Down-regulation Of L-selectin Research Articles

Kaposi's sarcoma-associated herpesvirus ubiquitin ligases downregulate cell surface expression of l-selectin.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic etiological factor for Kaposi's sarcoma and primary effusion lymphoma in immunocompromised patients. KSHV utilizes two immune evasion E3 ubiquitin ligases, namely K3 and K5, to downregulate the expression of antigen-presenting molecules and ligands of natural killer (NK) cells in the host cells through an ubiquitin-dependent endocytic mechanism. This allows the infected cells to evade surveillance and elimination by cytotoxic lymphocytes and NK cells. The number of host cell molecular substrates reported for these ubiquitin ligases is limited. The identification of novel substrates for these ligases will aid in elucidating the mechanism underlying immune evasion of KSHV. This study demonstrated that K5 downregulated the cell surface expression of l-selectin, a C-type lectin-like adhesion receptor expressed in the lymphocytes. Tryptophan residue located at the centre of the E2-binding site in the K5 RINGv domain was essential to downregulate l-selectin expression. Additionally, the lysine residues located at the cytoplasmic tail of l-selectin were required for the K5-mediated downregulation of l-selectin. K5 promoted the degradation of l-selectin through polyubiquitination. These results suggest that K5 downregulates l-selectin expression on the cell surface by promoting polyubiquitination and ubiquitin-dependent endocytosis, which indicated that l-selectin is a novel substrate for K5. Additionally, K3 downregulated l-selectin expression. The findings of this study will aid in the elucidation of a novel immune evasion mechanism in KSHV.

Ly9 (CD229) Antibody Targeting Depletes Marginal Zone and Germinal Center B Cells in Lymphoid Tissues and Reduces Salivary Gland Inflammation in a Mouse Model of Sjögren's Syndrome.

Sjögren's Syndrome (SjS) is a common chronic autoimmune disease characterized by the B cell hyperactivation, lymphocyte infiltration, and tissue damage of exocrine glands. It can also present life-threatening extraglandular manifestations, such as pulmonary and hepatic involvement, renal inflammation and marginal zone (MZ) B cell lymphoma. Several biologic agents have been tested in SjS but none has shown significant efficacy. Here, we report the effects of Ly9 (CD229) antibody targeting, a cell surface molecule that belongs to the SLAM family of immunomodulatory receptors, using NOD.H-2h4 mice as a model of SjS-like disease. Female mice were treated with anti-Ly9 antibody or isotype control at week 24, when all mice present SjS related autoantibodies, salivary gland infiltrates, and marginal zone (MZ) B cell pool enlargement. Antibody injection depleted key lymphocyte subsets involved in SjS pathology such as MZ, B1, and germinal center B cells in spleen and draining lymph nodes without inducing a general immunosuppression. Importantly, mice receiving anti-Ly9 mAb showed a reduced lymphocyte infiltrate within salivary glands. This reduction may be, in part, explained by the down-regulation of L-selectin and alfa4/beta7 integrin induced by the anti-Ly9 antibody. Furthermore, levels of anti-nuclear autoantibodies were reduced after anti-Ly9 treatment. These data indicate that Ly9 is a potential therapeutic target for the treatment of SjS.

Frontline Science: Myeloid-derived suppressor cells (MDSCs) facilitate maternal-fetal tolerance in mice.

During successful pregnancy, a woman is immunologically tolerant of her genetically and antigenically disparate fetus, a state known as maternal-fetal tolerance. How this state is maintained has puzzled investigators for more than half a century. Diverse, immune and nonimmune mechanisms have been proposed; however, these mechanisms appear to be unrelated and to act independently. A population of immune suppressive cells called myeloid-derived suppressor cells (MDSCs) accumulates in pregnant mice and women. Given the profound immune suppressive function of MDSCs, it has been suggested that this cell population may facilitate successful pregnancy by contributing to maternal-fetal tolerance. We now report that myeloid cells with the characteristics of MDSCs not only accumulate in the circulation and uterus of female mice following mating but also suppress T cell activation and function in pregnant mice. Depletion of cells with the phenotype and function of MDSCs from gestation d 0.5 through d 7.5 resulted in implantation failure, increased T cell activation, and increased T cell infiltration into the uterus, whereas induction of MDSCs restored successful pregnancy and reduced T cell activation. MDSC-mediated suppression during pregnancy was accompanied by the down-regulation of L-selectin on naïve T cells and a reduced ability of naïve T cells to enter lymph nodes and become activated. Because MDSCs regulate many of the immune and nonimmune mechanisms previously attributed to maternal-fetal tolerance, MDSCs may be a unifying mechanism promoting maternal-fetal tolerance, and their induction may facilitate successful pregnancy in women who spontaneously abort or miscarry because of dysfunctional maternal-fetal tolerance.

Superoxide anion mediates the L-selectin down-regulation induced by non-steroidal anti-inflammatory drugs in human neutrophils

Non-steroidal anti-inflammatory drugs (NSAIDs) induce the shedding of L-selectin in human neutrophils through a mechanism still not well understood. In this work we studied both the functional effect of NSAIDs on the neutrophils/endothelial cells dynamic interaction, and the potential involvement of reactive oxygen species (ROS) in the NSAIDs-mediated down-regulation of L-selectin. When human neutrophils were incubated with diclofenac, a significant reduction in the number of cells that rolled on activated endothelial cells was observed. Different NSAIDs (flufenamic acid, meclofenamic acid, diclofenac, indomethacin, nimesulide, flurbiprofen, meloxicam, phenylbutazone, piroxicam, ketoprofen and aspirin) caused variable increase in neutrophil intracellular ROS concentration, which was inversely proportional to the change produced in L-selectin surface expression. Pre-incubation of neutrophils with superoxide dismutase, but not with catalase, showed both a significant protective effect on the L-selectin down-regulation induced by several NSAIDs and a diminished effect of diclofenac on neutrophil rolling. Interestingly, diclofenac and flufenamic acid but not piroxicam significantly increased the extracellular superoxide anion production by neutrophils, and inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity with diphenyleneiodonium prevented the down-regulation of L-selectin by diclofenac. In accordance with these results, neutrophils from patients with chronic granulomatous disease, a hereditary disease in which neutrophils show a reduced capacity to form superoxide radicals, exhibited a lower down-regulation of L-selectin (IC50: 15.3μg/ml) compared to normal controls (IC50: 5.6μg/ml) in response to diclofenac. ConclusionA group of NSAIDs is capable of interfering with the ability of neutrophils to interact with endothelial cells by triggering L-selectin-shedding through the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane.

Humanised mice have functional human neutrophils

The differences between murine and human neutrophils mean that findings in mice may not translate to humans, and therefore an in vivo model with human neutrophils would be an important methodological advance. We generated humanised mice by injecting human cord blood derived CD34+ stem cells into irradiated NOD-scid-γc−/− mice. At least 3months after engraftment, treatment of mice with GCSF mobilised circulating human neutrophils, which comprised 2.6% of human leukocytes, and led to L-selectin shedding and upregulation of CD66b, CD11b and CD63. Subsequent in vivo LPS treatment led to further downregulation of L-selectin with upregulation of CD66b and CD63, and also resulted in human neutrophil sequestration in the lungs. Furthermore, human neutrophils from these mice were capable of robust functional responses. They were shown to undergo a respiratory burst, and to degranulate with upregulation of CD63 and CD66b, in response to fMLP and Escherichia coli. These data show that functional human neutrophils develop from CD34+ cord blood stem cells in NOD-scid-γc−/− mice. They suggest that this approach may facilitate the in vivo study of human neutrophils in clinically relevant models of infection and autoimmunity.

ADAM17 activation in circulating neutrophils following bacterial challenge impairs their recruitment

Neutrophil infiltration and bacterial clearance occur earlier in conditional knockout mice with leukocytes lacking the metalloprotease ADAM17 than in control mice. We investigated cell-intrinsic changes in neutrophils lacking ADAM17 and alterations in the inflammatory environment in conditional ADAM17 knockout mice to determine how the sheddase exerts its effects on neutrophil recruitment. In vivo analyses comparing control and ADAM17-deficient neutrophils revealed that the latter cells accumulated at increased levels in the inflamed mesenteric microvasculature and in the peritoneal cavity following bacterial challenge, indicating changes in their adhesive properties. Consistent with this, bacterial infection caused a marked down-regulation of L-selectin, an adhesion protein and substrate of ADAM17, from the surface of circulating neutrophils in control mice but not in conditional ADAM17 knockout mice. Neutrophils from gene-targeted mice with leukocytes expressing a noncleavable form of L-selectin also displayed a competitive advantage in the presence of control neutrophils when infiltrating a site of infection. Taken together, our findings reveal that impaired L-selectin shedding is a key mechanism underlying early neutrophil recruitment in conditional ADAM17 knockout mice during bacterial infection. Disrupting only the shedding of L-selectin, however, did not increase bacterial clearance, indicating that additional substrates also contribute to the detrimental role of ADAM17 during severe infection.

Acute intrahepatic CD8 T cell activation by plasmid DNA induces defective CD8 T cell immune response. (52.3)

Abstract The fate of CD8 T cells primed in the liver has been a subject of controversy in the attempt to explain the failure of immunity in chronic liver infections such as HBV, HCV and malaria. While most models show activation of CD8 T cells when liver resident cells present antigen, there are differences in opinion as to what becomes of these T cells after their initial priming. In this study, we used hydrodynamic injection that mimics acute HBV/HCV infection to express antigen in the liver, while taking advantage of the well-established LCMV P14 system, to induce acute intrahepatic T cells activation. We observed comparable CD8 T cells proliferation, up-regulation of CD44 and CD25, down-regulation of L-selectin, to the control (activation in lymphoid tissues). However, CD8 T cells primed in the liver expressed higher levels of pro-apoptotic markers such as Bim and active caspase 3, resulting in the loss of activated cells in all tissues. These liver activated CD8 T cells were incapable of protecting mice from lethal intracranial LCMV challenge. Our results confirm the up-regulation of ‘death markers’ during CD8 T cell priming in the liver, which leads to deletion of activated CD8 T cells needed to control viral infections in the liver. Thus, the failure of T cells to control chronic HBV/HCV is more likely due to defective liver priming of antigen-specific T cells.

Monocytes from sickle cell disease patients induce differential pulmonary endothelial gene expression via activation of NF-κB signaling pathway

Monocyte–endothelial interactions play an important role in inflammatory diseases and may modulate vasculopathy in sickle cell disease, a disorder with an important inflammatory component. We co-incubated normal and sickle monocytes, lymphocytes and TNF-α with pulmonary microvascular and arterial endothelial cells and compared the expression of genes coding for adhesion molecules and cytokines that might contribute to sickle vasoocclusion. Monocyte–endothelial cell co-incubation resulted in up-regulation of L-selectin, E-selectin, VCAM-1, ICAM-1, MCP-1, MMP-1, TNF-α, IL-6 and IL-1β and down-regulation of eNOS. Lymphocyte–endothelial cell co-incubations, induced similar effects restricted to pulmonary artery endothelial cells. TNF-α had similar effects on the endothelial cells as monocytes did, however monocyte induced gene expression in endothelial cells was not TNF-α dependent but was regulated through the NF-κB pathway. Sickle monocytes lead to altered expression of L-selectin, MCP-1 and MMP-1 in pulmonary vascular endothelium when compared with normal monocytes. The gene expression changes we observed could reflect pathological events of sickle vasoocclusion.

ADAM17 Activity and Other Mechanisms of Soluble L-selectin Production during Death Receptor-Induced Leukocyte Apoptosis

L-selectin is an adhesion molecule expressed by neutrophils that broadly directs their infiltration in to sites of inflammation. It is also present at relatively high levels in the serum of normal individuals. It is well established that L-selectin is efficiently shed from the surface of neutrophils upon their activation, a process that regulates its density and binding activity. Neutrophil programmed cell death is critical for the resolution of inflammation, and L-selectin downregulation is induced during this process as well. The mechanisms underpinning this latter process are much less understood, and were investigated in this study. Using a disintegrin and metalloprotease (ADAM)-17 radiation chimeric mice, we demonstrate for the first time that during early events of death receptor-mediated neutrophil apoptosis, L-selectin downregulation occurs primarily by ADAM17-mediated shedding. This was observed as well upon using shRNA to knock down ADAM17 expression in Jurkat cells, a well-studied cell line in terms of the molecular processes involved in the induction of apoptosis. These findings directly reveal that ADAM17 activity occurs during programmed cell death. Hence, the cleavage of particular ADAM17 substrates may be an additional component of the anti-inflammatory program initiated by apoptotic neutrophils. Of interest was that during later stages of induced leukocyte apoptosis, soluble L-selectin production occurred independent of ADAM17, as well as membrane events, such as blebbing and microparticle production. This process may provide an explanation for the lack of diminished serum L-selectin levels in ADAM17-null mice, and suggests a mechanism for the homeostatic maintenance of soluble L-selectin levels in the blood of healthy individuals.

Hydrogen Sulfide Improves Neutrophil Migration and Survival in Sepsis via K+ATPChannel Activation

Recovering the neutrophil migration to the infectious focus improves survival in severe sepsis. Recently, we demonstrated that the cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) pathway increased neutrophil recruitment to inflammatory focus during sterile inflammation. To evaluate if H(2)S administration increases neutrophil migration to infectious focus and survival of mice. Sepsis was induced by cecal ligation and puncture (CLP). The pretreatments of mice with H(2)S donors (NaHS or Lawesson's reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung lesions were prevented, and the survival rate increased from approximately 13% to approximately 80%. Even when treatment was delayed (6 h after CLP), a highly significant reduction in mortality compared with untreated mice was observed. Moreover, H(2)S pretreatment prevented the down-regulation of CXCR2 and l-selectin and the up-regulation of CD11b and G protein-coupled receptor kinase 2 in neutrophils during sepsis. H(2)S also prevented the reduction of intercellular adhesion molecule-1 expression in the endothelium of the mesenteric microcirculation in severe sepsis. Confirming the critical role of H(2)S on sepsis outcome, pretreatment with dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to the infectious focus, enhanced lung lesions, and induced high mortality in mice subjected to nonsevere sepsis (from 0 to approximately 80%). The beneficial effects of H(2)S were blocked by glibenclamide (a ATP-dependent K(+) channel blocker). These results showed that H(2)S restores neutrophil migration to the infectious focus and improves survival outcome in severe sepsis by an ATP-dependent K(+) channel-dependent mechanism.

Cyclical expression of L-selectin (CD62L) by recirculating T cells

L-Selectin (CD62L) mediates T-cell entry into lymph nodes. Whether the microenvironment modulates L-selectin expression of T cells during diapedesis and transit is unknown. Therefore, L-selectin expression was determined quantitatively on circulating T cells in blood, lymph nodes and thoracic duct by confocal laser scanning microscopy. We show that in contrast to leukocyte function-associated antigen-1 (CD11a/CD18) and ICAM-1 (CD54), L-selectin expression is cyclically expressed on recirculating T cells. It is reduced to approximately 30% of the blood value during entry across high endothelial venules. Within lymph nodes, CD4(+) T-cell subsets maintain reduced L-selectin expression at a similar level in all compartments (T-cell zone, B-cell zone and medulla). After exit, L-selectin is re-expressed to levels comparable to those of T cells in blood. Apparently, L-selectin levels are not only down-regulated during T-cell activation but also routinely reduced while transmigrating within lymph nodes. L-Selectin down-regulation seems to be ligand independent since it also occurs in the white pulp compartments of the spleen which lack classic L-selectin ligands such as GlyCAM-1 and CD34. In addition, T cells in non-lymphoid organs do not reveal reduced L-selectin levels. Thus, the ability of secondary lymphoid organs to reduce L-selectin expression of T cells prior to activation might be a prerequisite for their characteristic property to induce primary immune responses.

Leukocyte activation: The link between inflammation and coagulation during heatstroke. A study of patients during the 2003 heat wave in Paris*

The mechanisms linking severe inflammation and coagulation during heatstroke are poorly understood. Here, we examined the roles of the tissue factor pathway, leukocyte activation, and mediators of innate immunity in patients admitted to an intensive care unit for heatstroke during an intense heat wave in Paris. Retrospective observational study. Intensive care unit of a university medical center. Eighteen critically ill severe patients with heatstroke were enrolled in the study and 14 age-matched patients with severe sepsis as controls. None. High circulating levels of some inflammation and stress mediators (interleukin-6, -8, C5a, interleukin-1 receptor antagonist, heat shock protein 60 and 70) were observed. Blood leukocyte activation was shown by beta2 integrin up-regulation, L-selectin down-regulation, and strong production of reactive oxygen species and interleukin-8 ex vivo. High levels of circulating promatrix metalloproteinase-9 were detected in all the patients studied, and its active form was present in two patients. Overt disseminated intravascular coagulation according to the International Society of Thrombosis and Hemostasis score was present in five patients. Whole-blood tissue factor was present in all the patients and part of this activity was associated with microparticles in five patients. The degrees of inflammation and disseminated intravascular coagulation are correlated with clinical severity. These results suggest that neutrophil activation plays a key role in the acute activation of coagulation observed during severe heatstroke, despite a rapid and sustained antiinflammatory response. The comparison with a group of patients with severe sepsis suggests some common mechanisms, but more intense responses during heatstroke.

A comparative study of the effects of quercetin and its glucuronide and sulfate metabolites on human neutrophil function in vitro

Exposure of neutrophils to either lipopolysaccharide (LPS) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) is associated with changes in the expression of cell adhesion molecules and elevation of intracellular calcium ions. Although dietary flavonoids are reported to possess anti-inflammatory properties, little is known regarding the effect of their metabolites. We have investigated the effects of quercetin and its major metabolites on LPS and fMLP-stimulated human neutrophils using concentrations comparable to those reported in feeding studies on human volunteers. The metabolite quercetin 3-glucuronide caused a significant reduction in fMLP-evoked calcium influx in human neutrophils (approximately 35%), while neither quercetin 3′-sulfate nor quercetin produced a similar change. Acute exposure of human neutrophils to LPS altered cell shape and surface expression of CD16, but neither of these events were significantly altered by quercetin, quercetin 3-glucuronide nor quercetin 3′-sulfate. In addition, LPS caused a fivefold up-regulation in the expression of β 2-integrin (CD11b/Mac 1) and a concomitant 70% down-regulation of L-selectin (CD62L) adhesion molecule expression in human neutrophils. Neither effect was altered by quercetin, quercetin 3-glucuronide or quercetin 3′-sulfate. In conclusion, we found that acute exposure to quercetin and quercetin 3′-sulfate does not affect either expression of cell adhesion molecules or the elevation of intracellular calcium ions in response to LPS and fMLP in human neutrophils. However, quercetin 3-glucuronide reduced fMLP-evoked calcium responses. While this study highlights that metabolites of quercetin may possess different biological properties, dietary ingestion of quercetin is unlikely to exert a major effect on neutrophil function in vivo.

Selenium supplementation induces metalloproteinase-dependent L-selectin shedding from monocytes

Selenium therapy in patients with severe sepsis improves clinical outcome and has been associated with increased activity of the selenoprotein glutathione peroxidase. However, the mechanism of the observed beneficial effects remains unclear. We determined the effect of selenium treatment on the monocyte adhesion molecule L-selectin and L-selectin-related monocyte functions in vitro and transferred our findings to an in vivo mouse model. Monocytes were purified, cultured, and incubated in the presence or absence of supplemented selenium and metalloproteinase (MP) inhibitors for up to 16 h. Expression of L-selectin was unaffected after 2 and 6 h but decreased after 16 h of incubation in the presence of selenium. Soluble L-selectin (sL-selectin) in the supernatant was determined by ELISA. A 2.3-fold increase as a result of shedding of L-selectin was observed after 16 h of selenium treatment. Addition of the MP inhibitors GM6001, TNF-alpha-converting enzyme inhibitor 2, or GW280264X strongly reduced selenium-induced L-selectin shedding, indicating a MP-dependent mechanism. The functional consequences of L-selectin shedding were examined in a flow chamber model. Selenium-treated monocytes showed significantly decreased rolling and adhesion to the L-selectin ligand Sialyl-Lewis(a) under conditions of venous shear stress (0.5 dyne/cm(2)). Selenium treatment of C57BL6 mice led to increased serum levels of sL-selectin, underscoring the in vivo relevance of our findings. We describe a selenium-induced down-regulation of L-selectin on monocytes as a consequence of MP-dependent shedding of this membrane-anchored adhesion molecule. The impairment of monocyte adhesion by selenium supplementation may represent an important, underlying mechanism for the modulation of inflammatory reactions in patients with severe sepsis.

CD62L (L-Selectin) Down-Regulation Does Not Affect Memory T Cell Distribution but Failure to Shed Compromises Anti-Viral Immunity

The down-regulation of CD62L that accompanies T lymphocyte activation is thought to redirect cells away from lymph nodes to sites of infection. In this study, CD62L was maintained on Ag-activated T cells and their distribution, and ability to clear pathogen from peripheral sites determined. CD62L was down-regulated on Ag-specific CD8 T cells in lungs of C57BL/6 mice but maintained in CD62L transgenic mice at day 8 after influenza infection. However, the numbers of influenza-specific CD8 T cells recruited were similar in CD62L transgenic and C57BL/6 mice. Memory CD8 T cell numbers in the lungs and noninvolved organs 100 days after primary infection were similar in CD62L transgenic and C57BL/6 mice, despite differing CD62L expression. Transgenic mice expressing wild-type CD62L cleared a recombinant vaccinia virus expressing an influenza-derived CD8 T cell epitope as efficiently as C57BL/6 mice. However, transgenic mice expressing a protease resistant mutant of CD62L showed significantly delayed viral clearance, despite normal CTL generation and the presence of CD107a and IFN-gamma expressing influenza-specific CD8 T cells. These results demonstrate that CD62L down-regulation is not required for CD8 memory cells to home to sites of infection. However, their ability to clear virus is significantly compromised if CD62L shedding is abrogated.

High Efficiency Retro- and Lentiviral Transduction of Blood Endothelial Outgrowth Cells

Blood endothelial outgrowth cells (BEOC), bone marrow derived cells circulating in adult peripheral blood, may play an important role in postnatal vasculogenesis. Overexpression of antithrombotic genes in BOEC might enhance thromboresistance and open new avenues for the treatment of prosthetic and vascular thromboembolic disease. To genetically modify endothelial progenitor cells (EPC), we have used two different culture conditions for the generation of EPC from peripheral blood. Mononuclear cells harvested from peripheral blood of healthy volunteers by Ficoll-density gradient centrifugation were cultured (1) in fibronectin (FN)-coated plates with Endocult medium or (2) in collagen-coated plates with EBM2-MV medium. Colonies were counted at day 5 or between days 14 – 25, respectively. Cells were phenotypically analyzed on days 7, 14, 21, 28 or between days 30 – 60, respectively, for the following antigens: leukocyte markers CD45 and CD14, and endothelial markers CD31, CD34, CD105, CD141, CD144, CD146, and von-Willebrand (vW) antigen. Stem cell microarray hybridization was used to analyze the gene expression pattern. Proliferative potential was evaluated by determination of replating efficiency, growth kinetics, and CFSE dilution analysis. Using nonadherent cells cultured on FN-coated plates, colonies appeared on days 4–5. They consisted of central round cells with elongated cells sprouting at the periphery. These cells slowly disappeared after days 8–10. FACS analysis of these cells showed strong expression of CD45 and CD14, weak expression of CD31, but no expression of CD105, CD34, and vW-antigen. Using cells adherent to collagen, cells appeared at a median of 20 ± 5 days (range 9 – 29 days). These cells rapidly formed colonies with a cobblestone-like appearance and subsequently a confluent monolayer. After a lag phase, cell expanded exponentially (21 population doublings in 50–60 days). These cells showed strong expression of CD31, CD105, and CD146, intermediate expression of CD141, weak expression of CD34, and no expression of CD45 or CD14. Cells stained positive for VE-cadherin and vW-antigen. Microarray analysis displayed upregulated expression of endothelial genes such as PAI-1, uPA, eNOS, vWF, CD105, CD141, CD144, CD146, EphrinB4, TIE-1, VEGF-R2, endothelin and other genes such as CTGF, MMP2, ID1, ID3, TBX1, GATA2, FKHL16, ROBO4, but downregulation of CD11c, CD18, and L-selectin. An eGFP-encoding retroviral SFFV vector (cell free supernatant from PG13; titer 1.5x106 cfu) and a novel lentiviral LeGO vector expressing the red chomophore tdTomato (cell-free supernatant from PhoenixGP or 293T, packaging plasmids pMDLg/pRRE, pRSV-Rev; GALV env; 16x106 cfu) were used for centrifugation-enhanced transduction. BEOC were transduced with high efficiency, 62% – 80% and 91% – 99%, using the murine retroviral SF-eGFP and the human LeGo-T lentiviral vectors, respectively. Our results suggest that nonadherent cells cultured on FN-coated plates have a low proliferative potential and display an angiogenic macrophage-like phenotype. In contrast, adherent cells cultured on collagen-coated plates have a high proliferative potential, display an endothelial phenotype without coexpression of leukocyte antigens, and can be very efficiently transduced using retroviral vectors. Blood endothelial outgrowth cells may be an excellent autologous biomaterial source for vascular graft and device coatings, as well as for antithrombotic gene therapy.

Securinine, a GABAA receptor antagonist, enhances macrophage clearance of phase IIC. burnetii: comparison with TLR agonists

Innate immune cell stimulation represents a complementary approach to vaccines and antimicrobial drugs to counter infectious disease. We have used assays of macrophage activation and in vitro and in vivo phase II Coxiella burnetii infection models to compare and contrast the activity of a novel innate immune cell agonist, securinine, with known TLR agonists. As expected, TLR agonists, such as LPS (TLR4) and fibroblast-stimulating lipopeptide-1 (FSL-1; TLR2), induced macrophage activation and increased macrophage killing of phase II C. burnetii in vitro. FSL-1 also induced accelerated killing of C. burnetii in vivo. Securinine, a gamma-aminobutyric acid type A receptor antagonist, was found to induce TLR-independent macrophage activation in vitro, leading to IL-8 secretion, L-selectin down-regulation, and CD11b and MHC Class II antigen up-regulation. As seen with the TLR agonists, securinine also induced accelerated macrophage killing of C. burnetii in vitro and in vivo. In summary, as predicted by the literature, TLR agonists enhance macrophage killing of phase II C. burnetii in vitro, and at least for TLR2 agonists, this activity occurs in vivo as well. Securinine represents a novel macrophage agonist, which has similar effects as TLR agonists in this model yet apparently, does not act through known TLRs. Securinine has minimal toxicity in vivo, suggesting it or structurally similar compounds may represent novel, therapeutic adjuvants, which increase resistance to intracellular pathogens.

Chronic Immunodeficiency in Mice Lacking RasGRP1 Results in CD4 T Cell Immune Activation and Exhaustion

The Ras-guanyl nucleotide exchange factor RasGRP1 is an important link between TCR-mediated signaling and the activation of Ras and its downstream effectors. RasGRP1 is especially critical for the survival and differentiation of developing thymocytes whereas negative selection of thymocytes bearing an autoreactive TCR appears to be RasGRP1 independent. Despite apparently normal central tolerance, RasGRP1(-/-) mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion, including strong expression of programmed cell death-1. To elucidate the basis for RasGRP1(-/-) CD4 T cell immune activation, we initiated a series of adoptive transfer experiments. Remarkably, the copious amounts of cytokines and self-Ags present in hosts made lymphopenic through irradiation failed to induce the majority of RasGRP1(-/-) CD4 T cells to enter cell cycle. However, their infusion into either congenitally T cell- or T/B cell-deficient recipients resulted in robust proliferation and L-selectin down-regulation. These findings imply that the activation and proliferation of RasGRP1(-/-) CD4 T cells may be dependent on their residence in a chronically immunocompromised environment. Accordingly, bacterial and viral challenge experiments revealed that RasGRP1(-/-) mice possess a weakened immune system, exhibiting a T cell-autonomous defect in generating pathogen-specific T cells and delayed pathogen clearance. Collectively, our study suggests that chronic T cell immunodeficiency in RasGRP1(-/-) mice may be responsible for CD4 T cell activation, proliferation, and exhaustion.

Immunohistochemical localization of adhesion molecules (CD62 and CD18) in the mammary gland of dairy cows

Localization of the L-selectin (CD62L) and β2-integrin (CD18) bearing cells in different tissues of the bovine mammary gland was examined. Five dairy cows of Holstein-Friesian breed in the middle of their second and third lactation cycle were used in the study. Blood, milk and udder tissue samples were collected from each cow to estimate the milk somatic cell count (SCC) and bacteriological infection of the mammary gland. The expression of CD62L and CD18 on blood cells, milk cells and parenchymal tissues of udder, Fürstenberg’s rosette and the transverse section of the central part of the teat was tested. In the mammary gland quarters the value of SCC in milk secretion was also reflected in the presence of CD18<sup>+</sup> and CD62L<sup>+</sup> leukocytes in mammary tissues. In the quarters where SCC was higher than 10<sup>5</sup> a frequent incidence of L-selectin and β2-integrin cells was observed in the parenchyma and Fürstenberg’s rosette region, while in the quarters with low SCC, none or only a few reactive cells were found. In the mammary parenchymal tissue CD18 positive cells were present in both the epithelial and the connective tissue. In Fürstenberg’s rosette the cells were concentrated in the connective collagenous and loose tissue. None or only scattered L-selectin (CD62L) and β2-integrin (CD18) bearing cells were identified in the transverse section from the central part of the teat. When we compared the L-selectin and β2-integrin expression, the study revealed the down-regulation of L-selectin on the cells of mammary tissue.

Epinephrine Enhances Platelet-Neutrophil Adhesion in Whole Blood In Vitro

Previous studies showed that alpha- or beta-adrenoceptor stimulation by catecholamines influenced neutrophil function, cytokine liberation, and platelet aggregability. We investigated whether adrenergic stimulation with epinephrine also alters platelet-neutrophil adhesion. This might be of specific interest in the critically ill, because the increased association of platelets and neutrophils has been shown to be of key importance in inflammation and thrombosis. For this purpose, whole blood was incubated with increasing concentrations of epinephrine (10 nM, 100 nM, and 1 microM). To distinguish receptor-specific effects, a subset of samples was incubated with propranolol (10 microM) or phentolamine (10 microM) before exposure to epinephrine. After incubation, another subset of samples was also stimulated with 100 nM of N-formyl-methionyl-leucyl-phenylalanine. All samples were stained, and platelet-neutrophil adhesion and CD45, L-selectin, CD11b, P-selectin glycoprotein ligand-1, glycoprotein IIb/IIIa, and P-selectin expression were measured by two-color flow cytometry. Epinephrine significantly enhanced platelet-neutrophil adhesion and P-selectin and glycoprotein IIb/IIIa expression on platelets. CD11b and L-selectin expression on unstimulated neutrophils remained unchanged, whereas N-formyl-methionyl-leucyl-phenylalanine-induced upregulation of CD11b and downregulation of L-selectin were suppressed by epinephrine. beta-Adrenergic blockade before incubation with epinephrine increased platelet-neutrophil aggregates and adhesion molecule expression (CD11b, P-selectin, and glycoprotein IIb/IIIa) even further. These results demonstrate that epinephrine enhances platelet-neutrophil adhesion. The alpha-adrenergic receptor-mediated increase in P-selectin and glycoprotein IIb/IIIa expression on platelets may contribute substantially to this effect. Our study shows that inotropic support enhances the platelet-neutrophil interaction, which might be crucial for critically ill patients.