Lipopolysaccharide induces physical proximity between CD14 and toll-like receptor 4 (TLR4) prior to nuclear translocation of NF-kappa B

CD14, a GPI-linked protein, plays a pivotal role in LPS-mediated signaling by potentiating leukocyte adherence, activation, and cytokine production. Recent studies have identified the Toll-like receptor 4 (TLR4) as a membrane cofactor in LPS-mediated transmembrane signaling in cytokine induction, although the mechanism responsible for this cooperation is unknown. Using fluorescence resonance energy transfer (RET) techniques, we demonstrate that LPS triggers a physical association between CD14 and TLR4. Because LPS stimulation upregulates CD14 and TLR4 expression, it was necessary to control for the possibility that these newly expressed molecules were associated with one another independent of LPS stimulation. Although the calcium ionophore A23187 increased the expression of CD14 and TLR4, they did not exhibit energy transfer. However, following A23187 treatment, LPS promoted physical proximity between CD14 and TLR4. Therefore, we suggest that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NF-kappaB.     

Human toll-like receptor 2 mediates monocyte activation by Listeria monocytogenes, but not by group B streptococci or lipopolysaccharide

Human Toll like receptor (TLR) 2 has been implicated as a signaling receptor for LPS from Gram-negative bacteria and cell wall components from Gram-positive organisms. In this study, we investigated whether TLR2 can signal cell activation by the heat-killed group B streptococci type III (GBS) and Listeria monocytogenes (HKLM). HKLM, but not GBS, showed a time- and dose-dependent activation of Chinese hamster ovary cells transfected with human TLR2, as measured by translocation of NF-kappaB and induction of IL-6 production. A mAb recognizing a TLR2-associated epitope (TL2.1) was generated that inhibited IL-6 production from Chinese hamster ovary-TLR2 cells stimulated with HKLM or LPS. The TL2.1 mAb reduced HKLM-induced TNF production from human monocytes by 60%, whereas a CD14 mAb (3C10) reduced the TNF production by 30%. However, coadministrating TL2.1 and 3C10 inhibited the TNF response by 80%. In contrast to this, anti-CD14 blocked LPS-induced TNF production from monocytes, whereas anti-TLR2 showed no inhibition. Neither TL2.1 nor 3C10 affected GBS-induced TNF production. These results show that TLR2 can function as a signaling receptor for HKLM, possibly together with CD14, but that TLR2 is unlikely to be involved in cell activation by GBS. Furthermore, although LPS can activate transfected cell lines through TLR2, this receptor does not seem to be the main transducer of LPS activation of human monocytes. Thus, our data demonstrate the ability of TLR2 to distinguish between different pathogens.     

Engagement of CD14 on human monocytes terminates T cell proliferation by delivering a negative signal to T cells.

We have recently shown that engagement of the human monocytic Ag CD14 by murine mAb induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion. To determine whether CD14 plays a role in monocyte-T cell interactions, we tested the effect of anti-CD14 mAb on the proliferation of human T cells. Our results show that anti-CD14 mAb strongly inhibited T cell proliferation induced by Ag, anti-CD3 mAb, and mitogenic lectins. Inhibition by anti-CD14 mAb was epitope-dependent and required physical contact between monocytes and T cells. CD14 engagement did not affect IL-2R expression or IL-2 synthesis but induced a state of unresponsiveness that was not IL-2 specific; proliferation of anti-CD3-activated T cell blasts in response to both IL-2 and IL-4 was abrogated by addition of monocytes preincubated with anti-CD14 mAb. Inhibition of T cell proliferation after engagement of CD14 on monocytes was likely to result from delivery of a negative signal to T cells, rather than from disruption of a costimulatory monocyte-derived signal, because incubation of monocytes with anti-CD14 mAb also inhibited monocyte-independent T cell proliferation induced by PMA and ionophore. These results, together, point to a role of CD14 in the monocyte-dependent regulation of T cell proliferation.     

CD59、CD55在T细胞活化信号转导中的协同作用

目的:研究糖基磷脂酰肌醇(GPI)锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的协同效应。方法:应用siRNA技术,构建特异性针对CD55与CD59基因的重组载体pSUPER-siCD55,pSUPER-siCD59。实验分为未转染的Jurkat细胞组(Ⅰ组)、转染pSUPER空质粒的Jurkat细胞组(Ⅱ组)、转染pSUPER-siCD59重组质粒的Jurkat细胞组(Ⅲ组)及转染pSUPER-siCD55重组质粒的Jurkat细胞组(Ⅳ组)。RT-PCR检测转染细胞中CD55和CD59基因的表达噻唑蓝(MTT)比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对4组Jurkat细胞的增殖效应以及细胞内钙离子的变化、结果:稳定转染后,Ⅲ组细胞CD59分子的表达和Ⅳ组细胞CD55分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅲ组、Ⅳ组(P<0.05),Ⅰ组和Ⅱ组之间无差异结论:CD59和CD55在T细胞活化信号转导通路中存在协同效应。     

Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25- CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25-CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-gamma, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-kappaB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.     

