Lymphatic endothelial cell-targeting lipid nanoparticles delivering VEGFC mRNA improve lymphatic function after injury.

Dysfunction of the lymphatic system following injury, disease, or cancer treatment can lead to lymphedema, a debilitating condition with no cure. Advances in targeted therapy have shown promise for treating diseases where conventional therapies have been ineffective and lymphatic vessels have recently emerged as a new therapeutic target. Lipid nanoparticles (LNPs) have emerged as a promising strategy for tissue specific delivery of nucleic acids. Currently, there are no approaches to target LNPs to lymphatic endothelial cells, although it is well established that intradermal (ID) injection of nanoparticles will drain to lymphatics with remarkable efficiency. To design an LNP that would effectively deliver mRNA to LEC after ID delivery, we screened a library of 150 LNPs loaded with a reporter mRNA, for both self-assembly and delivery in vivo to lymphatic endothelial cells (LECs). We identified and validated several LNP formulations optimized for high LEC uptake when administered ID and compared their efficacy for delivery of functional mRNA with that of free mRNA and mRNA delivered with a commercially available MC3-based LNP (Onpattro™). The lead LEC-specific LNP was then loaded with VEGFC mRNA to test the therapeutic advantage of the LEC-specific LNP (namely, LNP7) for treating a mouse tail lymphatic injury model. A single dose of VEGFC mRNA delivered via LNP7 resulted in enhanced LEC proliferation at the site of injury, and an increase in lymphatic function up to 14-days post-surgery. Our results suggest a therapeutic potential of VEGFC mRNA lymphatic-specific targeted delivery in alleviating lymphatic dysfunction observed during lymphatic injury and could provide a promising approach for targeted, transient lymphangiogenic therapy.

Characterization of Atherosclerotic Mice Reveals a Sex-Dependent Susceptibility to Plaque Calcification but No Major Changes in the Lymphatics in the Arterial Wall.

Lymphatics participate in reverse cholesterol transport, and their presence in the arterial wall of the great vessels and prior experimental results suggest their possible role in the development of atherosclerosis. The aim of this study was to characterize the lymphatic vasculature of the arterial wall in atherosclerosis. Tissue sections and tissue-cleared aortas of wild-type mice unveiled significant differences in the density of the arterial lymphatic network throughout the arterial tree. Male and female Ldlr-/- and ApoE-/- mice on a Western diet showed sex-dependent differences in plaque formation and calcification. Female mice on a Western diet developed more calcification of atherosclerotic plaques than males. The lymphatic vessels within the aortic wall of these mice showed no major changes regarding the number of lymphatic junctions and end points or the lymphatic area. However, female mice on a Western diet showed moderate dilation of lymphatic vessels in the abdominal aorta and exhibited indications of increased peripheral lymphatic function, findings that require further studies to understand the role of lymphatics in the arterial wall during the development of atherosclerosis.

CCBE1 regulates the development and prevents the age-dependent regression of meningeal lymphatics.

Recent studies have described the importance of lymphatics in numerous organ-specific physiological and pathological processes. The role of meningeal lymphatics in various neurological and cerebrovascular diseases has been suggested. It has also been shown that these structures develop postnatally and are altered by aging and that the vascular endothelial growth factor C (VEGFC)/ vascular endothelial growth factor receptor 3 (VEGFR3) signaling plays an essential role in the development and maintenance of them. However, the molecular mechanisms governing the development and maintenance of meningeal lymphatics are still poorly characterized. Recent in vitro cell culture-based experiments, and in vivo studies in zebrafish and mouse skin suggest that collagen and calcium binding EGF domains 1 (CCBE1) is involved in the processing of VEGFC. However, the organ-specific role of CCBE1 in developmental lymphangiogenesis and maintenance of lymphatics remains unclear. Here, we aimed to investigate the organ-specific functions of CCBE1 in developmental lymphangiogenesis and maintenance of meningeal lymphatics during aging. We demonstrate that inducible deletion of CCBE1 leads to impaired postnatal development of the meningeal lymphatics and decreased macromolecule drainage to deep cervical lymph nodes. The structural integrity and density of meningeal lymphatics are gradually altered during aging. Furthermore, the meningeal lymphatic structures in adults showed regression after inducible CCBE1 deletion. Collectively, our results indicate the importance of CCBE1-dependent mechanisms not only in the development, but also in the prevention of the age-related regression of meningeal lymphatics. Therefore, targeting CCBE1 may be a good therapeutic strategy to prevent age-related degeneration of meningeal lymphatics.

The selective inhibition of the Syk tyrosine kinase ameliorates experimental autoimmune arthritis.

Autoimmune arthritis - such as rheumatoid arthritis - affect a significant proportion of the population, which can cause everyday joint pain, decreased mobility and reduced quality of life. Despite having more and more therapeutic options available, there are still a lot of patients who cannot reach remission or low disease activity by current therapies. This causes an urgent need for the development of new treatment options. The Syk tyrosine kinase plays an essential role in B cell receptor, Fc receptor and integrin signaling. It has been shown that the hematopoietic cell-specific deletion of Syk resulted in a complete protection against autoantibody-induced experimental arthritis. This prompted us to test the effect of entospletinib, a second generation, Syk-selective inhibitor, which has a tolerable safety profile according to hematological clinical trials, in experimental autoimmune arthritis. We found that entospletinib dose-dependently decreased the macroscopic signs of joint inflammation, while it did not affect the health status of the animals. In line with these findings, local neutrophil accumulation and cytokine levels were reduced compared to the vehicle-treated group, while macrophage accumulation and synovial fibroblast numbers were not significantly altered. Meanwhile, entospletinib dose-dependently decreased the cell responses of immune complex- or integrin ligand-activated neutrophils. Overall, we found that selective Syk inhibition by entospletinib reduced the activity of autoantibody-induced experimental arthritis, which seems to be based mainly on the effect of the inhibitor on neutrophil functions. Our data raise the possibility that entospletinib could be a good drug candidate in the treatment of human autoimmune arthritis.

Absence of Nkx2-3 induces ectopic lymphatic endothelial differentiation associated with impaired extramedullary stress hematopoiesis in the spleen.

The red and white pulps as two main parts of the spleen are arranged around distinct types of vasculature, and perform significantly different functions in both humans and mice. Previous observations indicated a profound alteration of the local vessel specialization in mice lacking Nkx2-3 homeodomain transcription factor, including contradictory results suggesting presence of an ectopic lymphatic vascular structure. Furthermore, how the absence of Nkx2-3 and the consequential changes in endothelial components affect the extramedullary hematopoietic activity restricted to the splenic red pulp is unknown. In this work, we investigated the role of Nkx2-3 homeodomain transcription factor as a major morphogenic determinant for vascular specification, and its effect in the extramedullary hematopoiesis following acute blood loss and pharmacological stimulation of megakaryocyte differentiation after treatment with thrombopoietin-receptor mimetic Romiplostim. We found that, in mice lacking Nkx2-3, Prox1-positive lymphatic capillaries containing gp38/CD31 double positive lymphatic endothelial cells develop, arranged into an extensive meshwork, while the Clever1-positive venous segments of red pulp blood vasculature are absent. This lymphatic endothelial shift is coupled with a severely compromised splenic erythropoiesis and a significantly reduced splenic megakaryocyte colony formation following Romiplostim treatment in mice lacking Nkx2-3. These findings indicate that the shift of microvascular patterning in the absence of Nkx2-3 includes the emergence of ectopic Prox1-positive lymphatic vessels, and that this pivoting towards lymph node-like vascular patterning is associated with an impaired reserve hematopoietic capacity of the splenic red pulp.

Fluorescence-Based Real-Time Analysis of Osteoclast Development.

