技术资料

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model,we investigated whether expression of CD1a and langerin,an LC-specific C-type lectin,imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin,placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease. View Publication

The deregulation of the immune response is a critical component in inflammatory disease. Recent in vitro data show that T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of cytokine signaling. Furthermore,tc-ptp(-/-) mice display immune defects and die within 5 weeks of birth. We report here that tc-ptp(-/-) mice develop progressive systemic inflammatory disease as shown by chronic myocarditis,gastritis,nephritis,and sialadenitis as well as elevated serum interferon-gamma. The widespread mononuclear cellular infiltrates correlate with exaggerated interferon-gamma,tumor necrosis factor-alpha,interleukin-12,and nitric oxide production in vivo. Macrophages grown from tc-ptp(-/-) mice are inherently hypersensitive to lipopolysaccharide,which can also be detected in vivo as an increased susceptibility to endotoxic shock. These results identify T-cell protein tyrosine phosphatase as a key modulator of inflammatory signals and macrophage function. View Publication

Inter-alpha inhibitor protein (IalphaIp) is an endogenous serine protease inhibitor in human plasma. Circulating IalphaIp levels were lower in 51 patients with severe sepsis than in healthy volunteers. Mean levels were 688+/-295 mg/L in patients with severe sepsis who survived (n=32),486+/-193 mg/L in patients with sepsis who died (n=19),and 872+/-234 mg/L in control subjects (n=25). IalphaIp levels were lower in patients with shock versus those without (540+/-246 [n=33] vs. 746+/-290 [n=18] mg/L; P=.0102). IalphaIp levels were inversely correlated with 28-day mortality rates and Acute Physiology and Chronic Health Evaluation II scores and directly correlated with antithrombin III,protein C,and protein S levels. The administration of IalphaIp (30 mg/kg body weight intravenously) increased the 50% lethal dose in mice by 100-fold after an intravenous challenge of Escherichia coli. Thus,human IalphaIp may be a useful predictive marker and potential therapeutic agent in sepsis. View Publication

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor,including ULBP-1,-2,and -3,but not MICA or MICB,in infected cells both in vitro and in vivo. However,the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G(2) cell-cycle arrest,conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4(+) T lymphocytes by a process that is Vpr dependent. Importantly,Vpr enhanced the susceptibility of HIV-1-infected cells to NK cell-mediated killing. Strikingly,Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell-mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells,suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall,these results indicate that Vpr is a key determinant responsible for HIV-1-induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced CD4(+) T-lymphocyte depletion but may also take part in HIV-1-induced NK-cell dysfunction. View Publication

Rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles that code a five-amino acid sequence motif in positions 70-74 of the HLA-DRbeta-chain,called the shared epitope (SE). The mechanistic basis of SE-RA association is unknown. We recently found that the SE functions as an allele-specific signal-transducing ligand that activates an NO-mediated pathway in other cells. To better understand the role of the SE in the immune system,we examined its effect on T cell polarization in mice. In CD11c(+)CD8(+) dendritic cells (DCs),the SE inhibited the enzymatic activity of indoleamine 2,3 dioxygenase,a key enzyme in immune tolerance and T cell regulation,whereas in CD11c(+)CD8(-) DCs,the ligand activated robust production of IL-6. When SE-activated DCs were cocultured with CD4(+) T cells,the differentiation of Foxp3(+) T regulatory cells was suppressed,whereas Th17 cells were expanded. The polarizing effects could be seen with SE(+) synthetic peptides,but even more so when the SE was in its natural tridimensional conformation as part of HLA-DR tetrameric proteins. In vivo administration of the SE ligand resulted in a greater abundance of Th17 cells in the draining lymph nodes and increased IL-17 production by splenocytes. Thus,we conclude that the SE acts as a potent immune-stimulatory ligand that can polarize T cell differentiation toward Th17 cells,a T cell subset that was recently implicated in the pathogenesis of autoimmune diseases,including RA. View Publication

The NKG2D receptor activates natural killer (NK) cell cytotoxicity and cytokine production on recognition of self-molecules induced by cellular stress under different conditions such as viral infections. The importance of NKG2D in the immune response to human cytomegalovirus (HCMV) is supported by the identification of several viral molecules that prevent the expression of NKG2D ligands by infected cells. In this study we report that,paradoxically,a significant,selective,and transient reduction of NKG2D expression on NK cells is detected during HCMV infection of peripheral blood mononuclear cells if needed. Antagonizing type I interferon (IFN),interleukin-12 (IL-12),and IFNgamma prevented HCMV-induced down-regulation of surface NKG2D. Moreover,treatment of purified NK cells with recombinant IFNbeta1 and IL-12 mimicked the effect,supporting a direct role of these cytokines in regulating NKG2D surface expression in NK cells. The loss of NKG2D expression selectively impaired NK-cell cytotoxicity against cells expressing NKG2D ligands but preserved the response triggered through other activating receptors. These results support that down-regulation of NKG2D expression on NK cells by cytokines with a key role in antiviral immune response may constitute a physiologic mechanism to control NK-cell reactivity against normal cells expressing NKG2D ligands in the context of inflammatory responses to viral infections. View Publication

The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-lambda1 (interleukin-29 [IL-29]),IFN-lambda2 (IL-28A),and IFN-lambda3 (IL-28B),each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-lambda producers among peripheral lymphocytes. Recently,it has been shown that IFN-lambda1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4(+) T cells and inhibiting the production of Th2-associated cytokines. Here,we asked whether Th2 cytokines exert reciprocal control over IFN-lambda production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-lambda1 transcription and secretion. However,pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments,we showed that IL-4-responsive monocytes are an intermediary cell,responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-lambda1 output. Thus,our experiments revealed a novel mechanism for regulation of both IFN-lambda1 production and pDC function,and suggests an expanded immunomodulatory role for Th2-associated cytokines. View Publication

Coinfection of HIV-1 patients with Plasmodium falciparum,the etiological agent of malaria,results in a raise of viral load and an acceleration of disease progression. The primary objective of this study was to investigate whether the malarial pigment hemozoin (HZ),a heme by-product of hemoglobin digestion by malaria parasites,can affect HIV-1 transmission by monocytes-derived dendritic cells (DCs) to CD4(+) T cells when HZ is initially internalized in monocytes before their differentiation in DCs. We demonstrate in this study that HZ treatment during the differentiation process induces an intermediate maturation phenotype when compared with immature and fully mature DCs. Furthermore,the DC-mediated transfer of HIV-1 is enhanced in presence of HZ,a phenomenon that may be linked with the capacity of HZ-loaded cells to interact and activate CD4(+) T cells. Altogether our findings suggest a new mechanism that could partially explain the increased HIV-1 virus production during a coinfection with P. falciparum. Understanding the multifaceted interactions between P. falciparum and HIV-1 is an important challenge that could lead to the development of new treatment strategies. View Publication

Macrophages have a central role in the pathogenesis of cryptococcosis since they are an important line of defense,serve as a site for fungal replication,and also can contribute to tissue damage. The objective of this study was to investigate the interaction of macrophages with cells from smooth-colony variants (SM) and mucoid-colony variants (MC) arising from phenotypic switching of Cryptococcus neoformans. Alveolar macrophages (AMs) isolated from SM- and MC-infected mice exhibited differences in gene and surface expression of PD-L1,PD-L2,and major histocompatibility class II (MHC-II). PD-L1 and PD-L2 are the ligands for PD1 and are differentially regulated in Th1- and Th2-type cells. In addition,macrophage activation in SM- and MC-infected mice was characterized as alternatively activated. Flow cytometric and cytokine analysis demonstrated that MC infection was associated with the emergence of Th17 cells and higher levels of interleukin-17 (IL-17) in lung tissue,which were reduced by AM depletion. In conclusion,our results indicate that macrophages play a significant role in maintaining damage-promoting inflammation in the lung during MC infection,which ultimately results in death. 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

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

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