CD59、CD55在T细胞活化信号转导中的协同作用

目的：研究糖基磷脂酰肌醇（GeD）锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的协同效应。方法：应用siRNA技术,构建特异性针对CD55与CD59基因的重组载体pSUPER—siCD55,pSUPER—siCD59。实验分为未转染的Jurkat细胞组（I组）、转染pSUPER空质粒的Jurkat细胞组（Ⅱ组）、转染pSUPER—siCD59重组质粒的Jurkat细胞组（Ⅲ组）及转染pSUPER—siCD55重组质粒的Jurkat细胞组（Ⅳ组）。RT—PCR检测转染细胞中CD55和CD59基因的表达。噻唑蓝（MTT）比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对4组Jurkat细胞的增殖效应以及细胞内钙离子的变化。结果：稳定转染后,Ⅲ组细胞CD59分子的表达和Ⅳ组细胞CD55分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅲ组、Ⅳ组（P〈0．05）,Ⅰ组和Ⅱ组之间无差异。结论：CD59和CD55在T细胞活化信号转导通路中存在协同效应。     

HUMAN B CELL DIFFERENTIATION: DEPENDENCE ON INTERACTIONS WITH MONOCYTES AND T LYMPHOCYTES VIA CD40, CD80 (B7.1), AND THE CD2-LIGANDS CD48 AND CD58 (LFA-3)

B cell differentiation depends on cellular interactions with T lymphocytes and monocytes via adhesion molecules (AM). In order to characterize AM which are required for B cell differentiation immunoglobulin production using unseparated peripheral blood mononuclear cells (PBMC) was studied. Unstimulated human PBMC were cultured for 9 days with mAb directed at CD2/CD48, /CD58, CD59, CD5/CD72, CD11a—CD18/CD54, CD28/CD80, CD86, CD40/CD40L, or rat CD2 (control). B cell differentiation was quantified measuring IgM and in some cases IgA, IgG, and IgE production. IgM levels were significantly reduced by mAb against CD40, CD48, CD58 and CD80. The reduction was not due to isotype switching to IgA, IgG or IgE. The role of CD40, CD48, CD58 and CD80 was further investigated after depletion of different cell types. Depletion of monocytes and NK cells resulted in no detectable IgM production irrespective of added mAbs. In contrast, IgM production was still present after depletion of T cells and NK cells. Only mAb against CD80 and CD48 significantly reduced IgM production, the reduction of IgM production by anti-CD40 mAb was less than in the presence of T cells. Importantly, anti-CD58 mAb had no effect on IgM production after T cell and NK cell depletion. Taken together, the AM CD40, CD48, CD58, and CD80 are involved in Ig production of unseparated PBMCs. In this model of B cell differentiation only the AM CD58 depend on the presence of T cells while CD48 and CD80 help was found to be T cell independent.     

Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes.

We used flow cytometry to determine how LPS-binding protein (LBP) effects the binding of fluorescein-labeled LPS to human monocytes via receptor-dependent mechanisms. The addition of human, rabbit, mouse, or FCS strikingly increased the binding of LPS to monocytes compared with controls incubated in serum-free medium. This binding was totally prevented by preincubation of monocytes with MY4, an anti-CD14 mAb, or by enzymatic removal of CD14 from monocytes. Depletion of LBP from rabbit serum with anti-LBP antibodies also produced a similar suppression. Solutions of albumin did not support the enhanced binding observed in serum but the addition of purified rabbit LBP to albumin solutions resulted in binding similar to that observed in serum-containing medium. When type-specific anti-LPS mAb was added to human serum, LPS binding to monocytes occurred but was only partly inhibited by anti-CD14 mAb, suggesting that receptors other than CD14 (presumably Fc or complement receptors) were involved. Serum increased by 100- to 1000-fold the sensitivity of monocytes to the triggering by LPS resulting in TNF secretion. TNF secretion was inhibited by anti-CD14 mAb up to 100 ng/ml of LPS and by anti-LPS mAb up to 1 to 10 ng/ml. The inhibition of TNF secretion by anti-LPS mAb appeared to be the result of directing LPS to monocyte receptors other than CD14. In contrast, in medium containing normal as well as acute serum and in the absence of anti-LPS antibodies, the binding of LPS to monocytes and the triggering of TNF secretion appeared to be mediated mainly by interactions between CD14 and LBP-LPS complexes.     

