Overview:  Macrophage Biology and Innate Immunity

    1.)  STK is expressed on macrophages and regulates innate immune recognition

    2.)  The MSP/STK signaling pathway regulates arginine metabolism in macrophages

    3.)  STK regulates adaptive immune responses and the progression of autoimmune disease

1.  STK is expressed on macrophages and regulates innate immune recognition

        Expression of the STK receptor by monocyte/macrophage populations is highly regulated.  STK/RON is not expressed on circulating monocytes, bone marrow-derived macrophages, splenic red pulp macrophages or alveolar macrophages, but is expressed on a number of specialized tissue macrophage populations, including peritoneal macrophages, osteoclasts, mesangial cells, Kupffer cells, dermal macrophages and marginal zone macrophages in the spleen.  Futhermore, LPS stimulation of macrophages in vitro or in vivo inhibits expression of STK, however the upregulation of STK expression on day 3 ConA elicited peritoneal macrophages and at sites of wounding has been demonstrated.  Our unpublished data indicate that IL-4, glucocorticoids and hypoxia (1% oxygen) induce the expression of STK on primary peritoneal macrophages, but not bone marrow-derived macrophages in vitro, suggesting that these factors may play a role in the induction of STK on elicited macrophages in vivo but that this response may be dependent upon the context of the tissue.  Interestingly, MSP synergizes with hypoxia to upregulate the expression of hypoxia-inducible genes such as VEGF and arginase, suggesting a potential role for this receptor in the regulation of macrophage activities during wound healing.  Based on the restricted expression of STK in cells of the monocyte/macrophage lineage, we speculate that the MSP/STK signaling pathway may play a role in regulating the recognition of exogenous and endogenous ligands by tissue resident macrophages as well as regulating the response of varying macrophage populations to these and other environmental stimuli.  This work is being performed by Dan Sharda.

        The murine STK/human RON receptor tyrosine kinase, a member of the MET family of RTKs, is composed of a disulfide linked extracellular a chain and transmembrane b chain the structure of which are poorly characterized.  Recent studies utilizing the MET receptor suggest that the c-terminal half of the extracellular domain contains four atypical Ig domains and the N-terminal ligand-binding region adopts a beta-propeller fold similar to that displayed by the alpha V integrin.  Consistent with the activation of nearly all RTKs, the ligands for MET and STK/RON (HGF and MSP), bind to the extracellular domain of their respective receptors resulting in the upregulation of kinase activity.  However, this family of receptors also appears to participate in the recognition of a wide range of both exogenous and endogenous ligands.  Listeria monocytogenes has been shown to gain entry into hepatocytes through the interaction of InlB with the Met receptor.  In addition, signaling through the STK receptor regulates the activation of the a_Mb₂ integrin, CR3, which recognizes a variety of exogenous and endogenous ligands including C3bi, ICAM-1, LPS and zymosan.  We have shown that CR3 activation by the MSP/STK signaling pathway results in the phagocytosis of C3bi-coated erythrocytes and enhanced macrophage binding to ICAM-1, via a PI3-kinase/PKCz-dependent mechanism.  In addition, MSP stimulation of primary peritoneal macrophages induces the expression of scavenger receptor A, which recognizes the exogenous ligands Lipid A and LTA, as well as oxLDL and apoptotic cells, resulting in enhanced uptake of acetylated LDL by these cells.  This work was performed by Michael Lutz.

        Kupffer cells account for 80-90% of total fixed tissue macrophages in the body.  These cells reside primarily in the lumen of hepatic sinusoids attached to endothelial cells where they clear bacteria and endotoxin from the blood transported from the gastrointestinal tract via the portal vein.  Effective clearance of Listeria monocytogenes is dependent on Kupffer cells as evidenced by increased Listeria in the blood of Kupffer cell-depleted mice.  Futhermore, this clearance is dependent on the interaction between Kupffer cells and neutrophils in an ICAM-1/CR3 dependent manner.  Mice with a targeted deletion in the gene encoding STK are more susceptible to infection with Listeria monocytogenes, harboring increased bacterial counts in the liver, but not the spleen.  As an intracellular pathogen, Listeria avoids detection by the innate immune system by residing in hepatocytes.  Therefore, the initial rapid clearance of these invaders by macrophages is essential in preventing infection.  STK may enhance initial clearance of Listeria by Kupffer cells through promoting the uptake of complement-opsonized bacterial products as well as augmenting CR3/ICAM-1-dependent interactions with neutrophils.  Alternatively, STK may enhance clearance of Listeria through the activation of other PRRs, such as SR-A, which has been shown to be critical in the innate response to infection with Listeria.  Subsequently, Kupffer cells lacking STK may be unable to efficiently control the initial infection, thus allowing for the dissemination of Listeria into the liver.  This work was performed in collaboration with Francine Gervais.

