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.

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.

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.

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.

Non-immunogenic murine hepatocellular carcinoma Hepa1-6 cells expressing the membrane form of macrophage colony stimulating factor are rejected in vivo and lead to CD8+ T-cell immunity against the parental tumor.

Hepatocellular carcinoma is a lethal disease and methods that develop effective cellular-based immunotherapy are needed. We retrovirally transduced non-immunogenic mouse Hepa1-6 hepatoma cells with the gene encoding the membrane form of macrophage colony stimulating factor (mM-CSF). Excess recombinant M-CSF and phagocytosis-inhibiting chemicals blocked macrophage-mediated killing of cloned mM-CSF transfected Hepa1-6 hepatoma cells. Macrophages derived from Hck(-/-)Fgr(-/-) and Lyn(-/-) triple knockout mice, which are incapable of performing phagocytosis, failed to kill the mM-CSF transduced cells. The mM-CSF transfected tumor clones failed to grow when injected into C57BL/6 or C57L/J mice. Splenocytes from these vaccinated mice displayed cytotoxicity against parental Hepa1-6 cells, but not against B16 and CT-26 tumor cells in vitro. Mice that rejected the mM-CSF transfected Hepa1-6 tumor subsequently rejected parental Hepa1-6 cells but not the B16 melanoma cells when rechallenged. Elimination of the CD8+ effector cells by an anti-CD8 antibody and complement treatment prevented the adoptive transfer of anti-Hepa1-6-specific immunity into naive animals. Thus, mM-CSF provides a method of generating effective anti-tumor immune responses by macrophages and cytotoxic T cells against the parental Hepa1-6 cells. Our work suggests that mM-CSF transduced hepatoma cells could be used as a tumor vaccine to stimulate immune responses against hepatocellular carcinoma.

Inhibition of beta 2 integrin receptor and Syk kinase signaling in monocytes by the Src family kinase Fgr.

While beta 2 integrin ligand-receptor recognition interactions are well characterized, less is known about how these events trigger signal transduction cascades to regulate the transition from tethering to firm adhesion, spreading, and transendothelial migration. We have identified critical positive and negative regulatory components of this cascade in monocytes. Whereas the Syk tyrosine kinase is essential for beta 2 integrin signaling and cell spreading, the Src family kinase Fgr is a negative regulator of this pathway. Fgr selectively inhibits beta 2 but not beta 1 integrin signaling and Syk kinase function via a direct association between the Fgr SH2 domain and Syk tyrosine Y342. The inhibitory effects of Fgr are independent of its kinase activity, are dose dependent, and can be overcome by chemokines and inflammatory mediators.

A CD19-dependent signaling pathway regulates autoimmunity in Lyn-deficient mice.

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.

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.

Fyn and Lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway.

Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor gamma (FcR gamma chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn(-/-) platelets, tyrosine phosphorylation of FcR gamma chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn(-/-) platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and alpha-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn(-/-)lyn(-/-) double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLC gamma 2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway. (Blood. 2000;96:4246-4253)

CD19 regulates Src family protein tyrosine kinase activation in B lymphocytes through processive amplification.

CD19 regulates constitutive and antigen receptor-induced signaling thresholds in B lymphocytes through its unique cytoplasmic domain. Herein, we demonstrate a novel molecular mechanism where interactions between CD19 and Lyn amplify basal and antigen receptor-induced Src family kinase activation. Lyn expression was required for CD19 tyrosine phosphorylation in primary B cells. Experiments with purified proteins demonstrated that CD19-Y513 was Lyn's initial phosphorylation and binding site. This led to processive phosphorylation of CD19-Y482, which recruited a second Lyn molecule, allowing for transphosphorylation and amplification of Lyn activation. In vivo, CD19 deficiency impaired, and CD19 overexpression enhanced, Lyn kinase activity. Thus, CD19 functions as a specialized adapter protein for the amplification of Src family kinases that is crucial for intrinsic and antigen receptor-induced signal transduction.

Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation.

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (FcepsilonRI). In this study, we have made the following observations on growth properties and FcepsilonRI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, FcepsilonRI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-gamma1 and C-gamma2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, FcepsilonRI-induced elevation of intracellular Ca2+ concentrations and activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-alpha and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase Calpha and protein kinase CbetaII, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components.

