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The response of innate immune cells to growth factors, immune complexes, extracellular matrix proteins, cytokines, pathogens, cellular damage, and many other stimuli is regulated by a complex net of intracellular signal transduction pathways. The majority of these pathways are either initiated or modulated by Src-family or Syk tyrosine kinases present in innate cells. The Src-family kinases modulate the broadest range of signaling responses, including regulating immunoreceptors, C-type lectins, integrins, G-protein-coupled receptors, and many others. Src-family kinases also modulate the activity of other kinases, including the Tec-family members as well as FAK and Pyk2. Syk kinase is required for initiation of signaling involving receptors that utilize immunoreceptor tyrosine activation (ITAM) domains. This article reviews the major activating and inhibitory signaling pathways regulated by these cytoplasmic tyrosine kinases, illuminating the many examples of signaling cross talk between pathways.
Innate immune cells, including macrophages, dendritic cells, granulocytes, and mast cells, function as the first line of defense against pathogens. These cells use a dizzying array of cell-surface receptors, which are connected to an equally complicated intracellular signal transduction network, to sense pathogen molecules and then orchestrate the appropriate immune response. Among the intracellular signaling molecules that are most crucial for innate immune cells are the cytoplasmic tyrosine kinases. Two major kinase families that operate in the proximal intracellular signaling pathways in innate cells are the Src-family kinases and the Syk-ZAP70 family. A third family of kinases, the Tek family, also have important roles in innate cells. They are not discussed in detail in this article, but are reviewed elsewhere in articles on the subject.
There are eight members of the Src family; innate immune cells primarily express Hck, Fgr, Lyn, and to a lesser extent, Src (Lowell 2004). The Syk-ZAP70 family has only two members and only Syk is found in innate cells. Most innate cell types express the same spectrum of kinases with some specific cellular differences. For example, mast cells express a broader range of Src-family kinases than macrophages or dendritic cells (Colgan and Hankel 2010). In general, Src-family and Syk kinases tend to operate together in signaling pathways, with the Src-family being “upstream” or activated first in response to pathogen detection. These enzymes then communicate downstream to Tec-family members. The Tec-family kinases expressed in innate cells include Btk, Bmx, and Tec (Koprulu and Ellmeier 2009; Tohyama and Yamamura 2009). Additionally, Src-family kinases activate yet another family of PTKs, the FAK/Pyk2 tyrosine kinases, which play a major role in integrin signaling (Hauck et al. 2000).
Though primarily studied in activating pathways, Src-family and Syk kinases also activate inhibitory signaling pathways (Nimmerjahn and Ravetch 2008). In many situations, inhibitory signaling often overrides the activating signal. Pathways can also be initiated at different times or rates. Finally, to add even more complexity, activating and inhibitory pathways often interact indirectly, for example, through the production of cytokines and growth factors and not through direct intracellular biochemical interactions; Hence the term signaling “cross talk,” which now appears commonly in the literature (O'Neill 2008; Ivashkiv 2009; Page et al. 2009).
In the prototypical immunoreceptor pathway, engagement of the receptor leads to activation of Src-family kinases, which in turn phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present on either the receptor or associated subunits (Fig. 1). This leads to recruitment of Syk, by binding of the Syk SH2 protein domain to the phospho-ITAM residues, and activation of Syk allowing it to phosphorylate downstream substrates. One of the enzymes activated downstream is phosphoinositide 3-kinase (PI3-kinase), which generates membrane-associated phosphatidylinositol (3,4,5)-triphosphate (PIP3). The FAK/Pyk2 kinases are activated directly via the Src-family kinases, where they contribute to downstream responses involving cell adhesion and migration. Though usually depicted as a linear signaling pathway with Src-kinases at the top and FAK/Pyk2 at the bottom, there are many points of interaction and cross regulation. Together, these pathways impinge on downstream factors, such as MAPK kinases, which have broad effects on gene transcription; the Rac/Rho pathway to modulate cytoskeletal function; the inositol trisphosphate (IP3), and diacylglycerol pathway (DAG), which regulates Ca2+ entry into cells and activation of various isoforms of protein kinase C (PKC). Overall, the outline of the prototypical immunoreceptor pathway as described here is similar in both innate and adaptive immune cells (Smith-Garvin et al. 2009; Kurosaki et al. 2010).
