The Src homology 2 domain-containing leukocyte protein of 76-kDa adaptor links integrin ligation with p44/42 MAPK phosphorylation and podosome distribution in murine dendritic cells

The Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an important molecular intermediate in multiple signaling pathways governing immune cell function. In this study, we report that SLP-76 is expressed in CD11c+ B220- dendritic cells (DCs) isolated from murine thymus or spleen, and that SLP-76 is rapidly phosphorylated on tyrosine residues upon plating of bone marrow-derived DCs (BMDCs) on integrin agonists. SLP-76 is not required for the in vitro or in vivo generation of DCs, but SLP-76-deficient BMDCs adhere poorly to fibronectin, suggesting impaired integrin function. Consistent with impaired adhesion, cutaneous SLP-76-deficient DCs leave ear tissue at an elevated frequency compared with wild-type DCs. In addition, the pattern and distribution of actin-based podosome formation are visibly altered in BMDCs lacking SLP-76 following integrin engagement. SLP-76-deficient BMDCs manifest multiple signaling defects following integrin ligation, including reduced global tyrosine phosphorylation and markedly impaired phosphorylation of p44/42 MAPK (ERK1/2). These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent DC functions, and add to the growing body of evidence that hemopoietic cells may use unique molecular intermediates and mechanisms for regulating integrin signaling.     

PRAM-1 is required for optimal integrin-dependent neutrophil function

PML-retinoic acid receptor alpha (RARalpha) regulated adaptor molecule 1 (PRAM-1) is an intracellular adaptor molecule that is upregulated during the induced granulocytic differentiation of promyelocytic leukemic cells and during normal human myelopoiesis. This report describes the generation of PRAM-1-deficient mice and an analysis of the function of this adaptor in neutrophil differentiation and mature neutrophil function. We demonstrate here that neutrophil differentiation is not impaired in PRAM-1-deficient mice and that PRAM-1-deficient neutrophils function normally following engagement of Fcgamma receptors. In contrast, mature PRAM-1-null neutrophils exhibit significant defects in adhesion-dependent reactive oxygen intermediate production and degranulation. Surprisingly, other integrin-dependent responses, such as cell spreading and activation of several signaling pathways, are normal. Together, these findings demonstrate the uncoupling of key integrin-dependent responses in the absence of PRAM-1 and show this adaptor to be critical for select integrin functions in neutrophils.     

Evidence for the requirement of ITAM domains but not SLP-76/Gads interaction for integrin signaling in hematopoietic cells

Syk tyrosine kinase and Src homology 2 (SH2) domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) are signaling mediators activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing immunoreceptors and integrins. While the signaling cascades descending from integrins are similar to immunoreceptors, the mechanism of Syk activation and SLP-76 recruitment remains unclear. We used an in vivo structure-function approach to study the requirements for the domains of Syk and SLP-76 in immunoreceptor and integrin signaling. We found that both SH2 domains and the kinase domain of Syk are required for immunoreceptor-dependent signaling and cellular response via integrins. While the Gads-binding domain of SLP-76 is needed for immunoreceptor signaling, it appears dispensable for integrin signaling. Syk and SLP-76 also are required for initiating and/or maintaining separation between the blood and lymphatic vasculature. Therefore, we correlated the signaling requirement of the various domains of Syk and SLP-76 to their requirement in regulating vascular separation. Our data suggest ITAMs are required in Syk-dependent integrin signaling, demonstrate the separation of the structural features of SLP-76 to selectively support immunoreceptor versus integrin signaling, and provide evidence that the essential domains of SLP-76 for ITAM signals are those which most efficiently support separation between lymphatic and blood vessels.     

