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RECEPTOR TYROSINE KINASES AND THE REGULATION OF MACROPHAGE ACTIVATION Correll PH, Morrison AC and Lutz MA Department of Veterinary Science The Pennsylvania State University University Park, PA 16802 Corresponding Author: Pamela H. Correll Department of Veterinary Science 115 Henning Building The Pennsylvania State University University Park, PA 16802-3500 Phone: 814-863-0128 FAX: 814-863-6140 Email: [email protected] Running Title: RTK activation of macrophages Key Words: Receptor tyrosine kinase, macrophage activation, STK/RON, Mer/Axl/Tyro3 It is becoming increasingly clear that macrophages are a diverse and dynamic population of cells that can be activated down a number of distinct developmental pathways (recently reviewed in [1, 2]). These cells have the capacity to perform a wide range of critical functions including; 1) the recognition, phagocytosis and clearance of invading pathogens through the expression of pattern recognition receptors (PRRs) and the upregulation of cytotoxic molecules, 2) immune modulation through the production of cytokines and chemokines, antigen presentation the regulation of T cell activation and differentiation, and 3) the resolution of inflammation and the promotion of healing through induction of matrix synthesis, fibroblast proliferation, angiogenesis and the clearance of cellular debris. In order to perform these disparate tasks, macrophages must be capable of inducing the expression of specific subsets of genes in a coordinated fashion in response to environmental stimuli. The basal activity of tissue resident macrophages, as well as their ability to respond to environmental stimuli, varies considerably. This response must be tightly controlled in order to stimulate immunity to infection, while at the same time, protecting host tissues from inflammatory damage and promoting normal tissue homeostasis. Here, we propose that receptor tyrosine kinases (RTKs) play a role in fine-tuning this system. Expression of at least three distinct families of RTKs has been reported in the monocyte/macrophage lineage. The receptor for macrophage colony-stimulating factor (c-fms, CSF-1R) is widely expressed in the monocyte/macrophage lineage[3], and is required for the development of a number of tissue-resident macrophage populations[4]. This receptor is a member of the PDGFR superfamily of RTKs , containing five immunoglobulin-like domains in the extracellular portion of the receptor and a split kinase domain (figure 1a). In contrast, the Axl/Tyro3/Mer family of receptors[5, 6] contains two immunoglobulin-like domains and two fibronectin type III repeats in the ectodomain and a contiguous kinase domain with two tandem tyrosines in the activation loop. The murine STK/human RON receptor[7, 8], a member of the Figure 1. Receptor Tyrosine Kinases Expressed in the Monocyte/Macrophage Lineage. A) Cartoon of the conserved structural features of the RON/STK, Axl/Tyro3/Mer and c-fms receptors and their family members. Green and light blue bars represent the kinase domain and fibronectin-like repeats, respectively. Half circles represent Ig domains B) Expression of the murine STK receptor in various cells of the monocyte/macrophage lineage. MET family of RTKs, is closely related to the Tyro3 family and shares a similar kinase structure. The ectodomain of STK, however, is composed of a disulfide linked extracellular α chain and transmembrane β 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[9]. While M-CSF has been implicated in the proliferation, but not the activity, of terminally differentiated macrophages[10], the Tyro3 family of receptors and STK appear to play a role in regulating the activation of these cells in response to environmental stimuli[11, 12]. Consistent with the activation of nearly all RTKs, the ligands for the Tyro3 family of RTKs (Gas6 and Protein S[13-15]) and STK (MSP, macrophage stimulating protein) [16], bind to the extracellular domain of their respective receptors resulting in the upregulation of kinase activity. However, RTKs also appear 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[17]. In addition, Tyro3 receptors play a direct role in the recognition of apoptotic cells through the interaction of Gas6 with phosphatidyl serine on the surface of apoptotic cells, and macrophages from Mer KO mice are defective in the clearance of apoptotic thymocytes[18]. Alternatively, these RTKs can also regulate the expression or activity of classic PRRs. For example, signaling through the STK receptor regulates the activation of the αMβ2 integrin, CR3, which recognizes a variety of exogenous and endogenous ligands including C3bi, ICAM-1, LPS and zymosan. 