Loss of neutrophil Shp1 produces hemorrhagic and lethal acute lung injury.

The acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1 knockout mice suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine-tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn.

Lipopolysaccharide (LPS) stimulates immune responses by interacting with the membrane receptor CD14 to induce the generation of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6. The mechanism by which the LPS signal is transduced from the extracellular environment to the nuclear compartment is not well defined. Recently, an increasing amount of evidence suggests that protein tyrosine kinases especially the Src-family kinases Hck, Fgr, and Lyn, play important roles in LPS signaling. To directly address the physiological function of Hck, Fgr and Lyn in LPS signaling, a genetic approach has been used to generate null mutations of all three kinases in a single mouse strain. hck-/-fgr-/-lyn-/- mice are moderately healthy and fertile; macrophages cultured from these mice express normal levels of CD14 and no other Src-family kinases were detected. Although the total protein phosphotyrosine level is greatly reduced in macrophages derived from hck-/-fgr-/-lyn-/- mice, functional analyses indicate that both elicited peritoneal (PEMs) and bone marrow-derived macrophages (BMDMs) from triple mutant mice have no major defects in LPS-induced activation. Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-alpha) are normal or even enhanced in hck-/-fgr-/-lyn-/- macrophages after LPS stimulation. The development of tumor cell cytotoxicity is normal in triple mutant BMDMs and only partially impaired in PEMs after LPS stimulation. Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription factor NF-kappaB, are the same in normal and mutant macrophages after LPS stimulation. The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not obligatory for LPS-initiated signal transduction.

Mapping of the Epstein-Barr virus and C3dg binding sites to a common domain on complement receptor type 2.

Complement receptor type 2 (CR2;CD21), a member of the superfamily of proteins containing short consensus repeats (SCRs), is the B cell receptor for both the gp350/220 envelope protein of Epstein-Barr virus (EBV), and for the C3dg protein of complement. By analysis of CR2 deletion mutants and chimeras formed with CR1 (CD35) we determined that of the 15 SCRs in CR2, the NH2-terminal two SCRs are necessary and sufficient to bind both gp350/220 and C3dg with affinities equivalent to those of the wild-type receptor. The epitope for OKB-7, a mAb that blocks binding of both EBV and C3dg and shares with these ligands B cell-activating capabilities, also requires both SCR-1 and SCR-2, whereas mAbs lacking these functions bind to other SCRs. Thus, EBV, a polyclonal activator of B cells, has selected a site that is proximate or identical to the natural ligand binding site in CR2, perhaps reflecting the relative immutability of that site as well as its signal transducing function.

Independent and opposing roles for Btk and lyn in B and myeloid signaling pathways.

Transphosphorylation by Src family kinases is required for the activation of Bruton's tyrosine kinase (Btk). Differences in the phenotypes of Btk-/- and lyn-/- mice suggest that these kinases may also have independent or opposing functions. B cell development and function were examined in Btk-/-lyn-/- mice to better understand the functional interaction of Btk and Lyn in vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk-/-lyn-/- mice. However, Btk-/-lyn-/- animals had a more severe immunodeficiency than Btk-/- mice. B cell numbers and response to T cell-dependent antigens were reduced. Btk and Lyn therefore play independent or partially redundant roles in the maintenance and function of peripheral B cells. Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn-/- mice. A transgene expressing Btk at approximately 25% of endogenous levels (Btklo) was crossed onto Btk-/- and Btk-/-lyn-/- backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.

Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn.