Recent and exciting developments in the immunoreceptor paradigm include recent progress in delineating how this pathway is connected to NF-κB and the demonstration that many innate immune receptors utilize the “immunoreceptor pathway” even though they lack ITAMs and therefore are not technically immunoreceptors.
The adapter protein CARD9, which contains a caspase-recruitment domain (CARD), has now been shown to be the link between a variety of ITAM-containing receptors involved in recognition of fungal and probably other pathogen structures and NF-κB (Fig. 2) (Gross et al. 2006; Gross et al. 2009). CARD9 is closely related to the lymphocyte protein CARMA-1, which forms a complex with the adapter proteins Bcl-10 and MALT1, and thus links the T- and B-cell receptors to the NF-κB pathway (Rawlings et al. 2006). CARD9 forms the same complex in innate cells. In lymphoid cells, CARMA-1 is activated by PKC isoforms (PKCθ in T-cells and PKCβ in B-cells), which phosphorylate CARMA-1, resulting in a conformational change that allows it to interact with IKK, leading to IκB turnover and NF-κB activation. In myeloid cells, it remains unclear if CARD9 activation is directly downstream of PKC activation (Hara and Saito 2009). Nevertheless, it is clear that in the absence of CARD9, receptors involved in fungal pathogen recognition (Dectin-1, Dectin-2) are unable to activate NF-κB, and more importantly, the entire repertoire of ITAM-containing receptors in myeloid cells are uncoupled from NF-κB (Robinson et al. 2006; Yamasaki et al. 2008). This results in profound defects in cytokine responses, which in vivo translates to poor responses to pathogens, specifically fungi such as Candida albicans, bacteria such as Listeria monocytogenes, and M. tuberculosis (Ruland 2008; Dorhoi et al. 2010).
Immunoreceptor signaling has also recently been shown to play a role downstream of innate receptors that lack ITAM sequences. This concept evolved from studies of innate cells lacking the ITAM-signaling adapters DAP12 and the FcεRIγ chain (referred to as FcRγ) (Nimmerjahn and Ravetch 2008; Lanier 2009). Typically, DAP12 and FcRγ are coupled to immunoreceptors through charged amino acid interactions within the transmembrane regions of each protein (Fig. 1). Surprisingly, a number of nonimmunoreceptor pathways, such as neutrophil integrin signaling or IL-3 responses in basophils, are lost in innate cells derived from mice lacking DAP12 or FcRγ (Mocsai et al. 2006; Hida et al. 2009). Examples of receptors that co-opt the classical immunoreceptor pathway are shown in Figure 1. The mechanisms by which these nonimmunoreceptors couple to DAP12 and/or FcRγ to initiate signaling remain unclear.
Most inhibitory signals are mediated in a fashion very similar to the immunoreceptor pathway, except that inhibitory pathways are initiated through phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) defined by the sequence amino acids I/V/L/SxYxxL/V (Fig. 3) (Munitz 2010). The Src-family kinase Lyn is primarily responsible for ITIM phosphorylation. Phospho-ITIM domains serve as docking sites for several types of phosphatases, such as SHP-1 (Src homology 2 domain containing protein tyrosine phosphatase-1/2) or SHIP-1 (SH-2-containing inositol phosphatase), which then down-modulate signaling responses by dephosphorylating downstream substrates. Like the activating pathways, this inhibitory pathway was first worked out in lymphocytes and NK cells, mainly through the study of the inhibitory Fc receptor, FcγRIIb (Ravetch and Lanier 2000). The number of newly recognized ITIM-containing inhibitory receptors in innate immune cells has grown to include a number of Ig superfamily molecules (PIR-B, Sirp-1α), sialic binding proteins, referred to as Siglecs, and a few C-type lectin receptors (Robinson et al. 2006; Crocker et al. 2007). In many cases, the ligands for these inhibitory receptors are unknown. In a broad sense, the inhibitory receptors function by limiting innate immune cell functions, including proliferation, cytokine responses, and cell adhesion. Since inhibitory pathways are often engaged at the same time as the activating pathways, the resulting cellular response depends on a balance between the activating signal and the inhibitory signal. Mutagenesis studies suggest that inhibitory signaling is usually dominant; for example, innate immune cells from Lyn kinase-deficient mice display hyperproliferative responses to cytokines and a hyperadhesive phenotype that correlates with the lack of phosphatase activation in these cells (reviewed by Scapini et al. 2009).