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/PKCζ-dependent mechanism[19]. In addition, MSP stimulation of primary peritoneal macrophages induces the expression of scavenger receptor A[20], 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. While the Tyro3 family of receptors is reported to be widely expressed by monocytes and their derivatives, 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 (figure 1b), including peritoneal macrophages, osteoclasts, mesangial cells, Kupffer cells, dermal macrophages and marginal zone macrophages in the spleen [21-23](and unpublished observations). Futhermore, LPS stimulation of macrophages in vitro or in vivo inhibits expression of STK[24], however the upregulation of STK expression on day 3 ConA elicited peritoneal macrophages and at sites of wounding has been demonstrated[21, 23]. 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 in the context of the peritoneal cavity or at sites of wounding. 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. 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 ICAM1/CR3 dependent manner[25]. 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[26]. 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[27]. Subsequently, Kupffer cells lacking STK may be unable to efficiently control the initial infection, thus allowing for the dissemination of Listeria into the liver. 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)[28, 29] and prostaglandin E2 (PGE2)[30] 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 NFκB in response to LPS in the presence of MSP/STK[29, 30]. STK-deficient mice exhibit increased susceptibility to LPSinduced septic shock[31-33] and enhanced inflammation in response to nickel-induced lung injury, associated with increased serum nitrite levels and pulmonary tyrosine nitrosylation[34]. 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[35], 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[36], 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 TNFα and display increased activation of NFκB in response to stimulation with LPS[37]. 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. 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[38]. We have shown that MSP induces expression of arginase I in primary peritoneal macrophages[20], and cooperates with IL-4 to enhance arginase activity in a Stat6-dependent manner (Wilson and Correll, submitted). 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 (Morrison and Correll, unpublished). 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[39]. 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 (figure 2). 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-γ 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 IFNγ and LPS, due to the inhibition of IL-12 p40 expression[40]. This inhibition appears to result from a block in Stat-1 tyrosine phosphorylation and ICSBP upregulation induced by IFN-γ. IL12 is a key regulator of IFN-γ production by NK and γδT 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 (reviewed in [41]). Pretreatment of macrophages with MSP also results in the inhibition of MHC Class II expression in response to both IFN-γ and IL-4, and reduced Th1/Th2 differentiation under polarizing conditions in vitro (Wilson and Correll, submitted). In vivo, STK-deficient mice exhibit increased inflammation in a Th1-mediated delayed-type hypersensitivity (DTH) response[31, 33]. 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 IL12[42]. 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 (figure 3, adapted from [1]). Figure 2. Activation of STK by MSP tips the balance of arginine metabolism toward ornithine. While pro-inflammatory cytokines and LPS induce expression of iNOS resulting in the metabolism of arginine to nitric oxide, Th2 cytokines induce expression of arginase resulting in the production of ornithine. MSP stimulation of the STK receptor inhibits iNOS expression in response to IFN-γ and LPS and induces the expression of arginase, thus favoring the metabolism of arginine to ornithine. Figure 3. MSP/STK limits IFN-γ-induced IL-12 production, MHC Class II expression and Th1 cell differentiation. IL-12 production by macrophages induces IFN-γ production by NK and γδ T cells and supports Th1 cell differentiation. IFN-γ also increases surface expression of MHC Class II on macrophages, resulting in enhanced antigen presentation. MSP/STK inhibits both IL-12 production and MHC Class II expression, and stimulation of primary peritoneal macrophages with MSP limits the ability of these cells to support Th1 differentiation. While signaling through RTKs has traditionally been shown to promote cell growth and survival, thus playing a critical role in differentiation and tumorigenesis, the studies described here have provided the first link between RTK signaling and immune regulation. However, the mechanism by which these