Neuroplastin splice variants Np55 and Np65: Who is doing the job in macrophages?

Neuroplastin, a paralog of CD147/Basigin, is known as a neuronal cell adhesion molecule and as an auxiliary subunit of plasma membrane calcium ATPases in both neurons and adaptive immune cells. Recently, an interesting study by Ren et al. (2022) provided evidence for an important role of neuroplastin in macrophages during bacterial infection. Here, we critically discuss one aspect of this study, the assignment of this role to Np65 as one of two prominent splice variants of neuroplastin.

Ca2+ Homeostasis by Plasma Membrane Ca2+ ATPase (PMCA) 1 Is Essential for the Development of DP Thymocytes.

The strength of Ca2+ signaling is a hallmark of T cell activation, yet the role of Ca2+ homeostasis in developing T cells before expressing a mature T cell receptor is poorly understood. We aimed to unveil specific functions of the two plasma membrane Ca2+ ATPases expressed in T cells, PMCA1 and PMCA4. On a transcriptional and protein level we found that PMCA4 was expressed at low levels in CD4-CD8- double negative (DN) thymocytes and was even downregulated in subsequent stages while PMCA1 was present throughout development and upregulated in CD4+CD8+ double positive (DP) thymocytes. Mice with a targeted deletion of Pmca1 in DN3 thymocytes had an almost complete block of DP thymocyte development with an accumulation of DN4 thymocytes but severely reduced numbers of CD8+ immature single positive (ISP) thymocytes. The DN4 thymocytes of these mice showed strongly elevated basal cytosolic Ca2+ levels and a pre-mature CD5 expression, but in contrast to the DP thymocytes they were only mildly prone to apoptosis. Surprisingly, mice with a germline deletion of Pmca4 did not show any signs of altered progression through the developmental thymocyte stages, nor altered Ca2+ homeostasis throughout this process. PMCA1 is, therefore, non-redundant in keeping cellular Ca2+ levels low in the early thymocyte development required for the DN to DP transition.

IKKγ/NEMO Localization into Multivesicular Bodies.

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3ß (GSK-3ß) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3ß leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3ß and V-ATPase in NF-κB signaling activation.

PLCG1 is required for AML1-ETO leukemia stem cell self-renewal.

In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

Re-evaluation of the immunohistochemical distribution of isoforms of nitric oxide synthase in the human prostate: A light and electron microscopical study.

Up until today, there are still uncertainties regarding the occurrence of isoforms of the nitric oxide synthase (eNOS, nNOS) in the human prostate. While nNOS was exclusively seen in slender nerve fibres branching within the transition zone, eNOS was reported in glandular structures and also in small vessels interspersing the tissue. This study aimed to re-evaluate by means of light and electron microscopy (LM, EM), the distribution of eNOS and nNOS in the transition zone of the human prostate. Tissue specimens were obtained from 16 patients who underwent surgery for pelvic malignancies. Using specific antibodies in conjunction with advanced fixation and staining procedures, the occurrence of eNOS and nNOS was investigated. nNOS was detected in nerve fibres interspersing the tissue and was also seen in glandular structures. EM revealed that in glandular epithelial cells immunoreaction for nNOS was limited to the cytoplasmic compartment. Vascular endothelial cells of small vessels transversing glandular structures significantly stained for eNOS, while epithelial layers of prostatic glandules appeared free of eNOS. The results implicate that, in the prostate, nNOS is a mediator of stromal and glandular tissue function, and counteract the assumption of eNOS activity in glandular epithelial cells as a source of NO synthesis.

Plasma membrane Ca2+ ATPase 1 (PMCA1) but not PMCA4 is critical for B-cell development and Ca2+ homeostasis in mice.