There is increasing evidence that ITAM-coupled activating receptors also contribute to inhibitory signaling (Fig. 3). This was first demonstrated in mast cell experiments that compared engagement of IgE-FcεR receptors by low versus high affinity haptens (Torigoe et al. 1998). The low affinity haptens suppressed the signaling responses. A similar observation was made by Pasquier et al., who found that a Fab fragment of an anti-FcαRI mAb inhibited IgG-mediated phagocytosis in a transfected macrophage cell line, suggesting that low affinity or partial activation of one ITAM-containing receptor can inhibit the function of another ITAM-containing immunoreceptor (Pasquier et al. 2005). Subsequently, this same group demonstrated that monovalent engagement of FcαRI inhibits chemotaxis to MCP-1 and TNF, as well as downstream signal transduction, in an SHP-1-dependent fashion, resulting in the attenuation of tissue injury in an Ab-induced glomerulonephritis model (Kanamaru et al. 2008). This has led to the model whereby low affinity or partial (i.e., monomeric) engagement of activating receptors results in only modest ITAM phosphorylation events, leading to the recruitment of phosphatases (SHP-1 in particular) instead of Syk kinase (Pinheiro da Silva et al. 2008), resulting in an overall diminution of cellular response (Fig. 3).
The activation of ITAM signaling pathways also functions to inhibit other signaling responses, such as TLR signaling (Hamerman et al. 2009). In particular, both macrophages and dendritic cells derived from DAP12 and FcRγ-deficient mice display increased responses to various TLR ligands, such as CpG, endotoxin (LPS), or yeast zymosan (Hamerman et al. 2005; Chu et al. 2008). In macrophages, this inhibitory signaling may be mediated through TREM-2, which is a DAP12 coupled receptor thought to be involved in recognition of various bacterial structures and potentially apoptotic cells (Hamerman et al. 2006; Takahashi et al. 2007; Hsieh et al. 2009; N'Diaye et al. 2009). Additional FcRγ and DAP12-associated receptors on both mouse and human plasmacytoid DCs have also been found to inhibit interferon production following TLR stimulation (Blasius et al. 2004; Fuchs et al. 2005; Swiecki and Colonna 2007).
The molecular mechanisms by which ITAM-based pathways cross-inhibit TLR responses remain unclear (reviewed by Ivashkiv 2008). In addition to phosphatase recruitment, activation of PI3-kinase-mediated pathways that both suppress NF-κB activation and alter recruitment of MyD88 and Mal adaptors to TLRs have been proposed (Kagan and Medzhitov 2006). Activation of calcineurin by ITAM signaling, leading to dephosphorylation of TLR signaling pathway molecules, may also be involved (Kang et al. 2007). Recently, Han et al. have provided an elegant model for ITAM-mediated down regulation of TLR responses (Han et al. 2010). Building on the observation that Mac-1-deficient mice (lacking the integrin αm protein that dimerizes with the β2 chain to form Mac-1) demonstrate increased inflammatory responses to dextran sodium sulfate-induced colitis (Abdelbaqi et al. 2006), this group and others (Wang et al. 2010) found that strong ligation of β2 integrins in macrophages and dendritic cells induces resistance to subsequent stimulation by various TLR ligands. Since signaling through β2 integrins proceeds in a DAP12/FcRγ to Syk pathway, Han et al. (2010) investigated potential intracellular Syk substrates. They found that Syk phosphorylates the TLR adapter proteins MyD88 and TRIF on specific tyrosine residues (Y277 and Y375, respectively), and that phosphorylation of these residues results in recruitment of the E3 ubiquitin ligase Cbl-b, which targets both the adapter proteins and Syk kinase itself for ubiquitin-mediated proteolysis, thus attenuating TLR responses (reviewed by Means and Luster 2010). This unique pathway provides a direct intracellular mechanism for signaling cross talk between ITAM and TLR pathways. It is likely that under physiologic situations of high affinity integrin ligation, such as during diapedisis through vascular endothelium, signaling through this pathway limits innate immune cell activation and reduces inadvertent immunopathology.