RTKs inhibit macrophage activation remains unclear. IL-10, a potent inhibitor of classical activation, induces Stat3 phosphorylation, and mice with a tissue-specific deletion of Stat3 in macrophages develop an intestinal inflammation similar to that observed in the IL-10 knockout animals and are more susceptible to septic shock[43]. Furthermore, microarray studies using macrophages from these mice have indicated that IL-10 exerts most, if not all, of its effects on macrophages through the activation of Stat3[44]. Activation of Stat3 by a number of wild-type and mutant RTKs, including those of the MET and Tyro3 families, has also been demonstrated[45-49]. While the effects of the MSP/STK signaling pathway on macrophage activation are similar to those observed for IL-10, we have demonstrated that STK does not enhance IL-10 production by peritoneal macrophages either alone or in combination with LPS. Futhermore, using peritoneal macrophages from IL-10 knockout mice, we have shown that the induction of arginase[20] and the inhibition of IL-12[40] by MSP are independent of IL-10. Alternatively, these signaling pathways may provide parallel but distinct functions in regulating macrophage activation during an immune response. For example, the expression of RTKs on macrophages could set a threshold which pro-inflammatory stimuli must overcome in order to induce classical macrophage activation, whereas the induction of IL-10 in response to LPS may provide an important negative feedback mechanism for limiting the extent and/or duration of this response. RTK signaling could ultimately inhibit LPS-induced NFκB activation and IFN-γ-induced Stat1 tyrosine phosphorylation through the induction of classical negative feedback pathways (figure 4). Suppressor of cytokine signaling (SOCS) proteins constitute a family of molecules that provide a negative feedback loop in the regulation of Jak/Stat signaling. IL-10 induces the Figure 4. Potential mechanisms by which RTKs could limit the activation of macrophages by IFN-γ and LPS. In other systems, RTKs have been shown to induce the tyrosine phosphorylation of Stat3, a critical negative regulator of macrophage activation, and induce expression of SOCS1 and SOCS3. Futhermore, RTKs have been shown to induce NFκB activation through a PI3K-dependent mechanism and to upregulate the expression of twist genes. Activation of these signaling pathways could induce negative-feedback inhibition of IFN-γ and LPS signaling. expression of SOCS1 and SOCS3 in a Stat3-dependent manner[50, 51] and studies of SOCS1 -/mice have demonstrated that SOCS1 is a critical regulator of both IFN-γ and LPS signaling in vivo[52, 53], while SOCS3 appears to prevent IFN-γ-like responses in cells stimulated with IL6[54, 55]. Several RTKs have also been shown to induce the expression of SOCS proteins, suggesting the possibility that RTK signaling could regulate macrophage activation through the induction of SOCS1 and SOCS3. RTK signaling, has been shown to activate NFκB signaling in several cell types through the PI3-kinase-, Akt-dependent phosphorylation of IKK[56, 57]. Conversely, PI3-kinase has been shown to be a negative regulator of iNOS expression in macrophages through sustained activation of NFκB[58], and the inhibition of iNOS by MSP has been shown to be PI3-kinase-dependent[59]. Furthermore, recent studies have demonstrated that PI3-kinase negatively regulates the production of IL-12 by dendritic cells and Th1 responses in vivo[60], and it has been suggested that PI3-kinase functions at the early phase of TLR signaling to modulate the magnitude of classical macrophage activation[61]. Negative feedback inhibition of the NFκB signaling pathway has also been demonstrated through the upregulation of IκB and, more recently, the p65-dependent expression of twist proteins. Twist 1 and Twist 2 are basic helix-loop-helix transcription factors that are upregulated in response to NFκB and studies from twist mutant mice demonstrate a role for twist 1 and 2 in the inhibition of p65 transactivation and the regulation of proinflammatory cytokine production in vivo[62]. Interestingly, the Met RTK has been shown to induce muscle differentiation through the upregulation of twist1 expression[63], and FGF2 induces expression of twist1 in osteoblasts[64]. 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-γ 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[65], 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[42]. While STK-deficient mice do not develop obvious signs of autoimmunity, the absence of STK on an MRL/Lpr background appears to exacerbate the pathogenesis of disease in these mice (Ray and Correll, unpublished observations). 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. 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