参考文献

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies,yet the molecular effectors of this process are relatively uncharacterized. For extravasation,studies based predominantly on static transwell assays support a general model,whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However,this model of chemotaxis cannot readily reconcile how chemokines influence intravasation,as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus,to analyze how T cells integrate perivascular chemokine signals under physiologic flow,we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment,transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However,when exposedto abluminal CXCL12,transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent,augmented by apical flow,blocked by antibodies to integrins,and reduced by AMD3100 in a dose-dependent manner. Moreover,CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream. View Publication

C57BL/6 (B6) mice are genetically highly susceptible to chronic type II Toxoplasma gondii infections that invariably cause lethal toxoplasmic encephalitis. We examined the ability of an attenuated type I vaccine strain to elicit long-term immunity to lethal acute or chronic type II infections in susceptible B6 mice. Mice immunized with the type I cps1-1 vaccine strain were not susceptible to a lethal (100-cyst) challenge with the type II strain ME49. Immunized mice challenged with 10 ME49 cysts exhibited significant reductions in brain cyst and parasite burdens compared to naive mice,regardless of the route of challenge infection. Remarkably,cps1-1 strain-immunized B6 mice chronically infected with ME49 survived for at least 12 months without succumbing to the chronic infection. Potent immunity to type II challenge infections persisted for at least 10 months after vaccination. While the cps1-1 strain-elicited immunity did not prevent the establishment of a chronic infection or clear established brain cysts,cps1-1 strain-elicited CD8(+) immune T cells significantly inhibited recrudescence of brain cysts during chronic ME49 infection. In addition,we show that uracil starvation of the cps1-1 strain induces early markers of bradyzoite differentiation. Collectively,these results suggest that more effective immune control of chronic type II infection in the genetically susceptible B6 background is established by vaccination with the nonreplicating type I uracil auxotroph cps1-1 strain. View Publication

Tandem pleckstrin homology domain proteins (TAPPs) are recruited to the plasma membrane via binding to phosphoinositides produced by phosphoinositide 3-kinases (PI3Ks). Whereas PI3Ks are critical for B-cell activation,the functions of TAPP proteins in B cells are unknown. We have identified 40 potential interaction partners of TAPP2 in B cells,including proteins involved in cytoskeletal rearrangement,signal transduction and endocytic trafficking. The association of TAPP2 with the cytoskeletal proteins utrophin and syntrophin was confirmed by Western blotting. We found that TAPP2,syntrophin,and utrophin are coexpressed in normal human B cells and B-chronic lymphocytic leukemia (B-CLL) cells. TAPP2 and syntrophin expression in B-CLL was variable from patient to patient,with significantly higher expression in the more aggressive disease subset identified by zeta-chain-associated protein kinase of 70 kDa (ZAP70) expression and unmutated immunoglobulin heavy chain (IgH) genes. We examined whether TAPP can regulate cell adhesion,a known function of utrophin/syntrophin in other cell types. Expression of membrane-targeted TAPP2 enhanced B-cell adhesion to fibronectin and laminin,whereas PH domain-mutant TAPP2 inhibited adhesion. siRNA knockdown of TAPP2 or utrophin,or treatment with PI3K inhibitors,significantly inhibited adhesion. These findings identify TAPP2 as a novel link between PI3K signaling and the cytoskeleton with potential relevance for leukemia progression. View Publication

TNF-like ligand 1A (TL1A),a member of the TNF superfamily,is the ligand of DR3 and DcR3. Several types of cells,such as endothelial cells,monocytes/macrophages,dendritic cells,and CD4 and CD8 T cells,are capable of producing this cytokine. In present study,we demonstrated that TL1A aggravated collagen-induced arthritis in mice. It increased collagen-induced arthritis penetrance and clinical scores as well as the severity of the pathological findings. TL1A administration led to the occurrence of multiple enlarged germinal centers in the spleen,and it boosted serum anti-collagen Ab titers in vivo. In vitro,TL1A augmented TNF-alpha production by T cells upon TCR ligation,and it greatly enhanced Th17 differentiation and IL-17 production. We further showed that human rheumatoid arthritis (RA) synovial fluids had elevated TL1A titers,and human chrondrocytes and synovial fibroblasts were capable of secreting TL1A upon TNF-alpha or IL-1beta stimulation. Taken together,these data suggest that TL1A secretion in lymphoid organs might contribute to RA initiation by promoting autoantibody production,and TL1A secretion stimulated by inflammatory cytokines in RA joints might be a part of a vicious circle that aggravates RA pathogenesis. View Publication

IFN-beta-1a has been used over the past 15 years as a primary therapy for relapsing-remitting multiple sclerosis (MS). However,the immunomodulatory mechanisms that provide a therapeutic effect against this CNS inflammatory disease are not yet completely elucidated. The effect of IFN-beta-1a on Th17 cells,which play a critical role in the development of the autoimmune response,has not been extensively studied in humans. We have investigated the effect of IFN-beta-1a on dendritic cells (DCs) and naive CD4(+)CD45RA(+) T cells derived from untreated MS patients and healthy controls in the context of Th17 cell differentiation. We report that IFN-beta-1a treatment down-regulated the expression of IL-1beta and IL-23p19 in DCs,whereas it induced the gene expression of IL-12p35 and IL-27p28. We propose that IFN-beta-1a-mediated up-regulation of the suppressor of cytokine signaling 3 expression,induced via STAT3 phosphorylation,mediates IL-1beta and IL-23 down-regulation,while IFN-beta-1a-induced STAT1 phosphorylation induces IL-27p28 expression. CD4(+)CD45RA(+) naive T cells cocultured with supernatants from IFN-beta-1a-treated DCs exhibited decreased gene expression of the Th17 cell markers retinoic acid-related orphan nuclear hormone receptor c (RORc),IL-17A,and IL-23R. A direct IFN-beta-1a treatment of CD45RA(+) T cells cultured in Th17-polarizing conditions also down-regulated RORc,IL-17A,and IL-23R,but up-regulated IL-10 gene expression. Studies of the mechanisms involved in the Th17 cell differentiation suggest that IFN-beta-1a inhibits IL-17 and induces IL-10 secretion via activated STAT1 and STAT3,respectively. IFN-beta's suppression of Th17 cell differentiation may represent its most relevant mechanism of selective suppression of the autoimmune response in MS. View Publication

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses,the function of some,like virion protein 11/12 (VP11/12),have not been clearly defined. Previously,we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis,M. J. Wagner,R. C. Doepker,J. M. Maciejko,C. M. Crider,K. R. Jerome,and J. R. Smiley,J. Virol. 82:6098-6108,2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here,we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394),a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However,we were unable to detect an interaction between ICP0 and Lck in T lymphocytes,and we show that ICP0,US3,and UL13 are not strictly required for Lck activation. In contrast,VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus,VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. View Publication

Mtb influences DC activity and T cell-mediated immune responses. We show that the treatment of immature monocyte-derived DC with Mtb elicited the formation of mature DC,producing TNF-alpha,IL-1beta,IL-6,and IL-23 and instructing CD4(+) cells to secrete IFN-gamma and IL-17. Mtb-induced cytokine release by DC depended on dectin-1 receptor engagement,whereas MR or DC-SIGN stimulation inhibited this process. A selective dectin-1 binding by the receptor agonist glucan was sufficient to enable DC to generate Th1/Th17 lymphocytes,showing features comparable with those induced by Mtb-treated DC. Interestingly,DC-SIGN or MR engagement inhibited Th17 and increased Th1 generation by glucan- or Mtb-treated DC. Our results indicate that Mtb modulates the lymphocyte response by affecting DC maturation and cytokine release. Dectin-1 engagement by Mtb enables DC to promote a Th1/Th17 response,whereas DC-SIGN and MR costimulation limits dectin-1-dependent Th17 generation and favors a Th1 response,probably by interfering with release of cytokines. View Publication

Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL),its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1),the AKT modulator and proto-oncogene,is differentially expressed in CLL and linked to its pathogenesis based on CD5(+) B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes,which further included the kinases LYN,SYK,ZAP70,and PKC. High TCL1 levels were also strongly associated with aggressive disease features,such as advanced clinical stage,higher white blood cell counts,and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie,shorter progression-free survival,P textless .001),regardless of therapy regimen,especially for ZAP70(+) tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful. View Publication

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium,following acute myocardial infarction,may be,in part,due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep,keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells,endothelial progenitor cells,T lymphocytes,B lymphocytes and NK cells using cell surface markers CD34(+),CD133(+)VEGFR2(+),CD45(+)CD3(+),CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+),respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells,granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep. View Publication

The type III family of IFNs displays immunomodulatory and antiviral activity. Each member (IFN-lambda1,-2,and -3) signals through the same heterodimeric receptor complex,which consists of the binding and signaling subunit (IL-28Ralpha) plus the IL-10Rbeta chain. Although the receptor has a wide tissue distribution,the direct effects of IFN-lambda on various immune cell subsets have not been fully characterized. We have identified high levels of IL-28Ralpha mRNA in pDC from peripheral blood and hypothesized that IFN-lambda plays an important role in pDC maturation and development. We show that stimulation of pDC with HSV or Imiquimod causes an increase in IL-28Ralpha mRNA. In these cells,IFN-lambda1 alters expression of the costimulatory molecules CD80 and ICOS-L and synergizes with IFN-alpha to up-regulate CD83. In addition,IFN-lambda1 has a variable effect on the homing molecule expression of pDC and mDC. IFN-lambda1-treated pDC display a marked difference in their ability to stimulate production of the signature cytokines IL-13,IFN-gamma,and IL-10 in a MLR. This work characterizes the variable effects of IFN-lambda on DC surface molecule expression and identifies a role in pDC activation and immunostimulatory potential. View Publication