Clare L. Abram

The Ins and Outs of Leukocyte Integrin Signaling

Integrins are the principal cell adhesion receptors that mediate leukocyte migration and activation in the immune system. These receptors signal bidirectionally through the plasma membrane in pathways referred to as inside-out and outside-in signaling. Each of these pathways is mediated by conformational changes in the integrin structure. Such changes allow high-affinity binding of the receptor with counter-adhesion molecules on the vascular endothelium or extracellular matrix and lead to association of the cytoplasmic tails of the integrins with intracellular signaling molecules. Leukocyte functional responses resulting from outside-in signaling include migration, proliferation, cytokine secretion, and degranulation. Here, we review the key signaling events that occur in the inside-out versus outside-in pathways, highlighting recent advances in our understanding of how integrins are activated by a variety of stimuli and how they mediate a diverse array of cellular responses.

Integrin signaling in neutrophils and macrophages uses adaptors containing immunoreceptor tyrosine-based activation motifs

At sites of inflammation, ligation of leukocyte integrins is critical for the activation of cellular effector functions required for host defense. However, the signaling pathways linking integrin ligation to cellular responses are poorly understood. Here we show that integrin signaling in neutrophils and macrophages requires adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs). Neutrophils and macrophages lacking two ITAM-containing adaptor proteins, DAP12 and FcRγ, were defective in integrin-mediated responses. Activation of the tyrosine kinase Syk by integrins required that DAP12 and FcRγ were first phosphorylated by Src family kinases. Retroviral transduction of neutrophils and macrophages with wild-type and mutant Syk or DAP12 demonstrated that the Src homology 2 domains of Syk and the ITAM of DAP12 were required for integrin signaling. Our data show that integrin signaling for the activation of cellular responses in neutrophils and macrophages proceeds by an immunoreceptor-like mechanism.

PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcRγ to induce slow leukocyte rolling

E-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) can activate the β2 integrin lymphocyte function-associated antigen-1 by signaling through spleen tyrosine kinase (Syk). This signaling is independent of Gαi-protein–coupled receptors, results in slow rolling, and promotes neutrophil recruitment to sites of inflammation. However, the signaling pathways linking E-selectin engagement of PSGL-1 to Syk activation are unknown. To test the role of Src family kinases and immunoreceptor tyrosine-based activating motif (ITAM)–containing adaptor proteins, we used different gene-deficient mice in flow chamber, intravital microscopy, and peritonitis studies. E-selectin–mediated phosphorylation of Syk and slow rolling was abolished in neutrophils from fgr−/− or hck−/− lyn−/− fgr−/− mice. Neutrophils from Tyrobp−/− Fcrg−/− mice lacking both DAP12 and FcRγ were incapable of sustaining slow neutrophil rolling on E-selectin and intercellular adhesion molecule-1 and were unable to phosphorylate Syk and p38 MAPK. This defect was confirmed in vivo by using mixed chimeric mice. Gαi-independent neutrophil recruitment into the inflamed peritoneal cavity was sharply suppressed in Tyrobp−/− Fcrg−/− mice. Our data demonstrate that an ITAM-dependent pathway involving the Src-family kinase Fgr and the ITAM-containing adaptor proteins DAP12 and FcRγ is involved in the initial signaling events downstream of PSGL-1 that are required to initiate neutrophil slow rolling.

Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice

Since the first example of conditional gene targeting in mice in 1994, the use of Cre recombinase and loxP flanked sequences has become an invaluable technique to generate tissue and temporal specific gene knockouts. The number of mouse strains expressing floxed-gene sequences, and tissue-specific or temporal-specific Cre-recombinase that have been reported in the literature has grown exponentially. However, increased use of this technology has highlighted several problems that can impact the interpretation of any phenotype observed in these mouse models. In particular, accurate knowledge of the specificity of Cre expression in each strain is critical in order to make conclusions about the role of specific cell types in the phenotypes observed. Cre-mediated deletion specificity and efficiency have been described in many different ways in the literature, making direct comparisons between these Cre strains impossible. Here we report crossing thirteen different myeloid-Cre mouse strains to ROSA-EYFP reporter mice and assaying YFP expression in a variety of naïve unstimulated hematopoietic cells, in parallel. By focusing on myeloid subsets, we directly compare the relative efficiency and specificity of myeloid deletion in these strains under steady-state conditions.

The Expanding Role for ITAM-Based Signaling Pathways in Immune Cells

The immunoreceptor tyrosine-based activation motif (ITAM) is the primary signaling domain used by classical immunoreceptors, such as the antigen receptors on B and T lymphocytes and the Fc receptors (FcRs) on myeloid cells. The ITAM is contained in the intracellular region of subunits associated with these receptors, often in pairs, or is part of the cytoplasmic domain of the receptors themselves. Data from many investigators have demonstrated that ITAMs are both necessary and sufficient for initiation of signaling downstream of all immunoreceptors. More recent reports indicate that ITAM signaling is used by additional receptors beyond the classical immunoreceptors: Cell adhesion molecules (integrins and PSGL-1), chemokine receptors (CXCR4), plexins, and lectin receptors all mediate immune cell function through ITAM-like signaling pathways. This convergence of intracellular signaling pathways in leukocytes illuminates the importance of tyrosine-based activation motifs in the immune system and suggests that inhibitors of ITAM signaling may have broader effects than originally envisioned. Cells of the immune system express a diverse number of cell surface receptors that recognize a diverse set of stimuli. Stimulation of these receptors by exogenous molecules initiates biochemical events in the immune cell that activate its ability to kill invading pathogens, produce antibodies, or secrete inflammatory cytokines. Understanding how these intracellular signals are transmitted from the cell surface is critical for intervening when dysregulation of these pathways leads to immune dysfunction, such as in autoimmune or inflammatory diseases. Although immune cells use various receptors to recognize exogenous stimuli, current research suggests that many of these receptors engage a common downstream intracellular signaling pathway. Central to this pathway are transmembrane adapter proteins that contain a dual tyrosine-containing motif (the immunoreceptor tyrosine-based activation motif, or ITAM) in their cytoplasmic tails. This protein domain serves as a target for protein phosphorylation events by tyrosine kinases that initiate the common downstream pathway. This STKE Review, with two figures, one table, and 39 references, describes how ITAM-dependent signaling pathways are used by a broad array of cell surface receptors.

Convergence of immunoreceptor and integrin signaling

Summary:  A common signaling pathway is known to operate downstream of immunoreceptors, such as the T‐cell, B‐cell, or Fc receptors, following engagement by their respective ligands. This pathway involves Src family kinase‐mediated tyrosine phosphorylation of immunoreceptor tyrosine‐based activation motifs (ITAMs) that recruit and activate spleen tyrosine kinase (Syk) or Zap70 (ζ‐associated protein of 70 kDa) kinases, which in turn activate a variety of downstream signals. Evidence has been building from a variety of sources, particularly mouse models, that molecules involved in the immunoreceptor signaling pathway are also required for signals initiated by integrins. Integrins are the major cell surface receptors that mediate adhesion of leukocytes to a variety of extracellular matrix proteins and counter‐receptors expressed on endothelial cells. Integrin ligation is a critical step in the activation of leukocyte effector functions (such as neutrophil degranulation or lymphocyte proliferation). Integrin signaling through pathways common to those utilized by immunoreceptors provides a mechanism by which leukocyte adhesion can regulate activation of cellular responses. In animal models, integrin‐mediated signal transduction plays a critical role in inflammatory disease. In this review, we discuss the convergence of immunoreceptor and integrin signaling, focusing on how these pathways modulate leukocyte activation.

Distinct Roles for Neutrophils and Dendritic Cells in Inflammation and Autoimmunity in motheaten Mice

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.

The diverse functions of Src family kinases in macrophages.

Macrophages are key components of the innate immune response. These cells possess a diverse repertoire of receptors that allow them to respond to a host of external stimuli including cytokines, chemokines, and pathogen-associated molecules. Signals resulting from these stimuli activate a number of macrophage functional responses such as adhesion, migration, phagocytosis, proliferation, survival, cytokine release and production of reactive oxygen and nitrogen species. The cytoplasmic tyrosine kinase Src and its family members (SFKs) have been implicated in many intracellular signaling pathways in macrophages, initiated by a diverse set of receptors ranging from integrins to Toll-like receptors. However, it has been difficult to implicate any given member of the family in any specific pathway. SFKs appear to have overlapping and complementary functions in many pathways. Perhaps the function of these enzymes is to modulate the overall intracellular signaling network in macrophages, rather than operating as exclusive signaling switches for defined pathways. In general, SFKs may function more like rheostats, influencing the amplitude of many pathways.

Leukocyte adhesion deficiency syndrome: a controversy solved.