Macrophage Fcgamma receptors (FcgammaRs) mediate the uptake and destruction of antibody-coated viruses, bacteria, and parasites. We examined FcgammaR signaling and phagocytic function in bone marrow-derived macrophages from mutant mice lacking the major Src family kinases expressed in these cells, Hck, Fgr, and Lyn. Many FcgammaR-induced functional responses and signaling events were diminished or delayed in these macrophages, including immunoglobulin (Ig)G-coated erythrocyte phagocytosis, respiratory burst, actin cup formation, and activation of Syk, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinases 1 and 2. Significant reduction of IgG-dependent phagocytosis was not seen in hck(-)(/)-fgr(-)(/)- or lyn(-)(/)- cells, although the single mutant lyn(-)(/)- macrophages did manifest signaling defects. Thus, Src family kinases clearly have roles in two events leading to FcgammaR-mediated phagocytosis, one involving initiation of actin polymerization and the second involving activation of Syk and subsequent internalization. Since FcgammaR-mediated phagocytosis did occur at modest levels in a delayed fashion in triple mutant macrophages, these Src family kinases are not absolutely required for uptake of IgG-opsonized particles.

The molecular mechanism of B cell activation by toll-like receptor protein RP-105.

The B cell-specific transmembrane protein RP-105 belongs to the family of Drosophila toll-like proteins which are likely to trigger innate immune responses in mice and man. Here we demonstrate that the Src-family protein tyrosine kinase Lyn, protein kinase C beta I/II (PKCbetaI/II), and Erk2-specific mitogen-activated protein (MAP) kinase kinase (MEK) are essential and probably functionally connected elements of the RP-105-mediated signaling cascade in B cells. We also find that negative regulation of RP-105-mediated activation of MAP kinases by membrane immunoglobulin may account for the phenomenon of antigen receptor-mediated arrest of RP-105-mediated B cell proliferation.

Negative regulation of phagocytosis in murine macrophages by the Src kinase family member, Fgr.

Ingestion of opsonized pathogens by professional phagocytes results in the generation and release of microbicidal products that are essential for normal host defense. Because these products can result in significant tissue injury, phagocytosis must be regulated to limit damage to the host while allowing for optimal clearance and destruction of opsonized pathogens. To pursue negative regulation of phagocytosis, we assessed the effect of the Src kinase family member, Fgr, on opsonin-dependent phagocytosis by mouse macrophages. We chose Fgr because it is present in high concentrations in circulating phagocytes but is not essential for Fcgamma receptor-mediated ingestion by mouse macrophages. Although expression of Fgr both in a macrophage cell line and in primary macrophages significantly attenuates ingestion mediated by Fcgamma receptors and CR3, it does not affect macropinocytosis or receptor-mediated endocytosis. This selective effect of Fgr is independent of its tyrosine kinase function. After Fcgamma receptor cross-linking, Fgr becomes associated with the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, SIRPalpha (a member of the signal-regulatory protein family, also known as Src homology 2 domain-containing protein tyrosine phosphatase [SHP] substrate 1 [SHPS-1], brain immunoglobulin-like molecule with tyrosine-based activation motifs [BIT], and P84) and potentiates the association of the phosphatase SHP-1 with SIRPalpha. This association is responsible, at least in part, for decreasing positive signaling essential for optimal phagocytosis. These data demonstrate an important negative regulatory role for this Src kinase family member and suggest that this homeostatic function must be overcome for optimal uptake and clearance of opsonized pathogens.

Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions.