A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase.

Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk(lo)) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk(-/-) mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk(lo) phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk(lo) phenotype in vitro but not in vivo, whereas CD19 and the p85alpha form of phosphoinositide 3-kinase behaved as Btk(lo) enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85alpha haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C beta, Fyn, CD22, Galphaq, or Galpha11 had no detectable effect on the function of Btk(lo) B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.

Regulation of protein kinase CbetaI by two protein-tyrosine kinases, Btk and Syk.

Two protein-tyrosine kinases, Bruton's tyrosine kinase (Btk) and Syk, and members of the protein kinase C (PKC) subfamily of serine/threonine kinases play crucial roles in signal transduction through antigen receptors in B lymphocytes and high-affinity IgE receptors (FcepsilonRI) in mast cells. The present study provides genetic, biochemical, and pharmacological evidence that, on FcepsilonRI stimulation, Syk regulates Btk, and Btk selectively regulates the membrane translocation and enzymatic activity of PKCbetaI among the conventional PKC isoforms (alpha, betaI, and betaII) expressed in mast cells. Syk/Btk-mediated PKCbetaI regulation is involved in transcriptional activation of the IL-2 and tumor necrosis factor alpha genes through the JNK pathway induced by FcepsilonRI stimulation. Accordingly, FcepsilonRI-induced production of these cytokines is inhibited by specific inhibitors of Btk and Syk, as well as broad-specificity inhibitors of PKC and a selective inhibitor of PKCbeta. Specific regulation of PKCbetaI by Btk is consistent with the selective association of Btk with PKCbetaI. Components of this signaling pathway may represent an attractive set of potential targets of pharmaceutical interference for the treatment of allergic and other immunologic diseases.

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.

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.

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.

Kinase pathways in chemoattractant-induced degranulation of neutrophils: the role of p38 mitogen-activated protein kinase activated by Src family kinases.

The aim of the present study was to investigate the role of tyrosine phosphorylation pathways in fMLP-induced exocytosis of the different secretory compartments (primary and secondary granules, as well as secretory vesicles) of neutrophils. Genistein, a broad specificity tyrosine kinase inhibitor, blocked the exocytosis of primary and secondary granules, but had only a marginal effect on the release of secretory vesicles. Genistein also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), raising the possibility that inhibition of ERK and/or p38 MAPK might be responsible for the effect of the drug on the degranulation response. Indeed, SB203580, an inhibitor of p38 MAPK, decreased the release of primary and secondary granules, but not that of secretory vesicles. However, blocking the ERK pathway with PD98059 had no effect on any of the exocytic responses tested. PP1, an inhibitor of Src family kinases, also attenuated the release of primary and secondary granules, and neutrophils from mice deficient in the Src family kinases Hck, Fgr, and Lyn were also defective in secondary granule release. Furthermore, activation of p38 MAPK was blocked by both PP1 and the hck-/-fgr-/-lyn-/- mutation. Taken together, our data indicate that fMLP-induced degranulation of primary and secondary granules of neutrophils is mediated by p38 MAPK activated via Src family tyrosine kinases. Although piceatannol, a reportedly selective inhibitor of Syk, also prevented degranulation and activation of p38 MAPK, no fMLP-induced phosphorylation of Syk could be observed, raising doubts about the specificity of the inhibitor.

Flow cytometric detection of CD10 (cALLA) on peripheral blood B lymphocytes of neonates.

CD10 (cALLA) was detected on the surface of CD19-positive circulating lymphocytes in the peripheral blood of 32/50 neonates tested. These cells are presumed to represent immature B cells, commonly referred to as haematogones, previously undescribed in peripheral blood. The CD10+/CD19+ cells expressed lower levels of CD22 consistent with these cells being immature B lymphocytes. The presence of CD10+/CD19+ cells in the blood was not significantly correlated with a leucoerythroblastic picture, adjusted gestational age, or the presence in blood smears of medium-to-large lymphocytes with an immature appearance that morphologically resembled classic bone-marrow haematogones.

Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages.

Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages.