ANALYSIS OF T CELL DEPENDENT NITRIC OXIDE PRODUCTION BY MURINE MACROPHAGES

Abstract

303 Nitric oxide (NO) plays an important regulatory and effector role in the development of inflammatory and immune responses. Recently several studies have implicated NO as an important effector molecule in allograft rejection. In contrast, other studies have indicated that NO products may serve as a mechanism to downregulate T cell proliferation during an allogeneic response. Several soluble and membrane bound T cell factors including IFNγ, TNFα, and CD40L have been reported to play an important role in regulating NO production by macrophages (Mφ). However, it remains unclear which of these factors are functionally important for NO production stimulated by activated T cells. The purpose of the current study was to investigate the regulation of T dependent NO production by thioglycollate elicited murine peritoneal Mφ in vitro. To determine if membrane bound factors are important for T cell dependent NO production by murine Mφ in vitro, split well chambers were used. DBA/2 (H-2d) T cells were cultured in contact with Mφ from either C57BL/6 (H-2b) wild type or C57BL/6 iNOS-/- mice in the upper chamber. Despite T cell cytokine (IFNγ and TNFα) production in the upper chamber, Mφ from C57BL/6 wild type mice in the lower chamber did not produce NO in the absence of direct contact with alloactivated T cells. This experiment demonstrated that cell-cell contact is critical for T-dependent NO production by murine Mφ. To determine the relative roles of IFNγ, TNFα, and CD40L in NO production, elicited peritoneal Mφ were cultured in vitro with recombinant cytokines or T cells in the presence of blocking monoclonal antibodies. These results indicate that IFNγ is an obligatory but not sufficient signal for maximal NO production. Either TNFα or CD40L were able to provide a second signal that in the presence of IFNγ stimulated NO production. Neither TNFα nor CD40L alone, or the combination of these two molecules elicited NO release. These results indicate that T dependent NO production requires two qualitatively different signals. IFNγ is the sole mediator of the first signal while either TNFα or CD40L can deliver the second signal. The observation that cell-cell contact is necessary for T-dependent NO production suggests that signals by CD40L and TNFα may be best delivered by membrane bound forms of these molecules.