Diphosphoryl lipid A derived from lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides inhibits activation of 70Z/3 cells by LPS.

Diphosphoryl lipid A derived from nontoxic lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides ATCC 17023 did not stimulate the murine pre-B cell line 70Z/3 to synthesize surface immunoglobulin or kappa mRNA. However, it effectively blocked Escherichia coli LPS-induced activation of 70Z/3 cells in a concentration-dependent manner. This inhibition was specific only to cells activated by LPS, since it did not inhibit activation of 70Z/3 cells by gamma interferon. Maximal inhibitory effect occurred when the antagonist was added within 2 h before adding the LPS. These results strongly suggested that R. sphaeroides diphosphoryl lipid A is competing with E. coli LPS for physiological lipid A receptors on the 70Z/3 cells.     

LPS-nonresponsive variants of mouse B cell lymphoma, 70Z/3: Isolation and characterization

We have used a genetic approach to study the differentiation of B lymphocytes. The cultured murine cell line 70Z/3 resembles pre-B cells in containing the heavy chain of the immunoglobulin IgM, , as an internal protein in the absence of light chain, L. However, overnight incubation with the B cell mitogen lipopolysaccharide (LPS) induces the cells to mature to a B lymphocyte-like state by the induction of L chain synthesis and the appearance of IgM on the cell surface. We have used immunoselection against surface-bound IgM to isolate LPS uninducible variants of 70Z/3. These fall into two complementation groups, LPS A and LPS B. LPS A variants predominated and were found at a frequency of 1/1200. These cells were completely unresponsive to LPS. LPS B was represented by a single variant in which a subset of cells was induced to display wild-type levels of membranebound IgM, and the proportion of induced cells increased with prolonged incubation with LPS. We detected no structural defects in either variant group, but LPS B may represent a defect in the decision to differentiate in response to LPS.     

PAX5 promotes pre-B cell proliferation by regulating the expression of pre-B cell receptor and its downstream signaling

PAX5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. Although previous studies have indicated that the Pax5-conditional-knockout mouse exhibited dedifferentiation of mature B cell and the development of aggressive lymphomas, the changes of Pax5 gene expressions in pre-B cells have not been analyzed. To understand the functional importance of Pax5 gene in the proliferation and survival of pre-B cells, we established a Pax5-knockdown model using 70Z/3 pre-B cell line. Pax5 knockdown 70Z/3 cells (70Z/3-KD cells) showed down-regulations of pre-BCR compounds such as CD19, BLNK, Id2 and λ5. The signaling via pre-BCRs was significantly diminished in the 70Z/3-KD cells, and this alteration was normalized by restored Pax5 gene expression. Loss of PAX5 reduced the growth rates in the 70Z/3-KD cells, compared to the mock cells. Meanwhile, the proliferation of pre-B cells was reduced by the knockdown of Pax5 gene. Moreover, further examinations showed that PAX5 was also activated in B cell acute lymphoblastic leukemia (B-ALL) as a cell proliferation enhancer. These findings suggested that pax5 is critically important for the proliferation and survival of pre-B cells.     

Soluble CD14 enhances membrane CD14-mediated responses to peptidoglycan: structural requirements differ from those for responses to lipopolysaccharide

The purpose of this study was to identify the functional significance of the binding of soluble CD14 (sCD14) to bacterial peptidoglycan (PGN) and to compare the structural requirements of sCD14 for the binding to PGN and lipopolysaccharide (LPS) and for sCD14-mediated enhancement of PGN- and LPS-induced cell responses. sCD14 did not facilitate the responses of membrane CD14 (mCD14)-negative pre-B 70Z/3 cells to PGN, although it facilitated the responses of these cells to LPS and although mCD14 facilitated the responses of 70Z/3 cells to PGN. sCD14 enhanced mCD14-mediated cell activation by both PGN and LPS, but only the responses to LPS, and not to PGN, were enhanced by LPS-binding protein. Four 4- or 5-amino-acid-long sequences within the 65-amino-acid N-terminal region of sCD14 were needed for binding to both PGN and LPS and for enhancement of cell activation by both PGN and LPS. However, deletions of individual sequences had different effects on the ability of sCD14 to bind to PGN and to LPS and on the ability to enhance the responses to PGN and to LPS. Thus, there are different structural requirements of sCD14 for binding to PGN and to LPS and for the enhancement of PGN- and LPS-induced cell activation.     