Angiostatin inhibits activation and migration of neutrophils

There is a critical need to identify molecules that modulate the biology of neutrophils because activated neutrophils, though necessary for host defense, cause exuberant tissue damage through production of reactive oxygen species and increased lifespan. Angiostatin, an endogenous anti-angiogenic cleavage product of plasminogen, binds to integrin α_vβ₃, ATP synthase and angiomotin and its expression is increased in inflammatory conditions. We test the hypothesis that angiostatin inhibits neutrophil activation, induces apoptosis and blocks recruitment in vivo and in vitro. The data show immuno-reactivity for plasminogen/angiostatin in resting neutrophils. Angiostatin conjugated to FITC revealed that angiostatin was endocytozed by activated mouse and human neutrophils in a lipid raft-dependent fashion. Co-immunoprecipitation of human neutrophil lysates, confocal microscopy of isolated mouse and human neutrophils and functional blocking experiments showed that angiostatin complexes with flotillin-1 along with integrin α_vβ₃ and ATP synthase. Angiostatin inhibited fMLP-induced neutrophil polarization, as well as caused inhibition of hsp-27 phosphorylation and stabilization of microtubules. Angiostatin treatment, before or after LPS-induced neutrophil activation, inhibited phosphorylation of p38 and p44/42 MAPKs, abolished reactive oxygen species production and released the neutrophils from suppressed apoptosis, as indicated by expression of activated caspase-3 and morphological evidence of apoptosis. Finally, intravital microscopy and myeloperoxidase assay showed inhibition of neutrophil recruitment in post-capillary venules of TNFα-treated cremaster muscle in mouse. These in vitro and in vivo data demonstrate angiostatin as a broad deactivator and silencer of neutrophils and an inhibitor of their migration. These data potentially open new avenues for the development of anti-inflammatory drugs.     

Receptor-stimulated oxidation of SHP-2 promotes T-cell adhesion through SLP-76-ADAP

Receptor-stimulated generation of intracellular reactive oxygen species (ROS) modulates signal transduction, although the mechanism(s) is unclear. One potential basis is the reversible oxidation of the active site cysteine of protein tyrosine phosphatases (PTPs). Here, we show that activation of the antigen receptor of T cells (TCR), which induces production of ROS, induces transient inactivation of the SH2 domain-containing PTP, SHP-2, but not the homologous SHP-1. SHP-2 is recruited to the LAT-Gads-SLP-76 complex and directly regulates the phosphorylation of key signaling proteins Vav1 and ADAP. Furthermore, the association of ADAP with the adapter SLP-76 is regulated by SHP-2 in a redox-dependent manner. The data indicate that TCR-mediated ROS generation leads to SHP-2 oxidation, which promotes T-cell adhesion through effects on an SLP-76-dependent signaling pathway to integrin activation.     

Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.     

Reduced neutrophil apoptosis in diabetic mice during staphylococcal infection leads to prolonged Tnfα production and reduced neutrophil clearance

Diabetes is a frequent underlying medical condition among individuals with Staphylococcus aureus infections, and diabetic patients often suffer from chronic inflammation and prolonged infections. Neutrophils are the most abundant inflammatory cells during the early stages of bacterial diseases, and previous studies have reported deficiencies in neutrophil function in diabetic hosts. We challenged age-matched hyperglycemic and normoglycemic NOD mice intraperitoneally with S. aureus and evaluated the fate of neutrophils recruited to the peritoneal cavity. Neutrophils were more abundant in the peritoneal fluids of infected diabetic mice by 48 h after bacterial inoculation, and they showed prolonged viability ex vivo compared to neutrophils from infected nondiabetic mice. These differences correlated with reduced apoptosis of neutrophils from diabetic mice and were dependent upon the presence of S. aureus and a functional neutrophil respiratory burst. Decreased apoptosis correlated with impaired clearance of neutrophils by macrophages both in vitro and in vivo and prolonged production of proinflammatory tumor necrosis factor alpha by neutrophils from diabetic mice. Our results suggest that defects in neutrophil apoptosis may contribute to the chronic inflammation and the inability to clear staphylococcal infections observed in diabetic patients.     

Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection

The appropriate regulation of neutrophil activation is critical for maintaining host defense and limiting inflammation. Polymorphonuclear neutrophils (PMNs) express a number of cytoplasmic tyrosine kinases that regulate signaling pathways leading to activation. One of the most highly expressed, but least studied, kinases in PMNs is proline rich kinase 2 (Pyk2). By analogy to the related focal adhesion kinase, Pyk2 has been implicated in regulating PMN adhesion and migration; however, its physiologic function has yet to be described. Using pyk2(-/-) mice, we found that this kinase was required for integrin-mediated degranulation responses, but was not involved in adhesion-induced cell spreading or activation of superoxide production. Pyk2-deficient PMNs also manifested reduced migration on fibrinogen-coated surfaces. The absence of Pyk2 resulted in a severe reduction in paxillin and Vav phosphorylation following integrin ligation, which likely accounts for the poor degranulation and cell migration. Pyk2(-/-) mice were unable to efficiently clear infection with Staphylococcus aureus in a skin abscess model, owing in part to the poor release of granule contents at the site of infection. However, Pyk2-deficient PMNs responded normally to soluble agonists, demonstrating that this kinase functions mainly in the integrin pathway. These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents.     

Olfactomedin 4 Inhibits Cathepsin C-Mediated Protease Activities, Thereby Modulating Neutrophil Killing of Staphylococcus aureus and Escherichia coli in Mice

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4(-/-) mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4(-/-) mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4(-/-) mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4(-/-) neutrophils. The bacterial killing and clearance capabilities observed in OLFM4(-/-) mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.     

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.     

Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III₂ and I-III₂-IV_1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function.     

The proline-rich tyrosine kinase Pyk2 modulates integrin-mediated neutrophil adhesion and reactive oxygen species generation

Neutrophils are first responders in infection and inflammation. They are able to roll, adhere and transmigrate through the endothelium to reach the site of infection, where they fight pathogens through secretion of granule contents, production of reactive oxygen species, extrusion of neutrophil extracellular traps, and phagocytosis. In this study we explored the role of the non-receptor focal adhesion kinase Pyk2 in neutrophil adhesion and activation. Using a specific Pyk2 pharmacological inhibitor, PF-4594755, as well as Pyk2-deficient murine neutrophils, we found that Pyk2 is activated upon integrin αMβ2-mediated neutrophil adhesion to fibrinogen. This process is triggered by Src family kinases-mediated phosphorylation and supported by Pyk2 autophosphorylation on Y402. In neutrophil adherent to fibrinogen, Pyk2 activates PI3K-dependent pathways promoting the phosphorylation of Akt and of its downstream effector GSK3. Pyk2 also dynamically regulates MAP kinases in fibrinogen-adherent neutrophils, as it stimulates p38MAPK but negatively regulates ERK1/2. Pharmacological inhibition of Pyk2 significantly prevented adhesion of human neutrophils to fibrinogen, and neutrophils from Pyk2-knockout mice showed a reduced ability to adhere compared to wildtype cells. Accordingly, neutrophil adhesion to fibrinogen was reduced upon inhibition of p38MAPK but potentiated by ERK1/2 inhibition. Neutrophil adherent to fibrinogen, but not to polylysine, were able to produce ROS upon lipopolysaccharide challenge and ROS production was completely suppressed upon inhibition of Pyk2. By contrast PMA-induced ROS production by neutrophil adherent to either fibrinogen or polylysine was independent from Pyk2. Altogether these results demonstrate that Pyk2 is an important effector in the coordinated puzzle regulating neutrophil adhesion and activation.     

The Src family kinases Hck and Fgr are dispensable for inside-out, chemoattractant-induced signaling regulating beta 2 integrin affinity and valency in neutrophils, but are required for beta 2 integrin-mediated outside-in signaling involved in sustained adhesion

Neutrophil beta(2) integrins are activated by inside-out signaling regulating integrin affinity and valency; following ligand binding, beta(2) integrins trigger outside-in signals regulating cell functions. Addressing inside-out and outside-in signaling in hck(-/-)fgr(-/-) neutrophils, we found that Hck and Fgr do not regulate chemoattractant-induced activation of beta(2) integrin affinity. In fact, beta(2) integrin-mediated rapid adhesion, in static condition assays, and neutrophil adhesion to glass capillary tubes cocoated with ICAM-1, P-selectin, and a chemoattractant, under flow, were unaffected in hck(-/-)fgr(-/-) neutrophils. Additionally, examination of integrin affinity by soluble ICAM-1 binding assays and of beta(2) integrin clustering on the cell surface, showed that integrin activation did not require Hck and Fgr expression. However, after binding, hck(-/-)fgr(-/-) neutrophil spreading over beta(2) integrin ligands was reduced and they rapidly detached from the adhesive surface. Whether alterations in outside-in signaling affect sustained adhesion to the vascular endothelium in vivo was addressed by examining neutrophil adhesiveness to inflamed muscle venules. Intravital microscopy analysis allowed us to conclude that Hck and Fgr regulate neither the number of rolling cells nor rolling velocity in neutrophils. However, arrest of hck(-/-)fgr(-/-) neutrophils to >60 microm in diameter venules was reduced. Thus, Hck and Fgr play no role in chemoattractant-induced inside-out beta(2) integrin activation but regulate outside-in signaling-dependent sustained adhesion.     

Altered CXCR2 signaling in beta-arrestin-2-deficient mouse models

CXCR2 is a G-protein-coupled receptor (GPCR) that binds the CXC chemokines, CXCL1-3 and CXCL5-8, and induces intracellular signals associated with chemotaxis. Many adaptor proteins are actively involved in the sequestration, internalization, and trafficking of CXCR2 and transduction of agonist-induced intracellular signaling. We have previously shown that adaptor protein beta-arrestin-2 (betaarr2) plays a crucial role in transducing signals mediated through CXCR2. To further investigate the role of betaarr2 on CXCR2-mediated signaling during acute inflammation, zymosan-induced neutrophils were isolated from peritoneal cavities of betaarr2-deficient (betaarr2(-/-)) and their wild-type (betaarr2(+/+)) littermate mice, and neutrophil CXCR2 signaling activities were determined by measurement of Ca(2+) mobilization, receptor internalization, GTPase activity, and superoxide anion production. The results showed that the deletion of betaarr2 resulted in increased Ca(2+) mobilization, superoxide anion production, and GTPase activity in neutrophils, but decreased receptor internalization relative to wild-type mice. Two animal models, the dorsal air pouch model and the excisional wound healing model, were used to further study the in vivo effects of betaarr2 on CXCR2-mediated neutrophil chemotaxis and on cutaneous wound healing. Surprisingly, the recruitment of neutrophils was increased in response to CXCL1 in the air pouch model and in the excisional wound beds of betaarr2(-/-) mice. Wound re-epithelialization was also significantly faster in betaarr2(-/-) mice than in betaarr2(+/+) mice. Taken together, the data indicate that betaarr2 is a negative regulator for CXCR2 in vivo signaling.     

Protein kinase C-θ is required for murine neutrophil recruitment and adhesion strengthening under flow

Protein kinase C (PKC)-θ is involved in T cell activation via regulating the avidity of the β(2) integrin LFA-1 in the immunological synapse. LFA-1 also mediates leukocyte adhesion. To investigate the role of PKC-θ in neutrophil adhesion, we performed intravital microscopy in cremaster venules of mice reconstituted with bone marrow from LysM-GFP(+) (wild-type [WT]) and PKC-θ gene-deficient (Prkcq(-/-)) mice. Following stimulation with CXCL1, both WT and Prkcq(-/-) cells became adherent. Although most WT neutrophils remained adherent for at least 180 s, 50% of Prkcq(-/-) neutrophils were detached after 105 s and most by 180 s. Upon CXCL1 injection, rolling of all WT neutrophils stopped for 90 s, but rolling of Prkcq(-/-) neutrophils started 30 s after CXCL1 stimulation. A similar neutrophil adhesion defect was seen in vitro, and spreading of Prkcq(-/-) neutrophils was delayed. Prkcq(-/-) neutrophil recruitment was impaired in fMLP-induced transmigration into the cremaster muscle, thioglycollate-induced peritonitis, and LPS-induced lung injury. We conclude that PKC-θ mediates integrin-dependent neutrophil functions and is required to sustain neutrophil adhesion in postcapillary venules in vivo. These findings suggest that the role of PKC-θ in outside-in signaling following engagement of neutrophil integrins is relevant for inflammation in vivo.     

Glutathione reductase facilitates host defense by sustaining phagocytic oxidative burst and promoting the development of neutrophil extracellular traps

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.     