While most studies have focused on intracellular mechanisms for inhibitory signaling, there is growing recognition that immunoreceptor activation can also be limited by post-transcriptional events. Following engagement of ITAM pathways in macrophages or dendritic cells, production of inhibitory cytokines such IL-10 and TGF-β can lead to subsequent down-modulation of signaling, both for the ITAM pathway itself and for other pathways, such as the TLRs. For example, engagement of the C-type lectin receptor DC-SIGN by ManLAM, a cell-wall component present in Mycobacterium tuberculosis, down-modulates subsequent stimulation of DCs by LPS (Geijtenbeek et al. 2003). The mechanism involves acetylation of the p65 subunit of NF-κB, leading to prolonged and increased production of IL-10, which in turn inhibits LPS and other TLR responses (Gringhuis et al. 2007). More recently, IL-10 production by neutrophils, following activation through C-type lectin receptors or other ITAM-Syk pathways, has been found to have an immunomodulatory effect on subsequent inflammatory responses (Zhang et al. 2009). Hence, in states of chronic mycobacterial infection, engagement of the C-type lectin/Syk pathway leads to enough neutrophil-derived IL-10 to limit responses of macrophages and DCs. Thus, neutrophil depletion can sometimes increase the immunopathology associated with infection. Similarly, high affinity engagement of β2 integrins on human macrophages can lead to sufficient production of IL-10 to down-modulate subsequent TLR stimulation (Wang et al. 2010). In all of these cases, the activating stimulation (either through the C-type lectins or the β2 integrins) needs to be present for hours to days to allow for sufficient production of IL-10 and other inhibitors. For integrin signaling, this indirect pathway is probably additive with the direct biochemical mechanism of inhibitory cross talk via MyD88/TRIF phosphorylation, but would dominate during prolonged periods of integrin activation potentially to limit chronic inflammation.
The history of tyrosine kinase involvement in innate immune responses to pathogen molecules (LPS, bacterial cell wall components, and foreign DNA) is long and confusing. Many of these studies pre-date the identification of the Toll-like receptors (TLRs) as the major receptors involved in recognition of pathogens. These studies were based on the findings that LPS treatment of innate immune cells increases overall protein tyrosine phosphorylation and various tyrosine kinase inhibitors, particularly those that broadly target Src-family kinases, and could block cellular responses to LPS (Orlicek et al. 1999; Smolinska et al. 2008). The hypothesis that TLRs could activate Src-family kinases were refuted by the finding that macrophages lacking Hck, Fgr, and Lyn displayed normal (or even enhanced) responses to LPS treatment (Meng and Lowell 1997). Indeed, recent studies have suggested that Lyn kinase functions as a negative regulator of TLR responses, based on the finding that Lyn-deficient macrophages show increased cytokine responses to TLR4 and TLR2 stimulation (Keck et al. 2010). The mechanism by which Lyn functions as an inhibitor of TLR responses is still unclear (Kagan and Medzhitov 2006).
The Src-family kinases make up the largest family of cytoplasmic tyrosine kinases expressed in innate cells. Of the nine Src-family kinases, most are present in one type of innate cell or another, with Hck, Fgr, and Lyn kinase being most heavily expressed in monocytes, macrophages, granulocytes, and DCs. These kinases have been implicated as primary signaling molecules downstream of a host of immune cell receptors, including immunoreceptors, cytokine receptors, integrins, and various pathogen receptors (TREMs and Dectins, as examples). Inhibitor studies and use of knockout mice have demonstrated a critical role for Src-family kinases in a variety of host defense and inflammatory conditions (Okutani et al. 2006; Abram and Lowell 2008; Ingley 2008). In some cases, these kinases play clear and direct roles in innate immune signaling, in both activating and inhibitory pathways (Figs. 1 and and3),3), but in other cases their role may be more indirect (for example, by modulating cytokine responses that impact TLR pathways). The Src-family kinases are also responsible for phosphorylation and direct activation of other cytoplasmic tyrosine kinases, in particular Tec-family and FAK/Pyk2 and less directly Syk; hence Src-family members are often at the top of most signaling pathways in innate cells.
The most well-understood function of these kinases is in the classical immunoreceptor activating (ITAM) pathway (Fig. 1) utilized by many immunoreceptors (Fc receptors, NK activating receptors, and pathogen recognition molecules such as TREM family members), where various Src-family members are known to phosphorylate residues on the receptor associated ITAM adapter to initiate downstream signaling responses. As stated above, the major new finding in this area has been the demonstration that many innate immune receptors that are not classical immunoreceptors in fact utilize this same ITAM-mediated mechanism for intracellular signaling, and hence loss of Src-family activity directly affects these pathways as well. The best example is the integrin pathway—loss of Src-family kinase activity (or removal of the ITAM adapters FcRγ and DAP12, the substrates of the kinases) results in a complete deficiency of β1, β2, and β3 integrin function in innate cells (Abram and Lowell 2009). In all of these pathways, it is uncertain how the Src-family kinases are activated following ligand binding by the receptor. Dephosphorylation of the regulatory C-terminal tyrosine in Src-family kinases, which activates these enzymes, occurs through the action of either receptor tyrosine phosphatases such as CD45 or CD148, or potentially other cytoplasmic tyrosine phosphatases (LMW-PTP and Lyp/PEP, as examples) (Hermiston et al. 2009; Saunders and Johnson 2010; Zambuzzi et al. 2010); however, whether these phosphatases are directly recruited to the signaling receptors remains unclear.
Src-family kinases also play important roles in pathways where ITAM/immunoreceptor molecules are not involved. A number of studies suggest that Src-family kinases work with Jak kinases in supporting cytokine responses, either by phosphorylation of receptor subunits or potentially phosphorylating Stat molecules (Reddy et al. 2000; Hayakawa and Naoe 2006). For the classical growth factor receptors, such as the G-CSF or the GM-CSF receptor, Src-kinases have been found to be physically associated with the receptor through SH3/receptor interactions involving membrane proximal regions (Sampson et al. 2007; Perugini et al. 2010). In other cytokine responses, Src-family kinases are thought to act more downstream of the receptor, often through interactions with TRAF signaling molecules. Such is the case in the RANK/RANKL signaling response, where interaction of Src and TRAF6 lead to enhancement of downstream responses (particularly activation of PI3-kinase activity and downstream Akt function) (Leibbrandt and Penninger 2008). Formation of TRAF6/Src signaling complexes has also been reported downstream of IL-1 and TNFα signaling pathways, as well as in CD40/CD40L interactions, again with the implication that this complex is involved in stimulating downstream PI3-kinase signaling (Mukundan et al. 2005; Wang et al. 2006).
Src-family kinases have also been shown to be involved in IL-6 signaling pathways, through a direct interaction with the IL-6 receptor signaling protein gp130 (Hallek et al. 1997; Hausherr et al. 2007). This signaling function of Src-family kinases may be particularly important in myeloma cells, where IL-6 serves as a growth factor for these cells. A number of studies have shown involvement of Src-family kinases downstream of the M-CSF receptor in macrophages and myeloid progenitors, with their function being to couple receptor activation to downstream PI3-kinase pathways (Lee and States 2000; Bourgin-Hierle et al. 2008). As in the case of the IL-3 receptor, the M-CSF receptor pathway may also co-opt ITAM-containing adapters to activate downstream functions, since DAP12 has been recently found to be required for optimal M-CSF receptor signaling (Zou et al. 2008). Finally, as in the immunoreceptor pathways, not all the signaling functions of Src kinases in cytokine pathways are activating, since the deficiency of Lyn kinase actually leads to enhanced responses to some cytokines, such as G-CSF, potentially through reduced recruitment of phosphatases (Mermel et al. 2006). The mechanism through which Lyn kinase functions as an inhibitor, while other Src-family members function as activators of cytokine signaling, remains unclear.
