated cultures in both the presence and absence of NMA. However, proliferation of the graft-infiltrating cells in response to these cytokines was observed only in the presence of NMA. The immunosuppressive macrolide FK506 was a potent inhibitor of .N = O production in these cultures, presumably acting by inhibiting the production of those cytokines that induce the oxidative L-arginine pathway.