Cross-linking of the neutrophil-beta 2- or beta 3-related leukocyte response integrins by extracellular matrix (ECM) proteins or monoclonal antibodies (mAb) stimulates cytoskeletal rearrangement leading to cell spreading and respiratory burst. Tyrosin phosphorylation of a variety of proteins and activation of the Src family kinases within polymorphonuclear leukocytes (PMN) have recently been implicated in the intracellular signaling pathways generated by leukocyte integrins (Yan, S.R., L. Fumagalli, and G Berton. 1995. J. Inflammation. 45:217-311.) To directly test whether these functional responses are dependent on the Src family kinases p59/61hck and p58c-fgr, we examined adhesion-dependent respiratory burst in PMNs isolated from hck -/-, fgr -/-, and hck -/- fgr -/- knockout mice. Purified bone marrow PMNS from wild-type mice released significant amounts of O2- when adherent to fibrinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl-methionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hck-/-fgr-/- double-mutant mic, however, failed to respond. This defect was specific for integrin signaling, since respiratory burst was normal in hck-/-fgr-/-PMNs stimulated by immune complexes or PMA. Stimulation of respiratory burst was observed in TNF-primed wild-type PMN plated on surfaces coated with murine intracellular adhesion molecule-1 (ICAM-1), while hck-/-fgr-/- PMNs, failed to respond. Direct cross-linking of the subunits of beta 2 and beta 2 integrins by surface-bound mAbs was elicited O2- production by wild-type PMNs, while the double-mutant hck-/-fgr-/- cells failed to respond. Photomicroscopy and cell adhesion assays revealed that the impaired functional responses of hck-/-fgr-/- PMNs were caused by defective spreading and tight adhesion on either ECM protein- or mAb-coated surfaces. In contrast, hck-/-or fgr-/-single mutant cells produced O2- at levels equivalent to wild-type cells on ECM protein, murine ICAM-1, and antiintegrin mAb-coated surfaces. Hence, either p59/61 hck and p 58c-fgr is required for signaling through leukocyte beta 2 and beta 3 integrins leading to PMN spreading and respiratory burst. This is the first direct genetic evidence of the importance of Src family kinases in integrin signaling within leukocytes, and it is also the best example of overlapping function between members of this gene family within a defined signal transduction pathway.

Early Targeting of L-Selectin on Leukocytes Promotes Recovery after Spinal Cord Injury, Implicating Novel Mechanisms of Pathogenesis.

L-selectin, a lectin-like receptor on all leukocyte classes, functions in adhesive and signaling roles in the recruitment of myeloid cells from the blood to sites of inflammation. Here, we consider L-selectin as a determinant of neurological recovery in a murine model of spinal cord injury (SCI). Spinal cord-injured, L-selectin knock-out (KO) mice (male) showed improved long-term recovery with greater white matter sparing relative to wild-type (WT) mice and reduced oxidative stress in the injured cord at 72 h post-SCI. There was a partial and transient reduction in accumulation of neutrophils in the injured spinal cords of KOs at 24 h post-injury. To complement these findings with KO mice, we sought a pharmacologic means for lowering L-selectin levels. We found that diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induced the shedding of L-selectin from the cell surface of myeloid subsets, specifically neutrophils and non-classical monocytes, in the blood and the injured spinal cord. Diclofenac administration to injured WT mice enhanced neurological recovery to a level comparable to that of KOs but did not improve recovery in KOs. While diclofenac treatment had no effect on myeloid cell accumulation, there was a reduction in oxidative stress at 72 h post-SCI. These findings implicate L-selectin in secondary pathogenesis beyond a role in leukocyte recruitment and raise the possibility of repurposing diclofenac for the treatment of SCI.

Defective negative regulation of antigen receptor signaling in Lyn-deficient B lymphocytes.

BACKGROUND: To elucidate the role of the Src family kinase Lyn in B cell receptor (BCR) signaling, we and others previously generated lyn-/- mice and analyzed their B cell responses. Although the initiation of BCR signaling in lyn-/- B cells is delayed, BCR-induced ERK2 activation and proliferation are enhanced. As the co-receptors Fc gamma RIIb1 and CD22 have been shown to be negative regulators of BCR signaling, we have now examined their functional roles in lyn-/- B cells. RESULTS: B cells from lyn-/- mice have increased expression of the protein product of the early response gene egr-1, enhanced activation of Jun N-terminal kinase (JNK), and elevated calcium responses upon BCR cross-linking. Tyrosine phosphorylation of Fc gamma RIIb1 in lyn-/- B cells was reduced but negative regulation of the BCR signal by Fc gamma RIIb1 was only modestly impaired. In contrast, tyrosine phosphorylation of CD22 was greatly decreased in lyn-/- B cells, correlating with the inability of CD22 to downregulate the BCR-induced calcium response in these cells. Surprisingly, CD22 remains capable of regulating the ERK2 and JNK pathways in lyn-/- B cells, which may relate to the small residual increase in BCR-induced CD22 phosphorylation. CONCLUSIONS: BCR signal initiation and negative regulation by Fc gamma RIIb1 is not critically dependent on Lyn. In contrast, Lyn plays a particularly important role in the tyrosine phosphorylation of CD22 and in the consequent inhibition of BCR-induced calcium influx. The net result of the Lyn deficiency in B cells is hyperresponsiveness to antigen stimulation, which may explain the autoimmunity observed in lyn-/- mice.