The amplitude and duration of Ca2+ signaling is crucial for B-cell development and self-tolerance; however, the mechanisms for terminating Ca2+ signals in B cells have not been determined. In lymphocytes, plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 (PMCA1 and PMCA4, aka ATP2B1 and ATP2B4) are the main candidates for expelling Ca2+ from the cell through the plasma membrane. We report here that Pmca4 (Atp2b4) KO mice had normal B-cell development, while mice with a conditional KO of Pmca1 (Atp2b1) had greatly reduced numbers of B cells, particularly splenic follicular B cells, marginal zone B cells, and peritoneal B-1a cells. Mouse and naïve human B cells showed only PMCA1 expression and no PMCA4 by western blot, in contrast to T cells, which did express PMCA4. Calcium handling was normal in Pmca4-/- B cells, but Pmca1 KO B cells had elevated basal levels of Ca2+ , elevated levels in ER stores, and reduced Ca2+ clearance. These findings show that the PMCA1 isoform alone is required to ensure normal B-cell Ca2+ signaling and development, which may have implications for therapeutic targeting of PMCAs and Ca2+ in B cells.

Deletion of the Nucleotide Exchange Factor Vav3 Enhances Axonal Complexity and Synapse Formation but Tampers Activity of Hippocampal Neuronal Networks In Vitro.

Vav proteins activate GTPases of the RhoA subfamily that regulate the cytoskeleton and are involved in adhesion, migration, differentiation, polarity and the cell cycle. While the importance of RhoA GTPases for neuronal morphology is undisputed, their regulation is less well understood. In this perspective, we studied the consequences of the deletion of Vav2, Vav3 and Vav2 and 3 (Vav2-/-, Vav3-/-, Vav2-/-/3-/-) for the development of embryonic hippocampal neurons in vitro. Using an indirect co-culture system of hippocampal neurons with primary wild-type (WT) cortical astrocytes, we analysed axonal and dendritic parameters, structural synapse numbers and the spontaneous network activity via immunocytochemistry and multielectrode array analysis (MEA). Here, we observed a higher process complexity in Vav3-/-, but not in Vav2-/- neurons after three and five days in vitro (DIV). Furthermore, an enhanced synapse formation was observed in Vav3-/- after 14 days in culture. Remarkably, Vav2-/-/3-/- double knockout neurons did not display synergistic effects. Interestingly, these differences were transient and compensated after a cultivation period of 21 days. Network analysis revealed a diminished number of spontaneously occurring action potentials in Vav3-/- neurons after 21 DIV. Based on these results, it appears that Vav3 participates in key events of neuronal differentiation.

A complex of Neuroplastin and Plasma Membrane Ca2+ ATPase controls T cell activation.

The outcome of T cell activation is determined by mechanisms that balance Ca2+ influx and clearance. Here we report that murine CD4 T cells lacking Neuroplastin (Nptn -/-), an immunoglobulin superfamily protein, display elevated cytosolic Ca2+ and impaired post-stimulation Ca2+ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca2+ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn -/- T cells, suggesting an explanation for altered Ca2+ handling. Supporting this, Ca2+ extrusion was impaired while Ca2+ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn -/- T cells. Consistent with sustained Ca2+ levels, differentiation of Nptn -/- T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.

Inflammatory cell infiltration and resolution of kidney inflammation is orchestrated by the cold-shock protein Y-box binding protein-1.

Tubular cells recruit monocytic cells in inflammatory tubulointerstitial kidney diseases. The cell-cell communication that establishes pro- or anti-inflammatory activities is mainly influenced by cytokines, reactive oxygen species, nitric oxide, and phagocytosis. Key proteins orchestrating these processes such as cold-shock proteins linked with chemoattraction and cell maturation have been identified. The prototypic member of the cold-shock protein family, Y-box binding protein (YB)-1, governs specific phenotypic alterations in monocytic cells and was explored in the present study. Following tubulointerstitial injury by unilateral ureteral obstruction, increased inflammatory cell infiltration and tubular cell CCL5 expression was found in conditional Ybx1 knockout animals with specific depletion in monocytes/macrophages (YB-1ΔLysM). Furthermore, YB-1ΔLysM mice exhibit enhanced tissue damage, myofibroblast activation, and fibrosis. To investigate relevant molecular mechanism(s), we utilized bone marrow-derived macrophage cultures and found that YB-1-deficient macrophages display defects in cell polarization and function, including reduced proliferation and nitric oxide production, loss of phagocytic activity, and failure to upregulate IL-10 and CCL5 expression in response to inflammatory stimuli. Co-culture with primary tubular cells confirmed these findings. Thus, monocytic YB-1 has prominent and distinct roles for cellular feed-forward crosstalk and resolution of inflammatory processes by its ability to regulate cell differentiation and cytokine/chemokine synthesis.

Maternal extracellular vesicles and platelets promote preeclampsia via inflammasome activation in trophoblasts.

Preeclampsia (PE) is a placenta-induced inflammatory disease associated with maternal and fetal morbidity and mortality. The mechanisms underlying PE remain enigmatic and delivery of the placenta is the only known remedy. PE is associated with coagulation and platelet activation and increased extracellular vesicle (EV) formation. However, thrombotic occlusion of the placental vascular bed is rarely observed and the mechanistic relevance of EV and platelet activation remains unknown. Here we show that EVs induce a thromboinflammatory response specifically in the placenta. Following EV injection, activated platelets accumulate particularly within the placental vascular bed. EVs cause adenosine triphosphate (ATP) release from platelets and inflammasome activation within trophoblast cells through purinergic signaling. Inflammasome activation in trophoblast cells triggers a PE-like phenotype, characterized by pregnancy failure, elevated blood pressure, increased plasma soluble fms-like tyrosine kinase 1, and renal dysfunction. Intriguingly, genetic inhibition of inflammasome activation specifically in the placenta, pharmacological inhibition of inflammasome or purinergic signaling, or genetic inhibition of maternal platelet activation abolishes the PE-like phenotype. Inflammasome activation in trophoblast cells of women with preeclampsia corroborates the translational relevance of these findings. These results strongly suggest that EVs cause placental sterile inflammation and PE through activation of maternal platelets and purinergic inflammasome activation in trophoblast cells, uncovering a novel thromboinflammatory mechanism at the maternal-embryonic interface.

Reduction of Airborne Bacterial Burden in the OR by Installation of Unidirectional Displacement Airflow (UDF) Systems.

BACKGROUND: Intraoperative bacterial contamination is a major risk factor for postoperative wound infections. This study investigated the influence of type of ventilation system on intraoperative airborne bacterial burden before and after installation of unidirectional displacement air flow systems. MATERIAL AND METHODS: We microbiologically monitored 1286 surgeries performed by a single surgical team that moved from operating rooms (ORs) equipped with turbulent mixing ventilation (TMV, according to standard DIN-1946-4 [1999], ORs 1, 2, and 3) to ORs with unidirectional displacement airflow (UDF, according to standard DIN-1946-4, annex D [2008], ORs 7 and 8). The airborne bacteria were collected intraoperatively with sedimentation plates. After incubation for 48 h, we analyzed the average number of bacteria per h, peak values, and correlation to surgery duration. In addition, we compared the last 138 surgeries in ORs 1-3 with the first 138 surgeries in ORs 7 and 8. RESULTS: Intraoperative airborne bacterial burden was 5.4 CFU/h, 5.5 CFU/h, and 6.1 CFU/h in ORs 1, 2, and 3, respectively. Peak values of burden were 10.7 CFU/h, 11.1 CFU/h, and 11.0 CFU/h in ORs 1, 2, and 3, respectively). With the UDF system, the intraoperative airborne bacterial burden was reduced to 0.21 CFU/h (OR 7) and 0.35 CFU/h (OR 8) on average (p<0.01). Accordingly, peak values decreased to 0.9 CFU/h and 1.0 CFU/h in ORs 7 and 8, respectively (p<0.01). Airborne bacterial burden increased linearly with surgery duration in ORs 1-3, but the UDF system in ORs 7 and 8 kept bacterial levels constantly low (<3 CFU/h). A comparison of the last 138 surgeries before with the first 138 surgeries after changing ORs revealed a 94% reduction in average airborne bacterial burden (5 CFU/h vs. 0.29 CFU/h, p<0.01). CONCLUSIONS: The unidirectional displacement airflow, which fulfills the requirements of standard DIN-1946-4 annex D of 2008, is an effective ventilation system that reduces airborne bacterial burden under real clinical conditions by more than 90%. Although decreased postoperative wound infection incidence was not specifically assessed, it is clear that airborne microbiological burden contributes to surgical infections.

CTLA-4 (CD152) enhances the Tc17 differentiation program.

Although CD8(+) T cells that produce IL-17 (Tc17 cells) have been linked to host defense, Tc17 cells show reduced cytotoxic activity, which is the characteristic function of CD8(+) T cells. Here, we show that CTLA-4 enhances the frequency of IL-17 in CD8(+) T cells, indicating that CTLA-4 (CD152) specifically promotes Tc17 differentiation. Simultaneous stimulation of CTLA-4(+/+) and CTLA-4(-/-) T cells in cocultures and agonistic CTLA-4 stimulation unambiguously revealed a cell-intrinsic mechanism for IL-17 control by CTLA-4. The quality of CTLA-4-induced Tc17 cells was tested in vivo, utilizing infection with the facultative intracellular bacterium Listeria monocytogenes (LM). Unlike CTLA-4(+/+) Tc17 cells, CTLA-4(-/-) were nearly as efficient as Tc1 CTLA-4(+/+) cells in LM clearance. Additionally, adoptively transferred CTLA-4(-/-) Tc17 cells expressed granzyme B after rechallenge, and produced Tc1 cytokines such as IFN-γ and TNF-α, which strongly correlate with bacterial clearance. CTLA-4(+/+) Tc17 cells demonstrated a high-quality Tc17 differentiation program ex vivo, which was also evident in isolated IL-17-secreting Tc17 cells, with CTLA-4-mediated enhanced upregulation of Tc17-related molecules such as IL-17A, RORγt, and IRF-4. Our results show that CTLA-4 promotes Tc17 differentiation that results in robust Tc17 responses. Its inactivation might therefore represent a central therapeutic target to enhance clearance of infection.

Multimolecular analysis of stable immunological synapses reveals sustained recruitment and sequential assembly of signaling clusters.

The formation of the immunological synapse between T cells and antigen-presenting cells (APC) begins within minutes of contact and can take hours for full T-cell activation. Although early phases of the synapse have been extensively studied for a select number of proteins, later phases have not yet been examined in detail. We studied the signaling network in stable synapses by measuring the simultaneous localization of 25 signaling and structural molecules over 2 h at the level of individual synapses using multi-epitope ligand cartography (MELC). Signaling proteins including phospho(p)ZAP70, pSLP76, pCD3ζ, and pLAT, along with proteins that influence synapse structure such as F-actin, tubulin, CD45, and ICAM-1, were localized in images of synapses and revealed the multidimensional construction of a mature synapse. The construction of the stable synapse included intense early TCR signaling, a phase of recruitment of structural proteins, and a sustained increase in signaling molecules and colocalization of TCR and pLAT signaling clusters in the center of the synapse. Consolidation of TCR and associated proteins resulted in formation of a small number of discrete synaptic microclusters. Development of synapses and cSMAC composition was greatly affected by the absence of Vav1, with an associated loss in PLCγ1 recruitment, pSLP76, and increased CXCR4. Together, these data demonstrate the use of multi-epitope ligand cartography to quantitatively analyze synapse formation and reveal successive recruitment of structural and signaling proteins and sustained phosphorylation at the mature synapse.

Toxoplasma gondii actively inhibits neuronal function in chronically infected mice.

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii-infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca(2+)) imaging studies revealed that tachyzoites actively manipulated Ca(2+) signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca(2+) uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca(2+) stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host.

Non-esterified polyunsaturated fatty acids distinctly modulate the mitochondrial and cellular ROS production in normoxia and hypoxia.

There is an intense discussion about the subcellular origin of the generation of reactive oxygen species (ROS) under hypoxia. Since this fundamental question can be addressed only in a cellular system, the O(2) -sensing rat pheochromocytoma (PC12) cells were used. Severe hypoxia is known to elevate non-esterified fatty acids. Therefore, the site(s) of ROS generation were studied in cells which we simultaneously exposed to hypoxia (1% oxygen) and free fatty acids (FFA). We obtained the following results: (i) at hypoxia, ROS generation increases in PC12 cells but not in mitochondria isolated therefrom. (ii) Non-esterified polyunsaturated fatty acids (PUFA) enhance the ROS release from PC12 cells as well as from mitochondria, both in normoxia and in hypoxia. (iii) PUFA-induced ROS generation by PC12 cells is not decreased either by inhibitors of the cell membrane NAD(P)H oxidase or inhibitors impairing the PUFA metabolism. (iv) PUFA-induced ROS generation of mitochondria is paralleled by a decline of the NADH-cytochrome c reductase activity (reflecting combined enzymatic activity of complex I plus III). (v) Mitochondrial superoxide indicator (MitoSOXred)-loaded cells exposed to PUFA exhibit increased fluorescence indicating mitochondrial ROS generation. In conclusion, elevated PUFA levels enhance cellular ROS level in hypoxia, most likely by impairing the electron flux within the respiratory chain. Thus, we propose that PUFAs are likely to act as important extrinsic factor to enhance the mitochondria-associated intracellular ROS signaling in hypoxia.

Loss of Lsc/p115 protein leads to neuronal hypoplasia in the esophagus and an achalasia-like phenotype in mice.

BACKGROUND & AIMS: Lsc/p115 originally was described as hematopoietic Ras homologous protein guanine exchange factor (Rho-GEF) regulating leukocyte migration, adhesion, and marginal zone B-cell homeostasis. Here we investigate the expression pattern of lsc/p115 in the gastrointestinal tract and the consequences of lsc/p115 deficiency in lsc/p115-knockout mice. METHODS: The phenotype of lsc/p115-deficient mice was analyzed in vivo with small-animal computed tomography scans and esophageal manometry. The morphology and myenteric plexus were evaluated with immunohistochemistry, morphometry, Western blot analyses, and quantitative reverse-transcription polymerase chain reaction. RESULTS: lsc/p115 is expressed in the gastrointestinal tract, sparing the segment of the small intestine. Immunohistochemical staining detects lsc/p115 in the muscle layer and the glial fibrillary acidic protein-positive glia in the esophagus. Esophageal manometry uncovers a severe motor dysfunction in lsc/p115-deficient mice. This achalasia-like phenotype is characterized by disturbed peristalsis, hypertension of the lower esophageal sphincter, and impaired relaxation of the lower esophageal sphincter. Lsc/p115-deficient mice develop a progressive dilatation of the esophagus and decrease of the muscle layer. The muscle cell differentiation is not altered in lsc/p115-deficient mice. However, the density of inhibitory and excitatory neurons and glia cells in the myenteric plexus and the muscle layer are reduced in morphometric analyses. This reduced number of glia cells is accompanied by reduced expression of the neurotrophic nerve growth factor. CONCLUSIONS: lsc/p115 deficiency results in impaired neuronal innervation and in motor dysfunction recapitulating several aspects of esophageal achalasia. Reduced expression of nerve growth factor and a reduced number of glia cells most likely contribute to this phenotype.

BOB.1/OBF.1 controls the balance of TH1 and TH2 immune responses.

BOB.1/OBF.1 is a transcriptional coactivator essential at several stages of B-cell development. In T cells, BOB.1/OBF.1 expression is inducible by co-stimulation. However, a defined role of BOB.1/OBF.1 for T-cell function had not been discovered so far. Here, we show that BOB.1/OBF.1 is critical for T helper cell function. BOB.1/OBF.1(-/-) mice showed imbalanced immune responses, resulting in increased susceptibility to Leishmania major infection. Functional analyses revealed specific defects in TH1 and TH2 cells. Whereas expression levels of TH1 cytokines were reduced, the secretion of TH2 cytokines was increased. BOB.1/OBF.1 directly contributes to the IFNgamma and IL2 promoter activities. In contrast, increased TH2 cytokine production is controlled indirectly, probably via the transcription factor PU.1, the expression of which is regulated by BOB.1/OBF.1. Thus, BOB.1/OBF.1 regulates the balance of TH1 versus TH2 mediated immunity.

Strongly reduced alloreactivity and long-term survival times of cardiac allografts in Vav1- and Vav1/Vav2-knockout mice.

Vav proteins mediate T- and B-cell activation by functioning as GTP/GDP exchange factors for small GTPases. We have studied the role of Vav1 and Vav2 in allogeneic T-cell activation, antibody responses and allograft rejection. Alloantigen-induced proliferation of T cells from Vav1- and Vav1/Vav2-knockout (ko) mice was decreased by >90% in a mixed lymphocyte reaction. In whole-blood cultures, Vav deficiency led to markedly impaired T- and B-cell activation. Expansion of Vav1- or Vav1/Vav2-ko T cells (C57BL/6) was reduced after transfer into severe combined immune deficiency/beige recipient mice (BALB/c). After priming with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin, T-cell-dependent anti-DNP IgM and IgG antibody levels were normal in Vav1-ko mice but undetectable in Vav1/Vav2-ko mice. The median survival time of BALB/c cardiac allografts transplanted into C57BL/6 Vav1-ko mice (n = 13) or Vav1/Vav2-ko mice (n = 5) was >100 and >77 days, compared with 8-9 days in the corresponding wild-type mice. Vav1/Vav2-ko mice with <100 days graft survival developed bacterial skin infections and were prematurely killed with beating cardiac allograft. Long-term surviving transplants of single and double ko mice showed mild cellular interstitial rejection and mild to severe vascular remodeling. In conclusion, our studies show for the first time that the absence of Vav1 and Vav1/Vav2 in ko mice strongly reduces alloreactivity and results in long-term allograft survival, whereas antibody responses were only affected in Vav1/Vav2 ko mice.

The granulocyte receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) directly associates with Vav to promote phagocytosis of human pathogens.

The human granulocyte-specific receptor carcinoembryonic antigen-related cell adhesion molecule (CEACAM)3 is critically involved in the opsonin-independent recognition of several bacterial pathogens. CEACAM3-mediated phagocytosis depends on the integrity of an ITAM-like sequence within the cytoplasmic domain of CEACAM3 and is characterized by rapid stimulation of the GTPase Rac. By performing a functional screen with CEACAM3-expressing cells, we found that overexpression of a dominant-negative form of the guanine nucleotide exchange factor Vav, but not the dominant-negative versions SWAP70, Dock2, or ELMO1 interfered with CEACAM3-initiated phagocytosis. Moreover, small interfering RNA-mediated silencing of Vav reduced uptake and abrogated the stimulation of Rac in response to bacterial CEACAM3 engagement. In Vav1/Vav2-deficient cells, CEACAM3-mediated internalization was only observed after re-expression of Vav. Vav colocalized with CEACAM3 upon bacterial infection, coimmunoprecipitated in a complex with CEACAM3, and the Vav Src homology 2 domain directly associated with phosphorylated Tyr(230) of CEACAM3. In primary human granulocytes, TAT-mediated transduction of dominant-negative Vav, but not SWAP70, severely impaired the uptake of CEACAM3-binding bacteria. These data support the view that, different from canonical ITAM signaling, the CEACAM3 ITAM-like sequence short-wires bacterial recognition and Rac stimulation via a direct association with Vav to promote rapid phagocytosis and elimination of CEACAM-binding human pathogens.

Mitogenic CD28 signals require the exchange factor Vav1 to enhance TCR signaling at the SLP-76-Vav-Itk signalosome.

Almost all physiological T cell responses require costimulation-engagement of the clonotypic TCR with MHC/Ag and CD28 by its ligands CD80/86. Whether CD28 provides signals that are qualitatively unique or quantitatively amplify TCR signaling is poorly understood. In this study, we use superagonistic CD28 Abs, which induce T cell proliferation without TCR coligation, to determine how CD28 contributes to mitogenic responses. We show that mitogenic CD28 signals require but do not activate the proximal TCR components TCRzeta and Zap-70 kinase. In cell lines lacking proximal TCR signaling, an early defect in the CD28 pathway is in phosphorylation of the adaptor molecule SLP-76, which we show is essential for recruitment of the exchange factor Vav leading to Ca(2+) flux and IL-2 production. Point mutations in CD28 that result in diminished Vav phosphorylation also result in defective Ca(2+) flux, IL-2 production, and Tec-kinase phosphorylation. Using Vav1-deficient mice, we further demonstrate the importance of Vav1 for efficient proliferation, IL-2 production, and Ca(2+) flux. Our results indicate that CD28 signals feed into the TCR signaling pathway at the level of the SLP-76 signalosome.