Toll-like receptor-2 is essential in murine defenses against Candida albicans infections

In this work, we studied the role of toll-like receptor-2 (TLR2) in murine defenses against Candida albicans. TLR2-deficient mice experimentally infected intraperitoneally (i.p.) or intravenously (i.v.) in vivo had very significant impaired survival compared with that of control mice. In vitro production of TNF-alpha and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2-/- mice in response to yeasts and hyphae of C. albicans were significantly lower (80% and 40%, respectively; P <0.05) than production by macrophages from wild-type mice. This impaired production of TNF-alpha and MIP-2 probably contributed to the 41% decreased recruitment of neutrophils to the peritoneal cavity of i.p. infected TLR2-/- mice. In contrast, in vitro phagocytosis of yeasts and production of reactive oxygen intermediates (ROI) were not affected in macrophages from TLR2-/- animals. Our data indicate that TLR2 plays a major role in the response of macrophages to C. albicans, triggering cytokine and chemokine expression, and it is essential for in vivo protection against infection.     

Cbl enforces Vav1 dependence and a restricted pathway of T cell development

Extensive studies of pre-TCR- and TCR-dependent signaling have led to characterization of a pathway deemed essential for efficient T cell development, and comprised of a cascade of sequential events involving phosphorylation of Lck and ZAP-70, followed by phosphorylation of LAT and SLP-76, and subsequent additional downstream events. Of interest, however, reports from our lab as well as others have indicated that the requirements for ZAP-70, LAT, and SLP-76 are partially reversed by inactivation of c-Cbl (Cbl), an E3 ubiquitin ligase that targets multiple molecules for ubiquitination and degradation. Analysis of signaling events in these Cbl knockout models, including the recently reported analysis of SLP-76 transgenes defective in interaction with Vav1, suggested that activation of Vav1 might be a critical event in alternative pathways of T cell development. To extend the analysis of signaling requirements for thymic development, we have therefore assessed the effect of Cbl inactivation on the T cell developmental defects that occur in Vav1-deficient mice. The defects in Vav1-deficient thymic development, including a marked defect in DN3-DN4 transition, were completely reversed by Cbl inactivation, accompanied by enhanced phosphorylation of PLC-γ1 and ERKs in response to pre-TCR/TCR cross-linking of Vav1^-/-Cbl^-/- DP thymocytes. Taken together, these results suggest a substantially modified paradigm for pre-TCR/TCR signaling and T cell development. The observed consensus pathways of T cell development, including requirements for ZAP-70, LAT, SLP-76, and Vav1, appear to reflect the restriction by Cbl of an otherwise much broader set of molecular pathways capable of mediating T cell development.     

Neutrophils and inducible nitric-oxide synthase are critical for early resistance to the establishment of Tritrichomonas foetus infection

Tritrichomonas foetus is the cause of trichomoniasis in cattle. Severe infection is often associated with heavy neutrophil and macrophage accumulation, although it is not known how this response protects during early parasite colonization. The goal of this study was to examine the effects of an early host response upon initial T. foetus colonization within the murine reproductive tract. Mice depleted of neutrophils before T. foetus infection had a significantly higher parasite burden within the reproductive tract compared with mock-depleted control mice. Additionally, gp91(phox-/-)/ iNOS(-/-), and iNOS(-/-) mice had substantially larger parasite burdens than C57BL/6 control mice, whereas gp91l(Phox-/-) mice had similar parasite burden to C57BL/6 control mice. Interestingly, phorbol 12-myristate 13-acetate-stimulated neutrophils and macrophages isolated from all groups of mice were unable to kill T. foetus in vitro. However, macrophages isolated from gp91l(phox-/-) and C57BL/6 mice stimulated with interferon-gamma and lipopolysaccharide were able to kill T. foetus in vitro, whereas macrophages isolated from gp91(phox(-/-)/ iNOS(-/-) and iNOS(-/-) mice were unable to kill T. foetus, suggesting the ability of macrophages to produce reactive nitrogen species but not reactive oxygen species (ROS) is critical for parasite killing during early infection in vivo and in vitro. Additionally, neutrophils seem to control early dissemination of T. foetus throughout the reproductive tract, although production of ROS is not critical for this process.