Expression and function of the murine CD95/FasR/APO-1 receptor in relation to B cell ontogeny

Mice defective in Fas-mediated apoptosis (lpr phenotype) have an intrinsic B cell abnormality that predisposes them to autoantibody production. To investigate potential roles for the Fas receptor (FasR) in B cell tolerance, FasR expression and function were evaluated at different stages of B cell development. FasR expression was very low or absent on pro- and pre-B cells, but was detected in early B cell lines and was up-regulated following IFN-γ-induced maturation of the pre-B cell line 70-Z. Whereas FasR expression was very low in resting mature sIgM⁺ B cells, expression was markedly increased following mitogen activation and was also elevated in two mature sIgG⁺ lymphoma lines. FasR expression correlated strongly with the ability of B cells to undergo Fas-mediated apoptosis. In addition, although Fas did not appear to play a direct role in apoptosis mediated by cross-linking of sIg with anti-IgM, anti-FasR and sublethal concentrations of anti-Ig were additive in the induction of apoptosis in the early B cell line WEHI 231. These findings suggest that the Fas pathway is not involved in the elimination of pro- and pre-B cells, but are compatible with an ancillary role for FasR in the elimination of early B cells and elimination of mature B cells following activation.     

Lipopolysaccharidelike immunological properties of cell wall glycoproteins isolated from Cytophaga johnsonae.

Glycoproteins (GP) previously shown to be involved in the gliding motility of Cytophaga johnsonae were examined for biological activities characteristic of lipopolysaccharide (LPS). These integral membrane proteins activated 70Z/3 pre-B cells to synthesize immunoglobulin M, induced B cells to synthesize non-antigen-specific polyclonal immunoglobulin, induced macrophages to produce tumor necrosis factor, and modulated the antibody response to type III pneumococcal polysaccharide in the absence of thymus-derived (T) lymphocytes. Except for the GP activity in the 70Z/3 assay, all activities of the GP were comparable to or greater than those of LPS. No LPS was detected in the preparations of GP used or in the phenol-water extracts of C. johnsonae. The mechanism by which these GP exerted their biological activities was distinct from that of LPS, since LPS-resistant C3H/HeJ mice responded to GP. Furthermore, biologically inactive diphosphoryl lipid A obtained from nontoxic LPS of Rhodopseudomonas sphaeroides (an analog of toxic lipid A), which is an antagonist of LPS, did not block the induction of tumor necrosis factor by GP in macrophages. These results showed that the cell surface GP from C. johnsonae are potent LPS-like activators of B cells and macrophages. We suggest that these GP might be good candidates for use in developing an effective adjuvant system.     

Differential activation of human and mouse Toll-like receptor 4 by the adjuvant candidate LpxL1 of Neisseria meningitidis

Neisseria meningitidis LpxL1 lipopolysaccharide (LPS) bearing penta-acylated lipid A is considered a promising adjuvant candidate for inclusion in future N. meningitidis vaccines, as it elicits a markedly reduced endotoxic response in human macrophages relative to that in wild-type (hexa-acylated) LPS, while it is an equally effective adjuvant in mice. As dendritic cells (DC) and Toll-like receptors (TLR) are regarded as central mediators in the initiation of an immune response, here we evaluated the ability of LpxL1 LPS to mature and to activate human DC and examined its TLR4-/MD-2-activating properties. Unexpectedly, purified LpxL1 LPS displayed minimal human DC-stimulating properties compared to wild-type LPS. Although whole bacteria induced DC maturation and activation irrespective of their type of LPS, the LpxL1 mutant failed to activate the human recombinant TLR4/MD-2 complex expressed in HeLa cells. Similarly, purified LpxL1 LPS was unable to activate human TLR4/MD-2 and it even acted as an antagonist of wild-type LPS. Both wild-type and LpxL1 LPSs activated the murine TLR4/MD-2 complex, consistent with their abilities to induce maturation and activation of murine DC. Assays with cells transfected with different combinations of human and murine TLR4 and MD-2 indicated that TLR4 was a more-major determinant of the LPS response than MD-2. The species-specific activation of the TLR4/MD-2 complex by LpxL1 LPS may have an impact on the use of LpxL1 LPS as an adjuvant and the use of murine immunization models in human meningococcal vaccine development.     