In vivo and in vitro regulation of type I IFN synthesis by synergistic effects of CD40 and type II IFN

During cognate interaction with CD40 ligand (CD154)-expressing T cells, Ag-presenting accessory cells are activated for increased cytokine synthetic and costimulatory function. We examined whether CD40 modulates in vivo innate immune function over time, hypothesizing that distinct cytokine responses evolve to delayed microbial exposure. C3H/HeN mice pretreated with activating anti-CD40 Ab (FGK45) produced 10-fold more serum IFN-gamma and IL-12 p70 to delayed, but not synchronous, challenge with LPS. A novel finding was that LPS-induced IFN-alpha increased by 20-fold in mice pretreated for 24 h, but not 6 h or less, with anti-CD40. Anti-CD40-pretreated C57BL/6 RAG-2(-/-) mice similarly increased IFN-alpha responses to delayed LPS challenge, confirming mediation by innate immunity. Type I IFNR- and IFN-gamma-deficient mice treated with anti-CD40 failed to expand serum IFN-alpha responses to LPS challenge. Combined pretreatment with anti-CD40 and anti-IFN-gamma mAb showed that IFN-gamma produced after anti-CD40 pretreatment, but before LPS challenge, was necessary for IFN-alpha synthetic enhancement. Anti-CD40 also increased polyinosinic-polycytidylic acid (poly(I:C))-inducible IFN-alpha by 5-fold in an IFN-gamma-dependent fashion, but did not significantly increase IFN-alpha production to CpG or Pam(3)Cys challenges. Poly(IC)-stimulated splenocytes from anti-CD40-pretreated mice produced 4-fold more IFN-alpha than controls and production associated with CD11c(+) cells. Finally, rIFN-gamma and anti-CD40 combined synergistically to increase poly(IC)-inducible IFN-alpha synthetic capacity in bone marrow dendritic cells. We conclude that innate immune production of IFN-alpha is cooperatively regulated by CD40 and IFN-gamma acting on dendritic cells, suggesting a unique mechanism by which innate immune function evolves in response to specific adaptive immune signals.     

CD4 ligation promotes the IL-4-independent development of IL-4-producing clones from naive CD4(+) T cells.

The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4(+) T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4(+) T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4(+) lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3epsilon mAb and IL-2, 65-85% formed clones over 12-14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-gamma and/or IL-3 regardless of the coactivating mAb. However, whereas 0-6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10-40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4(+) cells from BALB/c mice. Most IL-4-producing clones were derived from CD4(+) cells of naive (CD44(low) or CD62L(high)) phenotype and the great majority coproduced IFN-gamma and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4(+) T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.     

Engagement of the monocyte surface antigen CD14 induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion.

Murine anti-CD14 mAb which recognize different CD14 epitopes induced marked homotypic adhesion of normal human monocytes. Induction of aggregation by anti-CD14 mAb required Mg2+, occurred at an optimal temperature of 37 degrees C, but not at 4 degrees C, and exhibited a kinetics which differed from adhesion triggered by IFN-gamma and anti-CD43 mAb. Monocyte adhesion induced by anti-CD14 mAb required neither Fcy gamma R engagement nor cross-linking of CD14, because adhesion was induced by F(ab)'2 fragments, as well as by monovalent F(ab) fragments of anti-CD14 mAb. mAb to CD11a, CD18, and intercellular adhesion molecule-1 (ICAM-1), but not antibodies to CD11b and CD11c, inhibited monocyte adhesion induced by CD14 engagement. These results indicate that CD14-dependent adhesion is mediated by lymphocyte function-associated Ag-1/ICAM-1 interactions. This was confirmed by the absence of aggregation in anti-CD14-stimulated cells from a patient with leukocyte adhesion deficiency. Monocyte adhesion upon CD14 engagement was blocked by an inhibitor of protein kinases, sphingosine. This suggests that protein kinases play a role in the intracellular signaling pathway(s) which couple CD14 to lymphocyte function-associated Ag-1/ICAM-1.     

Involvement of CD14 in the inhibitory effects of dimethyl-alpha-cyclodextrin on lipopolysaccharide signaling in macrophages

The potential use of alpha-cyclodextrin and its hydrophilic alpha-cyclodextrin derivatives (alpha-CyDs) as antagonists against lipopolysaccharide (LPS), which stimulates the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production as well as nuclear factor-kappaB (NF-kappaB) activation in macrophages was examined. Of three alpha-CyDs used in the present study, 2,6-di-O-methyl-alpha-CyD (DM-alpha-CyD) had greater inhibitory activity than did the other CyDs against NO and TNF-alpha production through an impairment of gene expression in macrophage cell lines and primary macrophages stimulated with LPS and lipid A in a concentration-dependent manner. Concomitantly, DM-alpha-CyD inhibited NF-kappaB translocation into nucleus. These inhibitory effects of DM-alpha-CyD could be attributed to the release of CD14 from lipid rafts caused by an efflux of phospholipids, but not cholesterol. These results suggest that DM-alpha-CyD may have promise as a potent and unique antagonist for excess activation of macrophages stimulated with LPS.     