2.  The MSP/STK signaling pathway regulates arginine metabolism in macrophages

        While MSP, the ligand for STK, was originally isolated from serum due to its ability to induce chemotaxis and complement-mediated phagocytosis by primary peritoneal macrophages, studies in recent years have highlighted a pivotal role for the MSP/STK signaling pathway in regulating the effector functions of macrophages.  MSP stimulation of primary peritoneal macrophages inhibits the production of nitric oxide (NO)and prostaglandin E2 (PGE2) in response to pro-inflammatory cytokines and LPS, due to an inhibition of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (Cox-2), respectively.  This inhibition is associated with a decrease in the activation of NFkB in response to LPS in the presence of MSP/STK.  STK-deficient mice exhibit increased susceptibility to LPS-induced septic shock and enhanced inflammation in response to nickel-induced lung injury, associated with increased serum nitrite levels and pulmonary tyrosine nitrosylation.  MSP is a serum protein that is produced primarily in the liver and circulates in the serum in an inactive form.  The active form of MSP is generated by proteolytic cleavage by members of the coagulation cascade, and is found in the serum of patients with septic shock.  However, mice with a targeted deletion in MSP do not exhibit enhanced susceptibility to endotoxic shock, suggesting the presence of alternative ligands or ligand-independent activation of STK in vivo.  A deletion in the tyrosine kinase domain of the closely related Mer RTK also results in enhanced sensitivity to endotoxic shock, and macrophages from these animals produce elevated levels of TNFa and display increased activation of NFkB in response to stimulation with LPS.  Taken together, these data suggest that RTK expression on tissue-resident and exudate macrophages limits the activation of NFkB and the production of inflammatory mediators, including NO, by these cells in response to environmental stimuli, thus protecting host tissues from inflammatory damage.  This work was performed by Pamela Correll and QingPing Liu.

        In addition to its regulation at the level of iNOS transcription, NO production by activated macrophages is also regulated through substrate availability.  L-arginine is a substrate for both iNOS and arginase, which drives the production of ornithine, a precursor for polyamine and proline synthesis.  While the effects of NO are primarily cytotoxic, production of ornithine by macrophages promotes cell proliferation and matrix synthesis.  IL-4, a potent inhibitor of NO production, accomplishes this task by upregulating arginase expression through the activation of Stat6 and limiting the amount of L-arginine available to iNOS.  We have shown that MSP induces expression of arginase I in primary peritoneal macrophages, and cooperates with IL-4 to enhance arginase activity in a Stat6-dependent manner.  However, unlike IL-4, MSP inhibition of NO is independent of L-arginine availability, suggesting that the MSP/STK signaling pathway independently regulates both iNOS and arginase expression.  MSP-induced arginase I expression is dependent on the PI3-kinase and p38 MAPK signaling pathways.  Interestingly, resident peritoneal macrophages, but not bone marrow-derived macrophages, harvested from mice with a targeted mutation in the lipid phosphatase, SHIP-1, express elevated levels of arginase.  The ability of STK and PI3K to induce arginase I expression in resident macrophages suggests a potential dual role for RTK signaling in limiting tissue-destructive inflammation and promoting the wound healing process by tipping the balance of arginine metabolism in macrophages. This work was performed by Amy Morrison.

3.  STK regulates adaptive immune responses and the progression of autoimmune disease

        In addition to the regulation of macrophage effector functions, there is an emerging role for RTK signaling in regulating the immunomodulatory functions of macrophages.  While simultaneous stimulation of macrophages with MSP and IFN-g with or without LPS has little effect on cytokine production by macrophages, pretreatment of primary peritoneal macrophages with MSP for 2-4 hours results in the complete inhibition of IL-12 production in response to IFN-g and LPS, due to the inhibition of IL-12 p40 expression.  This inhibition appears to result from a block in Stat-1 tyrosine phosphorylation and ICSBP upregulation induced by IFN-g.  IL-12 is a key regulator of IFN-g production by NK and gdT cells in the early stages of an innate immune response, and serves to bridge innate and adaptive immunity by promoting the survival of Th1 cells.  Pretreatment of macrophages with MSP also results in the inhibition of MHC Class II expression in response to both IFN-g and IL-4, and reduced Th1/Th2 differentiation under polarizing conditions in vitro.  In vivo, STK-deficient mice exhibit increased inflammation in a Th1-mediated delayed-type hypersensitivity (DTH) response.  Furthermore, macrophages and dendritic cells from triple-mutant mice for the RTKs Tyro3, Axl and Mer express elevated levels of MHC Class II and the co-stimulatory molecule, B7-2, in response to LPS and produce increased levels of IL-12.   These data suggest that, in addition to regulating innate immune responses, the expression of RTKs on antigen presenting cells may play a key role in modulating T helper cell differentiation and thus regulating acquired immunity.  This work was performed by Amy Morrison and Caleph Wilson.

        The signals that regulate macrophage activation and function must be tightly controlled in order to promote immunity to infection, while protecting host tissues from inflammatory damage.  While these cells play a critical role in the host response to infection, macrophages are the primary effector cells in the tissue destruction phase of autoimmunity.  Cytotoxic effector molecules, such as NO, produced by activated macrophages have been implicated in the pathogenesis of organ-specific autoimmunity.  In addition, IL-12 has been shown to play an important role in sustaining autoimmunity in several mouse models, through its ability to mediate the production of IFN-g and favor a Th1 response.  Changes in the threshold of macrophage activation or reduced ability to clear apoptotic cells can predispose mice to autoimmunity.  The STK and Mer family of RTKs represent a new class of signaling molecules involved in the regulation of macrophages.  The expression of these RTKs on distinct populations of macrophages appears to play a role in innate recognition and regulate the threshold for classical macrophage activation.  In the absence of the Mer receptor, mice develop lupus-like autoimmunity, due at least in part to the inability of MER-deficient macrophages to efficiently clear apoptotic cells, while triple mutant Mer/Axl/Tyro3 mice develop a severe lymphoproliferative disorder accompanied by autoimmunity.  While STK-deficient mice do not develop obvious signs of autoimmunity, the absence of STK on an MRL/Lpr background profoundly exacerbates the pathogenesis of disease in these mice.  Due to the ability of RTKs to regulate innate immune recognition, production of cytotoxic effector molecules and T helper cell activation by macrophages, these receptors may provide a novel target for the treatment of a wide range of autoimmune disorders.  This work is being performed by Manujendra Ray.