Osteoclasts are multinucleated cells of hematopoietic origin which are critically involved in physiological and pathological bone resorption. They develop from myeloid progenitors through characteristic gene expression changes and intercellular fusion. This process is directed by M-CSF and RANKL which are also able to trigger osteoclast development from bone marrow cells in vitro. Osteoclasts are conventionally visualized by histochemical staining followed by manual counting, which hinders kinetic studies and automated quantification. Here we describe two fluorescence-based assays for the real-time analysis of myeloid cell to osteoclast development (FRAMCO) in primary mouse bone marrow cell cultures. Both assays rely on red-to-green fluorescence conversion of the membrane-targeted tdTomato/membrane-targeted eGFP (mTmG) transgene by Cre recombinase driven by the osteoclast-specific cathepsin K promoter (Ctsk-Cre). In the first assay (FRAMCO1.1), osteoclast-specific gene expression triggers red-to-green color conversion of cells carrying both the Ctsk-Cre and mTmG transgenes. In the second assay (FRAMCO1.2), red-to-green fluorescence conversion is triggered by fusion of neighboring co-cultured bone marrow cells separately carrying either the Ctsk-Cre or the mTmG transgenes. The two assays were tested using a high-content confocal fluorescence imaging system, followed by automated quantification. The FRAMCO1.1 assay showed robust red-to-green fluorescence conversion of more than 50% of the culture (including mononuclear cells) within 3 days under osteoclastogenic conditions. The FRAMCO1.2 assay showed a less robust but still readily measurable red-to-green color conversion in multinuclear cells within 5 days of differentiation. The assays required both the Ctsk-Cre and the mTmG transgenes and gave no signals in parallel macrophage cultures. The proper functioning of the two assays was also confirmed at the DNA, mRNA and bulk protein level. The assay systems were validated using lisophosphatidylcholine, a previously reported inhibitor of preosteoclast fusion. Taken together, our assays allow high-throughput automated real-time analysis of two critical aspects of osteoclast development, facilitating the screening for novel drug candidates for the pharmacological control of osteoclast-mediated bone resorption.

Foliate Lymphoid Aggregates as Novel Forms of Serous Lymphocyte Entry Sites of Peritoneal B Cells and High-Grade B Cell Lymphomas.

The cellular homeostasis of lymphoid tissues is determined by the continuous interactions of mobile hematopoietic cells within specialized microenvironments created by sessile stromal cells. In contrast to the lymph nodes and mucosal lymphoid tissues with well-defined entry and exit routes, the movement of leukocytes in the peritoneal cavity is largely unknown. In this study, we report that, in addition to the omental milky spots and fat-associated lymphoid clusters, in mice, the serous surface of the mesenteric adipose streaks contains lymphocyte-rich organoids comprised of a highly compacted leaf-like part connected to the adipose tissue that can also efficiently bind B cells and high-grade B cell lymphoma (diffuse large B cell lymphoma) cells. Denoted as foliate lymphoid aggregates (FLAgs), these structures show incomplete T/B segregation and a partially differentiated stromal architecture. LYVE-1-positive macrophages covering FLAgs efficiently bind i.p. injected normal B cells as well as different types of diffuse large B cell lymphoma cells. Within FLAgs, the lymphocytes compartmentalize according to their chemokine receptor pattern and subsequently migrate toward the mesenteric lymph nodes via the mesenteric lymphatic capillaries. The blood supply of FLAgs includes short vascular segments displaying peripheral lymph node addressin, and the extravasation of lymphocytes to the omental and mesenteric adipose tissues is partly mediated by L-selectin. The appearance of i.p. injected cells in mesenteric lymph nodes suggests that the mesentery-associated lymphatics may also collect leukocytes from the fat-associated lymphoid clusters and FLAgs, thus combining the mucosal and serous exit of mobile leukocytes and increasing the range of drainage sites for the peritoneal expansion of lymphoid malignancies.

