A1. Macrophages are central to the control of many immune responses, particularly at mucosal surfaces. The authors have demonstrated that macrophages express a receptor, relatively specific for substance P (NK1). Expression increases with activation. Substance P induces monokine production -- IL-1, IL-6, IL-12, IL-15, IL-18, and TNFalpha. All of these enhance Th1 responses. IL-10 is also induced by substance P and enhances Th2 cells. Another product, TGFbeta, is immunosuppressive. Although TGFbeta is induced by substance P alone, TGF-beta is inhibited by substance P during LPS induction. Levels of monokine secretion induced by substance P versus other activators are not discussed in detail. IL-12 levels are discussed in a paper in press. Induction of mRNA for other monokines in one animal is shown. The authors plan to describe the kinetics of substance P-induced monokine protein and mRNA in more detail. in vitro activation is followed by kinetic analysis ofcytokine mRNA and protein; in vitro activation , by mRNA content of gut and lymph node cells. A2. Substance P modestly induces MHC class II and B7-2 at 24 hours. This is not necessarily a direct effect, and could be from induced monokines. The mix of induced monokines also includes IL-10, a potent downregulator of MHC II and B7. Suppression will be quantitated after in vitro or in-vovo (by oral salmonella or a toxin based method) activation. A3. NK1 receptors are expressed by macrophages based on receptor binding assays, and recognition with an antiserum specific for NK1 receptors (generated in the author's lab). mRNA for the NK2 receptor is present after 24-hour LPS stimulation. It is at lower levels than the NK1 receptor and has different induction kinetics. These data will presumably will be published soon. Experiments are planned to more carefully define tachykinin receptor expression by macrophages. NK1 and NK2, after activation with substance P plus or minus co-activators, or in vivo, will be detected with RT-PCR, competitive RT-PCR, and by flow cytometry. A4. Macrophages + LPS activation, unlike monocytic cell lines, exhibit no Ca++ flux upon substance P stimulation. These experiments will be replicated with more extensive controls. This is an important observation/discrepancy, for the NK1 receptor is linked to G proteins in monocyte lines, and this suggests and alternate signalling pathway for substance P in macrophages. A5. In vivo, macrophages from gut-associated lymphoid tissue appear to secrete substance P, but only after activation with oral pathogens (salmonella) and immunogens. The authors will quantitate cell type versus cytokine content ex vivo with flow cytometry. This expands upon earlier demonstration of substance P mRNA in different cell types, and is an in vivo snapshot of amount of tachykinin production versus cell type.
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