Several Src-family kinases have been implicated in regulating chemokine or chemoattractant receptor signaling, which are mediated by G-protein-coupled receptors (GPCRs) (Luttrell and Luttrell 2004). Lyn kinase is activated in macrophages downstream of both CXCR4 and CCR5, and is thought to couple these GPCRs to the MAPK and PI3-kinase pathways (Ptasznik et al. 2002; Tomkowicz et al. 2006; Cheung et al. 2009). Inhibitor studies have also placed Src-family kinases downstream of monocyte-chemoattractant protein (MCP-1) signaling via CCR2 (Arefieva et al. 2005) and IL-8 signaling via CXCR1 (Sai et al. 2008). Similarly, neutrophils derived from hck−/−fgr−/− double mutant mice show significant functional defects following formyl peptide (fMLF) stimulation, which involves signaling through GPCR coupled formyl peptide receptors (Fumagalli et al. 2007). But even in the GPCR signaling pathway, the same paradigm of Src-kinases acting both in a positive and inhibitory fashion seems to be established. While Hck/Fgr-deficient neutrophils have reduced responses to foryml peptide stimulation, they manifest hyper-responsive signaling to chemokines that signal through CCR1 and CXCR2 (Zhang et al. 2005). This inhibitory function in the CCR1/CXCR2 pathways involves phosphorylation of the ITIM-containing inhibitory receptor PIR-B, which in turn is involved in recruiting tyrosine phosphatases that modulate downstream responses from the receptors.
Src-family kinases have been implicated in the innate immune cell response to selectin engagement, primarily recognition of E-selectin by leukocyte PSGL-1 and CD44 counter-receptors. Engagement of these receptors leads to activation of tyrosine phosphorylation that is inhibited by PP2 and other relative Src-family-kinase-specific inhibitors (Hidari et al. 1997; Kumar et al. 2001). The Fgr kinase seems dominant in this signaling response (Zarbock et al. 2008), although there is clearly some redundancy with other Src-family members (Yago et al. 2010). However, like the integrin pathway, it appears that selectin recognition also depends on ITAM adapters, since this signaling is reduced in DAP12-deficient neutrophils (Zarbock et al. 2008).
A number of GPI-linked proteins in innate cells are known to signal through Src-family kinases, including TLR4-associated CD14 and the urokinase plasminogen activator receptor (uPA-R) (Stefanova et al. 1993; D'Alessio and Blasi 2009). In innate immune cells (mainly macrophages), engagement of these receptors leads to increased tyrosine phosphorylation and subsequent signaling events that affect adhesion and migration. It is likely that clustering of these receptors leads to aggregation of lipid raft membrane structures, through the GPI linkages in the receptors, which in turn brings the kinases together, since they too are located in the raft structures.
Besides their function in TLR pathways, Tec-family kinases also play important roles in downstream immunoreceptor signaling, where their activation is mediated both through PIP3 generation by PI3-kinase and by direct phosphorylation by Src-family kinases. Monocytes from patients with Bruton's agammaglobulinemia show reduced uptake of both Ig and complement opsonized particles (Amoras et al. 2003; Jongstra-Bilen et al. 2008). Studies in macrophage cell lines suggest that Btk and Tec communicate to the actin polymerization pathway, since these kinases localize to phagocytic cups near sites of actin polymerization. Btk and Tec also play a significant role in macrophage/osteoclast RANK/RANKL signaling. Remarkably, like Src or Src/Hck-deficient mice, the double mutant Tec−/−Btk−/− mice have severe osteopetrosis due to impaired osteoclast maturation (Shinohara et al. 2008). Since it is known that RANK signaling is mediated, in part, through DAP12 (Koga et al. 2004), the effect of Tec/Btk deficiency may reflect impaired activation of downstream actin polymerization responses.