Lymphatic mimicry in maternal endothelial cells promotes placental spiral artery remodeling.

Molecular heterogeneity of endothelial cells underlies their highly specialized functions during changing physiological conditions within diverse vascular beds. For example, placental spiral arteries (SAs) undergo remarkable remodeling to meet the ever-growing demands of the fetus - a process which is deficient in preeclampsia. The extent to which maternal endothelial cells coordinate with immune cells and pregnancy hormones to promote SA remodeling remains largely unknown. Here we found that remodeled SAs expressed the lymphatic markers PROX1, LYVE1, and VEGFR3, mimicking lymphatic identity. Uterine natural killer (uNK) cells, which are required for SA remodeling and secrete VEGFC, were both sufficient and necessary for VEGFR3 activation in vitro and in mice lacking uNK cells, respectively. Using Flt4Chy/+ mice with kinase inactive VEGFR3 and Vegfcfl/fl Vav1-Cre mice, we demonstrated that SA remodeling required VEGFR3 signaling, and that disrupted maternal VEGFR3 signaling contributed to late-gestation fetal growth restriction. Collectively, we identified a novel instance of lymphatic mimicry by which maternal endothelial cells promote SA remodeling, furthering our understanding of the vascular heterogeneity employed for the mitigation of pregnancy complications such as fetal growth restriction and preeclampsia.

Hematopoietic or Osteoclast-Specific Deletion of Syk Leads to Increased Bone Mass in Experimental Mice.

Syk is a non-receptor tyrosine kinase critically involved in signaling by various immunoreceptors including B-cell-receptors and activating Fc-receptors. We have previously shown that Syk also mediates immunoreceptor-like signals required for the in vitro development and function of osteoclasts. However, the perinatal lethality of Syk-/- mice precluded the analysis of the role of Syk in in vivo bone metabolism. To overcome that problem, we generated mice with osteoclast-specific (SykΔOC ) or hematopoietic (SykΔHaemo ) Syk deficiency by conditional deletion of Syk using Cre recombinase expressed under the control of the Ctsk or Vav1 promoter, respectively. Micro-CT analysis revealed increased bone trabecular density in both SykΔOC and SykΔHaemo mice, although hematopoietic Syk deficiency caused a more severe phenotype than osteoclast-specific Syk deficiency. Osteoclast-specific Syk deficiency reduced, whereas hematopoietic Syk deficiency completely blocked in vitro development of osteoclasts. Both interventions inhibited the resorptive activity of osteoclasts and osteoclast-specific gene expression. Kinetic analysis of Syk protein levels, Cre expression and the genomic deletion of the Sykflox allele revealed complete and early deletion of Syk from SykΔHaemo osteoclasts whereas Syk was incompletely deleted at a later stage of osteoclast development from SykΔOC cultures. Those results provide an explanation for the in vivo and in vitro difference between the SykΔOC and SykΔHaemo mutant strains and suggest late activation of, and incomplete target gene deletion upon, osteoclast-specific Cre expression driven by the Ctsk promoter. Taken together, our results indicate that Syk plays an indispensable role in osteoclast-mediated in vivo bone resorption and suggest that Syk-specific inhibitors may provide therapeutic benefit in inflammatory and other diseases characterized by excessive osteoclast-mediated bone resorption.

The Src family kinases Hck, Fgr, and Lyn are critical for the generation of the in vivo inflammatory environment without a direct role in leukocyte recruitment.

Although Src family kinases participate in leukocyte function in vitro, such as integrin signal transduction, their role in inflammation in vivo is poorly understood. We show that Src family kinases play a critical role in myeloid cell-mediated in vivo inflammatory reactions. Mice lacking the Src family kinases Hck, Fgr, and Lyn in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and skin blistering disease, as well as from the reverse passive Arthus reaction, with functional overlap between the three kinases. Though the overall phenotype resembled the leukocyte recruitment defect observed in ß2 integrin-deficient (CD18(-/-)) mice, Hck(-/-)Fgr(-/-)Lyn(-/-) neutrophils and monocytes/macrophages had no cell-autonomous in vivo or in vitro migration defect. Instead, Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fcγ receptor signal transduction. Our results suggest that infiltrating myeloid cells release proinflammatory chemokine, cytokine, and lipid mediators that attract further neutrophils and monocytes from the circulation in a CD18-dependent manner. Src family kinases are required for the generation of the inflammatory environment but not for the intrinsic migratory ability of myeloid cells.