Lupus-like kidney disease in mice deficient in the Src family tyrosine kinases Lyn and Fyn.

Systemic lupus erythematosus (SLE) is an autoimmune disease whose cause is poorly understood. Mice rendered deficient in specific genes have served as useful animal models in deciphering the genetic control of the disease [1]. We [2] and others [3, 4] previously demonstrated that mice deficient in the Src family tyrosine kinase Lyn developed a mild lupus-like disease with high survival rates. During the course of investigating the functional interaction of Src family kinases, we generated a mouse strain deficient in both Lyn and Fyn. The double-mutant mice died at relatively young ages and developed a severe lupus-like kidney disease. Unlike the double-mutant mice, single mutants deficient in either Lyn or Fyn lived longer and had distinct subsets of the symptoms found in the former. Lyn deficiency led to high levels of autoantibody production and glomerulonephritis, as previously reported [2--4], whereas loss of Fyn contributed to proteinuria by a B and T lymphocyte-independent mechanism. Our data suggest that the severe kidney disease in the double-mutant mice results from a combination of immunological and kidney-intrinsic defects. This new animal model may be informative about the causes of human SLE.

Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport.

Src-family kinases play a central role in regulation of hematopoietic cell functions. We found that mouse erythrocytes express the Src-family kinases Fgr and Hck, as well as Lyn. To directly test whether Fgr and Hck play any role in erythrocyte function, we analyzed red cells isolated from fgr-/-, hck-/-, and fgr-/- hck-/- knock-out mice. Mean corpuscular hemoglobin concentration and median density are increased, while K content is decreased, in fgr-/- hck-/- double-mutant erythrocytes compared with wild-type, fgr-/-, or hck-/- erythrocytes. Na/K pump and Na/K/Cl cotransport were not altered, but K/Cl cotransport activity was significantly and substantially higher (approximately three-fold) in fgr-/- hck-/- double-mutant erythrocytes. This enhanced K/Cl cotransport activity did not depend on cell age. In fact, in response to bleeding, K/Cl cotransport activity increased in parallel with reticulocytosis in wild-type erythrocytes, while abnormal K/Cl cotransport did not change as a consequence of reticulocytosis in fgr-/- hck-/- double-mutant erythrocytes. Okadaic acid, an inhibitor of a phosphatase that has been implicated in activation of the K/Cl cotransporter, inhibited K/Cl cotransport in wild-type and fgr-/- hck-/- double-mutant erythrocytes to a comparable extent. In contrast, staurosporine, an inhibitor of a kinase that has been suggested to negatively regulate this same phosphatase enhanced K/Cl cotransport in wild-type but not in fgr-/- hck-/- double-mutant erythrocytes. On the basis of these findings, we propose that Fgr and Hck are the kinases involved in the negative regulation of the K/Cl cotransporter-activating phosphatase. Abnormality of erythrocyte K/Cl cotransport in fgr-/- hck-/- double-mutant animals represents the first demonstration that Src-family kinases may be involved in regulation of membrane transport.

Integrin signalling in neutrophils and macrophages.

Integrins have been characterized extensively as adhesion receptors capable of transducing signals inside the cell. In myelomonocytic cells, integrin-mediated adhesive interactions regulate different selective cell responses, such as transmigration into the inflammatory site, cytokine secretion, production or reactive oxygen intermediates, degranulation and phagocytosis. In the last few years, great progress has been made in elucidating mechanisms of signal transduction by integrins in neutrophils and macrophages. This review summarises the current information on the role of integrins in regulating myelomonocytic cell functions and highlights the signalling pathways activated by integrin engagement in these cells. Also, exploiting the current knowledge of mechanisms of integrin signal transduction in other cell types, we propose a model to explain how integrins transduce signals inside neutrophils and macrophages, and how signaling pathways leading to regulation of selective cell functions may be coordinated.