Triggering of co-mitogenic signals in T cell proliferation by anti-LFA-1 (CD18, CD11a), LFA-3, and CD7 monoclonal antibodies

Proliferative T cell responses were elicited in a comitogenic assay when purified mAb against CD 18, CD11a, LFA-3, and CD7 were immobilized onto solid plastic surfaces together with submitogenic doses of mAb against the CD3 complex. The proliferative response was associated to the production of IL-2 and to the expression of IL-2R. We explored the possibility that a second signal provided by either PMA or a Ca2+ ionofore could replace the anti-CD3 mAb in the comitogenic assay. Interestingly, our data clearly indicate that PMA but not the ionofore was capable of mediating the co-mitogenic effect in conjunction with solid-bound mAb (CDw18, CD11a, LFA-3, and CD7). We also demonstrate that the mAb (anti-CD4 and anti-CD2) which have been previously described as co-mitogenic in combination with anti-CD3 are capable of eliciting this activating signal in the presence of PMA. These data indicate that mAb to certain cell surface differentiation Ag that in soluble form inhibit T cell function such as LFA-1, LFA-3, and CD2 can under appropriate conditions induce co-mitogenic signals on T cells. Our results support the hypothesis that several cell surface differentiation Ag may participate in conjunction with the T3-Ti complex in the transmembrane signal transduction leading to T cell activation.     

Glutamine decreases lipopolysaccharide-induced IL-8 production in Caco-2 cells through a non-NF-kappaB p50 mechanism

Glutamine (Gln) supplementation has been shown to decrease production of pro-inflammatory cytokines by the human intestinal mucosa. The mechanism of this is poorly understood. We hypothesize that Gln down-regulates lipopolysaccharide (LPS)-stimulated pro-inflammatory cytokine production in Caco-2 cells by nuclear factor-kappa B (NF-kappaB). Caco-2 cells were incubated with different concentrations of Gln with or without methionine sulfoximine (MS, an inhibitor of glutamine synthetase) before stimulation with LPS. IL-6, IL-8, IL-10 and TNF-alpha protein and mRNA level were determined. NF-kappaB translocation was determined using an ELISA-based kit. IL-8 was the only detectable cytokine/chemokine. The largest amount of IL-8 was secreted by cells in the presence of MS with no Gln in the medium after exposure to LPS. LPS increased IL-8 production, peaking 10h after LPS administration. The addition of Gln (0.5 or 5.0mM) decreased IL-8 peptide and mRNA expression. LPS increased NF-kappaB nuclear translocation in the presence or absence of MS. Neither Gln nor MS altered NF-kappaB nuclear translocation. These results indicate that the lack of glutamine increases IL-8 production by Caco-2 cells after LPS stimulation. However, the glutamine-mediated decrease in LPS-stimulated IL-8 production is not associated with NF-kappaB p50 nuclear binding.     

Identification of the anti-CD3-unresponsive subpopulation of CD4+, CD45RA+ peripheral T lymphocytes.

The majority of peripheral CD4+ T lymphocytes proliferate in vitro in response to anti-CD3 in presence of autologous APC. The present study describes a subpopulation of CD4+ T cells that cannot be activated and progress into cell cycle by stimulation with anti-CD3 plus APC or with mitogenic combinations of anti-CD2. The in vitro responses of these anti-CD3-unresponsive CD4+ T cells were investigated with a panel of mAb to CD2, CD3, and CD28, and found to be similar to those previously observed for mature thymocytes: only the combination of anti-CD2 plus anti-CD28 produced cell proliferation. Anti-CD3-unresponsive T cells were CD45RA+, but represented only 14 to 22% of the CD4+, CD45RA+ T cell population. Activation with anti-CD2 plus anti-CD28 mAb resulted in major changes in the cell surface phenotype and functional properties: a loss of CD45RA+ occurred and an increased expression of CD45RO, CD29, and CD58 (LFA3), as well as a gain in responsiveness to anti-CD3 and anti-CD2. This change in CD45 phenotype from CD45RA to CD45RO occurs in both the anti-CD3-responsive and in the anti-CD3-unresponsive subsets of the CD45RA+, CD4+ cells after cell proliferation. The anti-CD3-unresponsive subset may represent a pool of not yet fully differentiated peripheral T cells. The acquisition of anti-CD3 responsiveness could occur as a consequence of Ag priming or by an Ag-independent mechanism. Involvement of the CD28 Ag in this process is suggested from the present study.