Bruton’s tyrosine kinase deficiency in macrophages inhibits nitric oxide generation leading to enhancement of IL-12 induction

Abstract

We show that macrophages of X-linked immunodeficient mice with a mutant nonfunctional Bruton’s tyrosine kinase produce less NO than wild-type macrophages in response to a variety of stimuli. Induction of the inducible NO synthase (iNOS) protein, the transcription factor IFN regulatory factor-1 involved in iNOS expression and the transcription factor STAT-1 involved in regulating IFN regulatory factor-1 induction are all poorer in X-linked immunodeficient than in wild-type macrophages. On the other hand, induction of IL-12 is higher in X-linked immunodeficient than in wild-type macrophages. Macrophage IL-12 induction is enhanced by iNOS inhibitors such as aminoguanidine and thiocitrulline and is inhibited by NO generation via sodium nitroprusside. There is relative enhancement of IFN-γ production by immune T cells from mice immunized under aminoguanidine cover. Our data thus suggest that Bruton’s tyrosine kinase participates in signaling for iNOS induction via IFN regulatory factor-1 in macrophages and that NO is an inhibitor of IL-12 induction. The cytokine effector molecules produced by effector T cells generated by immunization in vivo are centrally involved in optimal clearance of infections. Macrophage activating proinflammatory “type 1” cytokines such as IFN-γ are essential for clearance of intracellular bacterial infections (1, 2), while “type 2” cytokines involved in B cell help such as IL-4 and IL-5 are more relevant for extracellular infections (3, 4). The differential commitment of responding T cells to producing either the type 1 (IFN-γ, TNF-β) or the type 2 (IL-4, IL-5, IL-10) cytokine groups (5, 6, 7) is regulated by both cognate and noncognate signals to T cells (8, 9, 10). Cytokine-mediated signals of APC origin are prominent among these. IL-6, IL-10 and IL-12 can all be produced by macrophages (11, 12, 13). While IL-6 and IL-10 drive T cells to the type 2 effector pathway (14, 15), IL-12 is the major APC cytokine known to drive them to IFN-γ production as well as enhance T cell proliferation (16, 17, 18). All these APC-derived cytokines are inducible by a variety of environmental, potentially pathogen-derived stimuli (19, 20, 21). Thus, the induction of costimulatory cytokines on APCs appears to be a major factor in T cell priming, and the regulation of induction of these cytokines becomes an issue of interest in understanding how T cell responses are controlled. This induction of costimulatory cytokines from APCs is commonly seen to occur in response to the same stimuli that lead to APC activation for effector purposes, such as macrophage activation for phagocytosis and pathogen clearance. Molecules induced during such effector activation of macrophages prominently include generators of free radicals such as NO (22, 23). The connections between the induction of costimulatory and effector molecules in activated APCs are likely to be a point of immunoregulatory control. Signal transduction events in APCs are one target of studies involving the regulation of induction of costimulatory and effector cytokines and their effects on each other (24, 25). Nonreceptor-associated tyrosine kinases have been shown to be important components of many signaling cascades. One such enzyme, Bruton’s tyrosine kinase (Btk),3 is expressed in both B cell and myeloid APC lineages (26, 27), but its functional significance has so far been examined mainly in the context of B cell activation (28, 29). We report here its role in regulating NO induction and IL-12 production in macrophages using mice carrying a mutation in the btk gene and demonstrate the cross-regulatory control of IL-12 production by NO.