Integrin signal transduction in myeloid leukocytes.

Integrin-mediated adhesion serves as a powerful costimulus for neutrophil activation. Clustering of integrins at the leukocyte membrane by interaction with surface-bound ligands (extracellular matrix proteins or endothelial cell counter-receptors) leads to a number of signaling events that culminate in actin cytoskeletal rearrangement and neutrophil functional responses such as migration, degranulation, and respiratory burst. Although the signaling reactions elicited by integrin ligation are complex and the relative contribution of each pathway to neutrophil function is unclear, a large body of evidence suggests that activation of tyrosine kinases is a very proximal event in these signaling cascades. This review summarizes the role of adhesion in activating neutrophil functional properties and the contribution of leukocyte tyrosine kinases to regulation of integrin signaling in myeloid cells. Significant advances in our understanding of leukocyte integrin signaling have been afforded by studies using knockout mice lacking members of the Src-family of tyrosine kinases normally expressed in myeloid cells. These studies have demonstrated that these kinases (Hck, Fgr, and Lyn) are not required for myeloid cell development or for many of the functional properties of myeloid cells but are critical in controlling integrin-mediated signaling events. Absence of these kinases results in impaired adhesion-dependent neutrophil activation both in vivo and in vitro. These studies suggest that leukocyte-specific tyrosine kinases may be good therapeutic targets for controlling myeloid cell-dependent inflammatory disease.

Role of Src kinases and Syk in Fcgamma receptor-mediated phagocytosis and phagosome-lysosome fusion.

Phagocytosis is increased by Fcgamma receptors (FcgammaRs), and studies with syk(-/-) macrophages demonstrated that Syk kinase is required for FcgammaR phagocytosis. Similar studies with macrophages lacking the Src family kinases Hck, Fgr, and Lyn showed that these kinases are not required for phagocytosis but that they enhance the rate of particle engulfment. In this report we show that both wild-type and hck(-/-)fgr(-/-) macrophages expressed Fyn, Src, and Yes and that these kinases were activated on ingestion of immunoglobulin G (IgG)-coated particles and redistributed, together with Syk, to actin-rich phagocytic cups and the phagosomal membrane. At doses blocking IgG-dependent phagocytosis, the tyrosine kinase inhibitors PP1 and piceatannol inhibited both Src family kinase and Syk activities, as well as their redistribution to actin-rich phagocytic cups. Hck, Fgr, and Lyn were dispensable for lysosome-phagosome fusion (PLF) induced by IgG-coated particles. However, PP1 or piceatannol hampered unopsonized yeast-induced PLF despite the fact that they did not block yeast internalization.

Phosphatidylinositol 3-kinase and mTOR mediate lipopolysaccharide-stimulated nitric oxide production in macrophages via interferon-beta.

Bacterial lipopolysaccharide (LPS) elicits responses by macrophages that help the body repel infections. Recent evidence indicates that phosphatidylinositol 3-kinase (PI 3-kinase) may mediate some of these responses. Here, we show that exposing macrophages to LPS rapidly increased membrane-associated PI 3-kinase activity and also elevated p70 S6 kinase activity. Inhibitors of PI 3-kinase or the mammalian target of rapamycin (mTOR) fully blocked p70 S6 kinase activation, implying that this kinase is controlled by PI 3-kinase and mTOR. These inhibitors also substantially reduced LPS-induced nitric oxide (NO) production. This inhibition was, in part, attributable to impaired LPS-stimulated secretion of interferon-beta, an autocrine co-factor for NO production. However, the addition of exogenous interferon-beta did not fully restore NO production, indicating that the NO response was being inhibited by another mechanism as well. Together, these data suggest that PI 3-kinase, mTOR, and possibly p70 S6 kinase mediate LPS-induced NO production by regulating the secretion of interferon-beta and by a second undefined mechanism.

Regulation of synthesis of amyloid A-related protein.