Lymphatic function is required prenatally for lung inflation at birth.

Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants.

The secreted lymphangiogenic factor CCBE1 is essential for fetal liver erythropoiesis.

The secreted protein CCBE1 is required for lymphatic vessel growth in fish and mice, and mutations in the CCBE1 gene cause Hennekam syndrome, a primary human lymphedema. Here we show that loss of CCBE1 also confers severe anemia in midgestation mouse embryos due to defective definitive erythropoiesis. Fetal liver erythroid precursors of Ccbe1 null mice exhibit reduced proliferation and increased apoptosis. Colony-forming assays and hematopoietic reconstitution studies suggest that CCBE1 promotes fetal liver erythropoiesis cell nonautonomously. Consistent with these findings, Ccbe1(lacZ) reporter expression is not detected in hematopoietic cells and conditional deletion of Ccbe1 in hematopoietic cells does not confer anemia. The expression of the erythropoietic factors erythropoietin and stem cell factor is preserved in CCBE1 null embryos, but erythroblastic island (EBI) formation is reduced due to abnormal macrophage function. In contrast to the profound effects on fetal liver erythropoiesis, postnatal deletion of Ccbe1 does not confer anemia, even under conditions of erythropoietic stress, and EBI formation is normal in the bone marrow of adult CCBE1 knockout mice. Our findings reveal that CCBE1 plays an essential role in regulating the fetal liver erythropoietic environment and suggest that EBI formation is regulated differently in the fetal liver and bone marrow.

Phosphoinositide 3-OH kinase regulates integrin-dependent processes in neutrophils by signaling through its effector ARAP3.

ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased ß2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of ß2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3.

Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Phospholipase Cγ2 is required for basal but not oestrogen deficiency-induced bone resorption.

BACKGROUND: Osteoclasts play a critical role in bone resorption under basal conditions, but they also contribute to pathological bone loss during diseases including postmenopausal osteoporosis. Phospholipase Cγ2 (PLCγ2) is an important signalling molecule in diverse haematopoietic lineages. Here, we tested the role of PLCγ2 in basal and ovariectomy-induced bone resorption, as well as in in vitro osteoclast cultures using PLCγ2-deficient (PLCγ2(-/-) ) mice. MATERIALS AND METHODS: The trabecular architecture of long bone metaphyses was tested by micro-CT and histomorphometric analyses. Postmenopausal osteoporosis was modelled by surgical ovariectomy. Osteoclast development and function, gene expression and PLCγ2 phosphorylation were tested on in vitro osteoclast and macrophage cultures. RESULTS: PLCγ2(-/-) mice had significantly higher trabecular bone mass under basal conditions than wild-type mice. PLCγ2 was required for in vitro development and resorptive function of osteoclasts, but not for upregulation of osteoclast-specific gene expression. PLCγ2 was phosphorylated in a Src-family-dependent manner upon macrophage adhesion but not upon stimulation by M-CSF or RANKL. Surprisingly, ovariectomy-induced bone resorption in PLCγ2(-/-) mice was similar to, or even more robust than, that in wild-type animals. CONCLUSIONS: Our results indicate that PLCγ2 participates in bone resorption under basal conditions, likely because of its role in adhesion receptor signalling during osteoclast development. In contrast, PLCγ2 does not appear to play a major role in ovariectomy-induced bone loss. These results suggest that basal and oestrogen deficiency-induced bone resorption utilizes different signalling pathways and that PLCγ2 may not be a suitable therapeutic target in postmenopausal osteoporosis.

