Regulation of macrophage arginase expression and tumor growth by the Ron receptor tyrosine kinase.

M1 activation of macrophages promotes inflammation and immunity to intracellular pathogens, whereas M2 macrophage activation promotes resolution of inflammation, wound healing, and tumor growth. These divergent phenotypes are characterized, in part, by the expression of inducible NO synthase and arginase I (Arg1) in M1 versus M2 activated macrophages, respectively. In this study, we demonstrate that the Ron receptor tyrosine kinase tips the balance of macrophage activation by attenuating the M1 phenotype while promoting expression of Arg1 through a Stat6-independent mechanism. Induction of the Arg1 promoter by Ron is mediated by an AP-1 site located 433 bp upstream of the transcription start site. Treatment of primary macrophages with macrophage stimulating protein, the ligand for Ron, induces potent MAPK activation, upregulates Fos, and enhances binding of Fos to the AP-1 site in the Arg1 promoter. In vivo, Arg1 expression in tumor-associated macrophages (TAMs) from Ron(-/-) mice was significantly reduced compared with that in TAMs from control animals. Furthermore, we show that Ron is expressed specifically by Tie2-expressing macrophages, a TAM subset that exhibits a markedly skewed M2 and protumoral phenotype. Decreased Arg1 in TAMs from Ron(-/-) mice was associated with reduced syngeneic tumor growth in these animals. These findings indicate that Ron induces Arg1 expression in macrophages through a previously uncharacterized AP-1 site in the Arg1 promoter and that Ron could be therapeutically targeted in the tumor microenvironment to inhibit tumor growth by targeting expression of Arg1.

Inhibition of TLR4-induced IκB kinase activity by the RON receptor tyrosine kinase and its ligand, macrophage-stimulating protein.

The RON receptor tyrosine kinase regulates the balance between classical (M1) and alternative (M2) macrophage activation. In primary macrophages, the ligand for Ron, macrophage-stimulating protein (MSP), inhibits the expression of inducible NO synthase, a marker of classically activated macrophages, whereas promoting the expression of arginase I, a marker of alternative activation. Ron(-/-) mice express increased levels of IL-12, a product of classically activated macrophages, after endotoxin administration, resulting in increased serum IFN-γ levels and enhanced susceptibility to septic shock. In this study, we demonstrate that MSP inhibits LPS-induced IL-12p40 expression, and this inhibition is dependent on the docking site tyrosines in Ron. To further define this inhibition, we examined the effect of Ron on signaling pathways downstream of Ron. We found that MSP does not inhibit the MyD88-independent activation of IFN regulatory factor 3 and production of IFN-ß in response to LPS, nor does it inhibit MyD88-dependent TGF-ß-activated kinase phosphorylation or MAPK activation in primary macrophages. However, the induction of IκB kinase activity, IκB degradation, and DNA binding of NF-κB after LPS stimulation is delayed in the presence of MSP. In addition, Ron inhibits serine phosphorylation of p65 and NF-κB transcriptional activity induced by LPS stimulation of TLR4. Finally, MSP inhibits the NF-κB-dependent upregulation of the nuclear IκB family member, IκBζ, a positive regulator of secondary response genes including IL-12p40. LPS also induces expression of Ron and an N-terminally truncated form of Ron, Sf-Ron, in primary macrophages, suggesting that the upregulation of Ron by LPS could provide classical feedback regulation of TLR signaling.

The RON receptor tyrosine kinase regulates IFN-gamma production and responses in innate immunity.

Receptor tyrosine kinases are emerging as a class of key regulators of innate immune responses. We have shown previously that the RON receptor tyrosine kinases (murine Stk), expressed on tissue-resident macrophages, inhibit classical macrophage activation while promoting hallmarks of alternative activation, thus regulating the critical balance between the inflammatory and wound-healing properties of activated macrophages. We have also shown previously that RON(-/-) mice are more susceptible to in vivo endotoxin challenge than wild-type mice, suggesting that the expression of this receptor confers a degree of endotoxin resistance to these animals. Here we demonstrate that, in response to in vivo LPS challenge, RON(-/-) mice harbor significantly increased systemic levels of IFN-gamma and IL-12p70 and increased levels of IL-12p40 transcript in their spleen. This elevation of IFN-gamma can be attributed to splenic NK cells responding to the elevated levels of IL-12. Analysis of RON and IFN-gamma receptor double-knockout mice indicates that the enhanced susceptibility of RON(-/-) mice to endotoxin challenge is dependent on IFN-gamma-mediated signals. In vitro studies demonstrate that stimulation of primary peritoneal macrophages with macrophage-stimulating protein, the ligand for RON, inhibits IFN-gamma-induced STAT1 phosphorylation and CIITA expression, resulting in reduced surface levels of MHC class II. Further studies demonstrating the induction of suppressor of cytokine signaling 1 via macrophage-stimulating protein/RON signaling provide a potential mechanistic insight into this regulatory pathway. These results indicate that the RON receptor regulates both the production of and response to IFN-gamma, resulting in enhanced susceptibility to endotoxin challenge.

Case report: anaphylactic reaction to guaifenesin.

Adverse drug reactions lead to a significant number of hospital admissions each year and thus contribute to the overall financial burden of health care. Some of these drug reactions are allergic responses. As the overall predictability of allergic responses to drugs remains low, efforts to improve our understanding of the processes underlying these responses continue as we strive toward the ultimate goal of primary prevention. Allergic reactions range from mild pruritic to severe systemic anaphylactic responses. We report a case of a young healthy man who developed an anaphylactic reaction to an over-the-counter expectorant. A skin test showed that the patient had an immunoglobulin E-mediated allergic response to guaifenesin, one of the components of commonly available cough medications. Our review of published literature showed that this is the first report of a severe allergic response to guaifenesin.

Macrophage-stimulating protein, the ligand for the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase, inhibits IL-12 production by primary peritoneal macrophages stimulated with IFN-gamma and lipopolysaccharide.

IL-12, produced by APCs during the initial stages of an immune response, plays a pivotal role in the induction of IFN-gamma by NK and gammadeltaT cells and in driving the differentiation of Th1 cells, thus providing a critical link between innate and acquired immunity. Due to the unique position occupied by IL-12 in the regulation of immunity, many mechanisms have evolved to modulate IL-12 production. We have shown previously that macrophage-stimulating protein (MSP), the ligand for the stem cell-derived tyrosine kinase/recepteur d'origine nantais (RON) receptor, inhibits NO production by macrophages in response to IFN-gamma and enhances the expression of arginase. Mice lacking RON exhibit increased inflammation in a delayed-type hypersensitivity reaction and increased susceptibility to endotoxic shock. In this study we demonstrate that pretreatment of macrophages with MSP before IFN-gamma and LPS results in the complete inhibition of IL-12 production due to suppression of p40 expression. This response is mediated by the RON receptor, and splenocytes from RON(-/-) animals produce increased levels of IFN-gamma. MSP pretreatment of macrophages resulted in decreased tyrosine phosphorylation of Stat-1 and decreased expression of IFN consensus sequence binding protein in response to inflammatory cytokines. In addition to IL-12, the expression of IL-15 and IL-18, cytokines that are also dependent on IFN consensus sequence binding protein activation, is inhibited by pretreatment with MSP before IFN-gamma and LPS. We also show that the ability of MSP to inhibit IL-12 production is independent of IL-10. Taken together, these results suggest that MSP may actively suppress cell-mediated immune responses through its ability to down-regulate IL-12 production and thus inhibit classical activation of macrophages.