The concentration of serum amyloid A polypeptide (SAAL) increases greatly during the acute phase responses to infection or inflammation. We find that SAAL synthesis comprises 2.5% of murine hepatic protein synthesis after lipopolysaccharide (LPS) administration, but much less in normal liver. SAAL messenger RNA (mRNA) in liver increases at least 500-fold above the normal level. A recombinant plasmid homologous to SAAL mRNA has been isolated, as has most of the mouse genome DNA encoding the plasmid's nucleotide sequence. This gene is transcribed into RNA much more frequently after LPS administration than it is in normal liver. In a number of other mammalian genes, cytosine methylation is inversely related to the rate of transcription. Methylation of CCGG sequences in hepatic DNA homologous to the recombinant plasmid has been examined. Little or no change is found after LPS administration. This suggests that other factors are responsible for the increase in SAAL mRNA in the acute phase response.

Distinct and overlapping functional roles of Src family kinases in mouse platelets.

BACKGROUND AND OBJECTIVES: Src family kinases (SFKs) play a critical role in initiating and propagating signals in platelets. The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbß3, and in vivo. METHODS AND RESULTS: Mouse platelets express four SFKs, Fgr, Fyn, Lyn and Src, with Lyn expressed at a considerably higher level than the others. Using mutant mouse models, we demonstrate that platelet activation by collagen-related peptide (CRP) is delayed and then potentiated in the absence of Lyn, but only marginally reduced in the absence of Fyn or Fgr, and unaltered in the absence of Src. Compound deletions of Lyn/Src or Fyn/Lyn, but not of Fyn/Src or Fgr/Lyn, exhibit a greater delay in activation relative to Lyn-deficient platelets. Fibrinogen-adherent platelets show reduced spreading in the absence of Src, potentiation in the absence of Lyn, but no change in the absence of Fyn or Fgr. In mice double-deficient in Lyn/Src or Fgr/Lyn, the inhibitory role of Lyn on spreading on fibrinogen is lost. Lyn is the major SFK-mediating platelet aggregation on collagen at arterial shear and its absence leads to a reduction in thrombus size in a laser injury model. CONCLUSION: These results demonstrate that SFKs share individual and overlapping roles in regulating platelet activation, with Lyn having a dual role in regulating GPVI signaling and an inhibitory role downstream of αIIbß3, which requires prior signaling through Src.

Evaluation of the physiological significance of botrocetin/ von Willebrand factor in vitro signaling.

BACKGROUND: A signaling pathway is difficult, if not impossible, to elucidate in platelets using only in vivo studies. Likewise, the physiological significance of signaling information obtained exclusively from in vitro observations is unknown. Therefore, both in vitro and in vivo experiments are required to establish the physiological significance of a signaling pathway. OBJECTIVE: To evaluate the physiological significance of signaling data obtained from botrocetin (bt)/von Willebrand factor (VWF)-stimulated washed platelets. METHOD: Stable thrombus formation in response to FeCl(3)-induced injury of the mouse carotid artery was used to evaluate the physiological significance of signaling data obtained from bt/VWF-stimulated washed platelets. RESULTS: Syk, PLCgamma2, Galphaq and P2Y12, but not LAT, were found either to be required for or to affect stable thrombus formation. Prior in vitro studies had demonstrated that LAT is not required for bt/VWF-induced platelet aggregation in the presence of exogenous fibrinogen. These data provide the first demonstration of the in vivo role for these signaling molecules in GPIb-dependent/initiated signal transduction and are consistent with the signaling pathway deduced from in vitro studies of bt/VWF-stimulated washed platelets using metabolic inhibitors and knockout mice. CONCLUSION: The broad agreement between the in vitro and the in vivo results establish that bt/VWF stimulation of washed platelets can provide physiologically significant glycoprotein Ib-dependent/initiated signaling data.

Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr.

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61(hck) (Hck) and p58(c-fgr) (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink beta2 and beta3 integrins. To determine if the defective function of hck-/-fgr-/- neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck-/-fgr-/- mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor alpha and interleukin 1alpha were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.