Sadly, mistakes of nature are often our greatest learning tools. Such is the case with the recent description by three independent groups of the molecular etiology of leukocyte adhesion deficiency (LAD) syndrome III.1–3 The discovery that mutations in kindlin-3 are responsible for this rare genetic disorder teaches us a great deal about how leukocytes regulate adhesion and trafficking through their primary adhesive receptors—the b1-, b2and b3-integrin receptors. This story also teaches us how mistakes can be made (and avoided) in science. There are three types of LAD syndromes, LADI, II and III (also referred to as variant LADI).4 As the name implies, these diseases are characterized by an inability of leukocytes to adhere and migrate during inflammatory and host defense reactions. As a result, patients with these autosomal recessive syndromes present with repeated bacterial and fungal infections in the absence of pus formation. Hundreds of LADI patients have been described worldwide, all with mutations in the gene encoding the b2 leukocyte integrin. Less than 10 patients have been described with LADII, which is a clinically milder form of the disease, and is caused by mutations in a fucose transporter protein leading to poor formation of Sialyl Lewis X (CD15), the ligand for L-selectin. LADIII is also rare (approximately 20 patients worldwide, with the largest kindred of Turkish origin), but presents with both the immunodeficiency of LADI as well as severe bleeding disorders resembling Glanzman’s thrombasthenia—a known deficiency of the platelet aIIbb3-integrin. Unlike LADI, the characterized LADIII patients have normal cell surface expression of all the major leukocyte integrins; however, these receptors fail to become activated to mediate leukocyte and platelet adhesion and cell spreading. Hence, these patients must have a defect in the intracellular signaling pathways that regulate integrin activation. The signaling networks that regulate b1-, b2and b3-integrin function are complex.5 In resting leukocytes and platelets, the integrins are held in a bent/inactive conformation that limits their ability to interact with ligands (endothelial ICAMs or extracellular matrix proteins such as fibrinogen or collagen). This conformation is maintained by a salt bridge between the cytoplasmic tails of the a and b chains of the integrin heterodimer, and through association with proteins such as talin. Following cellular activation (by agonists including chemokines, growth factors, antigens or selectin ligands), the cytoplasmic tails of the integrins separate, transmitting a conformational change throughout the integrin heterodimer that results in unfolding of the extracellular domains to allow high-affinity ligand binding. The processes that mediate integrin activation are referred to as ‘inside–out’ signaling (detailed in Figure 1). The fact that LADIII patients fail to activate high-affinity integrin ligand binding led Alon et al.7 to focus on each of the molecules involved in the inside–out pathway. Initial reports suggested that Rap1 failed to become activated in leukocytes from several patients. The group investigated the upstream regulator of Rap1, CalDAG-GEF1, and in fact they identified a potential mutation in a splice acceptor site for exon16 of the CalDAGGEF1 gene in two of the LADIII patients.6 Leukocytes from these patients had low levels of CalDAG-GEF1 mRNA and the protein was not detected in cell lysates. The patients showed complete failure to activate b1 and b2-integrins on leukocytes, as well as b3integrins on platelets. Altogether, this explains the full clinical spectrum of infections and bleeding suffered by these children.8 The fact that CalDAG-GEF1-deficient mice manifest an overall syndrome extremely similar to human LADIII9 buttressed this group’s assertion that LADIII is caused by loss of CalDAG-GEF1 leading to impaired Rap1 activation and failure to activate leukocyte integrins. This view was reflected in a number of reviews of integrin signaling, including our own.5 Yet, when more LADIII patients were examined, in particular patients from other families, impairment of Rap1 activation and reduced CalDAG-GEF1 expression was not observed.3,10 Whereas some of these patients had the splice acceptor ‘mutation’ described by Alon’s group, others did not. These disparate results initiated the controversy as to the molecular nature of LADIII syndrome. To identify the genetic defect in these patients Kuijpers et al.1 carried out a complete homozygosity mapping experiment on a larger number of LADIII samples, using single nucleotide polymorphism oligonucleotide arrays. Although these experiments localized the causative genetic mutation to a region containing the CalDAG-GEF1 gene, not all patients had the splice acceptor mutation and no defects in CalDAG-GEF1 expression were found. Instead mutations were mapped to the FERMT3 gene, which encodes kindlin-3, located a mere 500 kb away from the CalDAG-GEF1 gene. Mutations creating premature stop codons (Arg509X, Arg573X and Trp229X) were found in the different families. Leukocytes from patients with these mutations lacked kindlin-3 protein by immunoblotting. These results appear to rule out CL Abram and CA Lowell are at the Department of Laboratory Medicine, University of California, San Francisco, CA, USA. E-mail: Clifford.lowell@ucsf.edu Immunology and Cell Biology (2009), 1–3 & 2009 Australasian Society for Immunology Inc. All rights reserved 0818-9641/09

DAP12 Is Required for Macrophage Recruitment to the Lung in Response to Cigarette Smoke and Chemotaxis toward CCL2

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