Based on studies in nonimmune cells, Src-family kinases can directly modulate the activity of FAK/Pyk2 kinases (Fig. 1). FAK is widely expressed in most cells of the body, while Pyk2 is expressed mainly in the nervous system, T-cells, and various innate cells. FAK kinase has been very extensively studied in fibroblasts and various tumor cell types, where it plays an important role in cell adhesion signaling downstream of integrin activation (Tomar and Schlaepfer 2009). More limited studies of Pyk2 suggest a similar function. Both of these kinases are substrates for Src-family kinases following integrin ligation. Phosphorylation of FAK or Pyk2 by Src-family members leads to protein unfolding and activation of their enzymatic activity. In innate cells, FAK and Pyk2 are found in podosomes, which are the main contact sites in leukocytes (Calle et al. 2006). Macrophages lacking FAK exhibit elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, and elevated basal Rac1 activity, leading to a marked inability to form stable lamellipodia necessary for directional locomotion (Okigaki et al. 2003; Owen et al. 2007a). These defects point to an alteration in integrin outside-in signaling, consistent with the role of this kinase in fibroblasts and tumor cells. As a result of this defect, recruitment of FAK-deficient monocytes to sites of inflammation is impaired. A similar phenotype is observed in macrophages derived from Pyk2-deficient animals (Okigaki et al. 2003). The involvement of FAK and Pyk2 in macrophage integrin signaling may also affect bacterial phagocytosis by these receptors, since siRNA-mediated knockdown of FAK and/or Pyk2 can reduce uptake of various Yersina strains of bacteria (Owen et al. 2007b). FAK deficiency also reduces neutrophil adhesion, migration, and antibacterial uptake, again potentially through defects in integrin-mediated signaling events (Kasorn et al. 2009). Inhibition of Pyk2 function in neutrophils, through use of inhibitory peptides transduced into the cells, reduces cell adhesion and spreading following integrin activation, again suggesting a similar role for this kinase in neutrophil integrin signaling (Han et al. 2003). Pyk2 has also been implicated in the integrin signaling responses leading to cytokine (in particular IL-10) production (Wang et al. 2010). Despite these initial studies, the specific individual functions of FAK versus Pyk2 in innate immune integrin signaling remains to be defined.
The repertoire of signaling pathways that Syk has been implicated in (reviewed by Mocsai et al. 2010) is more restricted than Src-family kinases. Syk kinase is activated by engagement of its two SH2 domains by phospho-ITAM domains; hence it functions only in ITAM-like pathways. This includes, of course, the novel “inhibitory ITAM” pathway described above. It is likely that Syk is only involved in the subset of signaling pathways that also depend on Src-family kinases. However, this doesn't mean that blockade of Syk kinases always produces the same phenotype in a given immunoreceptor signaling response that blockade of Src-family members does. For example, the defect in macrophage phagocytosis in Src-family-kinase-deficient cells is different than in Syk-deficient cells. Lack of Src-family kinases produces a moderate to severe defect in FcR-mediated particle uptake due to a reduction in initial actin polymerization at the phagocytic cup (Fitzer-Attas et al. 2000). However, Syk-deficient cells show a complete block in FcR-mediated particle uptake due to a block in fusion of the arms of the phagocytic cup, a step subsequent to actin polymerization events (Crowley et al. 1997). Hence, it is likely that the more proximal Src-family kinases signal to many other pathways besides just ITAM-mediated Syk activation. Finally, it should be noted that Syk has been implicated in some pathways in which the role of Src-family kinases are unknown. This is particularly true for the C-type lectin receptors in innate cells.