PI3Kß plays a critical role in neutrophil activation by immune complexes.

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the ß isoform of phosphoinositide 3-kinase (PI3Kß) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kß alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kß and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kß by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the ß isoform of PI3K. PI3Kß-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kß and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kß as a potential therapeutic target in inflammatory disease.

Genetic deficiency of Syk protects mice from autoantibody-induced arthritis.

OBJECTIVE: The Syk tyrosine kinase plays an important role in diverse functions in hematopoietic lineage cells. Although previous in vitro and pharmacologic analyses suggested Syk to be a possible player in the development of autoimmune arthritis, no in vivo genetic studies addressing that issue have yet been reported. The aim of the present study was to test whether genetic deficiency of Syk affects autoantibody-induced experimental arthritis in the K/BxN serum-transfer model. METHODS: Syk(-/-) bone marrow chimeras carrying a Syk-deficient hematopoietic system were generated by transplanting Syk(-/-) fetal liver cells into lethally irradiated wild-type recipients. After complete repopulation of the hematopoietic compartment, autoantibody-mediated arthritis was induced by injection of arthritogenic K/BxN serum. Arthritis development was monitored by macroscopic and microscopic observation of the ankle joints, micro-computed tomography of bone morphology, as well as a joint function assay. RESULTS: Genetic deficiency of Syk in the hematopoietic compartment completely blocked the development of all macroscopic and microscopic signs of arthritis. The Syk(-/-) mutation also prevented the appearance of periarticular bone erosions. Finally, Syk(-/-) bone marrow chimeras were completely protected from arthritis-induced loss of articular function. CONCLUSION: Our results indicate that Syk is critically involved in the development of all clinically relevant aspects of autoantibody-mediated K/BxN serum-transfer arthritis in experimental mice. These results provide the first in vivo genetic evidence of the role of Syk in the development of autoimmune arthritis.

Critical role of phospholipase Cgamma2 in integrin and Fc receptor-mediated neutrophil functions and the effector phase of autoimmune arthritis.

beta(2) integrins and Fcgamma receptors are critically involved in neutrophil activation at the site of inflammation. Both receptor types trigger a receptor-proximal tyrosine phosphorylation cascade through Src family kinases and Syk, but further downstream signaling events are poorly understood. We show that phospholipase C (PLC) gamma2 is phosphorylated downstream of Src family kinases and Syk during integrin or Fc receptor-mediated activation of neutrophils. PLCgamma2(-/-) neutrophils are completely defective in beta(2) integrin or Fcgamma receptor-mediated functional responses such as respiratory burst, degranulation, or cell spreading in vitro and show reduced adhesion/spreading in inflamed capillary venules in vivo. However, PLCgamma2(-/-) neutrophils respond normally to various other agonists, including chemokines, bacterial formyl peptides, Toll-like receptor ligands, or proinflammatory cytokines, and migrate normally both in vitro and in vivo. To confirm the in vivo relevance of these observations, the effect of the PLCgamma2(-/-) mutation was tested in the K/BxN serum transfer arthritis model, which is known to require beta(2) integrins, Fcgamma receptors, and neutrophils. PLCgamma2 deficiency completely protected mice from clinical signs and histological features of arthritis as well as from arthritis-induced loss of articular function. These results identify PLCgamma2 as a critical player of integrin and Fc receptor-mediated neutrophil functions and the neutrophil-mediated effector phase of autoimmune arthritis.

Syk-mediated translocation of PI3Kdelta to the leading edge controls lamellipodium formation and migration of leukocytes.

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in beta(2) integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during beta2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I(A). Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110delta of PI3K class I(A) as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110delta to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of beta2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110delta signaling for beta2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.

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 FcRgamma, were defective in integrin-mediated responses. Activation of the tyrosine kinase Syk by integrins required that DAP12 and FcRgamma 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.