Syk kinase plays a critical role in all innate immune cell signaling pathways involving ITAM or ITAM-like signaling adapters. This includes signaling from classical immunoreceptors (FcRs, TREMs, Dectin-2, and others), nonimmunoreceptors that co-opt the ITAM adapters for signaling (integrins, selectins, IL-3 receptor), and those C-type lectin receptors that have hemi-ITAM sequences embedded in their C-terminal tails (Dectin-1) (Fig. 1). Since many of the C-type lectin and other immunoreceptor-like pathways converge on Syk, loss of Syk activity produces a more profound block than loss of any specific receptor (LeibundGut-Landmann et al. 2007). These same C-type lectin receptors also are involved in innate immune recognition of mycobacterial and viral pathogen molecules, all of which are affected by Syk blockade in either macrophages or dendritic cells (Chen et al. 2008; Werninghaus et al. 2009).
In addition to signaling downstream through CARD9 to NF-κB, Syk-dependent signaling events have been linked to activation of intracellular pattern recognition receptors, which in turn activate the inflammasome complex leading to IL-1β production. For example, activation of the Nod-like receptor protein NLRP3 requires upstream Syk activity during innate immune cell responses to fungal molecules (Gross et al. 2009). Similarly, Syk-deficient dendritic cells fail to respond to monosodium urate crystals (present in the joints of gout patients), a pathway also known to require NLRP3 (Ng et al. 2008; Martinon 2010). It remains unclear how Syk activation is coupled to the NLRP3/inflammasome complex.
In contrast to Src-family kinases, it is likely that Syk does not signal in cytokine or GPCR-linked pathways. Lack of Syk has no impact on neutrophil, macrophage, or mast cell recognition of various cytokine growth factors or GPCR agonists, such as formyl peptides, ATP, or other agents (Mocsai et al. 2003).
The roles of cytoplasmic tyrosine kinases in innate immune responses are complex, ranging from direct signaling involvement in very defined pathways (such as Syk in C-type lectin receptor signaling) to more diffuse interactions (Src-family kinases somehow regulating GPCR responses) to indirect secondary effects (Src-family and Syk function in immunoreceptor pathways leading to IL-10 production that feeds back on TLR-mediated responses). It is clear that Src-family members have the broadest effects on overall signaling, while Syk has more defined roles. This diffuse, versus very defined, functional role is mirrored in the fact that deficiencies in single Src-family kinases tend to produce limited signaling defects in innate cells, while loss of Syk has very defined broad functional effects. Because of redundancy, studying the role of Src-kinases in any pathway often requires blocking multiple family members, after which one often finds that many signaling responses are affected. This overall broad function for Src-family members is analogous to how a rheostat controls lighting: It dials up and dials down responses in a graded fashion. In contrast, Syk acts more like a signaling switch: It is critically required in an absolute way in a limited number of pathways.
When viewed in this fashion, it becomes obvious that if we are to design therapeutics that target these kinases for use in inflammatory or autoimmune disease, we are better off focusing on the switch kinases (Syk) rather than the rheostat kinases (Src-family), since targeting the former will produce defined effects. Indeed, a number of companies are close to producing/releasing highly active Syk kinase inhibitors that have strong potential for treatment of immune-mediated disease (Cohen and Fleischmann 2010; Colonna et al. 2010). In contrast, most of the Src-family inhibitors produced and used clinically so far are rather broadly acting, and hit enzymes besides just Src-family members. This may be useful for when treating cancer, which is where most of the anti-Src kinase drugs have been used (Kim et al. 2009), but is likely problematic for chronic treatment of immune-mediated disease. Moreover, distinguishing between individual Src-family members may be chemically impossible to achieve. Nevertheless, given our ever-expanding understanding of kinase-mediated signaling pathways in innate cells, it is quite likely that in the very near future we will see highly active drugs that block these pathways, resulting potentially in clinical benefit.
CAL is supported by NIH grants AI065495 and AI068150. We thank Clare Abram, Patrizia Scapini, and Chrystelle Lamagna for critical reading of the manuscript.
Editors: Lawrence E. Samelson and Andrey S. Shaw
Additional Perspectives on Immunoreceptor Signaling available at www.cshperspectives.org