Fut8基因通过促进VLA-4/VCAM-1表达影响B细胞发育

目的探讨核心岩藻糖基化修饰对VLA-4/VCAM-1表达及B细胞发育的影响。方法通过RNA干扰技术使前B细胞(70Z/3)和基质细胞(ST2)的Fut8基因沉默,免疫沉淀和Western blot检测Fut8基因沉默效率,以细胞黏附实验和克隆形成实验分别研究Fut8基因对VLA-4/VCAM-1之间亲和力和对B细胞分化发育的影响。结果 Fut8基因沉默使VLA-4/VCAM-1之间的亲和力以及前B细胞和基质细胞之间的黏附作用降低,并使前B细胞的克隆形成能力明显下降。流式细胞仪分析结果也表明,Fut8-/-祖B细胞向前B细胞分化过程受阻。结论本实验揭示了Fut8基因调节VLA-4/VCAM-1之间结合能力以及前B细胞克隆形成的机理,为B细胞分化发育研究提供理论依据。     

Cell activation by Porphyromonas gingivalis lipid A molecule through Toll-like receptor 4- and myeloid differentiation factor 88-dependent signaling pathway

Porphyromonas gingivalis lipopolysaccharide (LPS) and its bioactive center, lipid A, are known to exhibit very low endotoxic activities and activate LPS-hyporesponsive C3H/HeJ mice that have a point mutation in the cytoplasmic portion of Toll-like receptor (TLR) 4, in contrast to classical enterobacterial LPS and their lipid A. In the present study, we attempted to determine which TLR mediates the response to lipid A from P. gingivalis strain 381. P. gingivalis LPS and its natural lipid A fraction induced NF-kappa B activation primarily in Ba/F3 cells expressing mouse TLR 2 (Ba/mTLR2), rather than in those expressing mouse TLR4 and its accessory protein MD2 (Ba/mTLR4/mMD2). Further purification of the natural lipid A fraction resulted in a significant decrease of NF-kappa B activation in Ba/mTLR2, although not in Ba/mTLR4/mMD2. The synthetic counterpart of P. gingivalis strain 381-lipid A (compound PG-381) also elicited NF-kappa B activation in Ba/mTLR4/mMD2, but not Ba/mTLR2. Furthermore, P. gingivalis purified natural lipid A and compound PG-381 lacked the ability to activate gingival fibroblasts from C3H/HeJ, TLR4 knockout (KO) and myeloid differentiation factor 88 (MyD88) KO mice. These findings demonstrate that the P. gingivalis lipid A molecule induces cell activation via a TLR4/MD2-MyD88-dependent pathway, and suggest the possibility that unknown bacterial components in P. gingivalis LPS and its lipid A may induce cell activation via TLR2.     

Lipopolysaccharide stimulation converts vigorously washed dendritic cells (DCs) to nonexhausted DCs expressing CD70 and evoking long-lasting type 1 T cell responses

A great variety of in vitro culture protocols for human monocyte-derived dendritic cells (mo-DCs) has been used to generate DCs suitable for use in immunotherapy. It is thought that activated DCs undergo one-way differentiation into "exhausted" DCs. In the present study, we contrived an in vitro method for facilitating expression of CD70 by mature DCs. This was achieved by vigorous washing of mo-DCs before exposure to lipopolysaccharide (LPS). Unexpectedly, these mature DCs retain expression of some interleukin (IL)-12 family members after extended periods and maintain their ability to stimulate type 1 T cell responses. In contrast, DCs exposed to IL-4 before LPS stimulation or LPS-stimulated DCs not exposed to washing stress before activation failed to express CD70 and did differentiate into exhausted DCs. It is interesting that DCs expressing CD70 (CD70+ DCs) induced interferon-gamma production from purified, allogeneic CD8+ T cells through a direct CD27-CD70 interaction. This is evidence for a pathway resulting in generation of CD8 T effectors by B7-independent mechanisms. These data suggest that exposure of immature DCs to LPS stimulation contributes to their terminal differentiation into CD70+ DCs, which have potent ability to prolong type 1 T cell responses through alternative pathways.     

Microbial induction of co-stimulatory activity for CD4 T-cell growth.

The activation of naive CD4 T cells by antigen is a critical step in the initiation of an immune response; it requires both ligation of the T-cell receptor (TCR) and the delivery of co-stimulatory factors by accessory cells. We have examined the role of syngeneic accessory cells in the response of purified normal CD4 T cells to anti-CD3 antibody as ligand. We show that the ability to deliver co-stimulatory signals is inducible in B cells by microbial products such as bacterial lipopolysaccharide (LPS), mitogenic influenza viruses, and synthetic polyinosinic-polycytidylic acid (poly-I:C) as a mimic of viral infection. LPS stimulation for 16 h allows the co-stimulatory activity of B cells to become resistant to paraformaldehyde fixation. LPS induction of fixation-resistant co-stimulator activity requires new protein synthesis, as it is inhibited by cycloheximide. Using the anti-CD45RB mAb 16A as marker for naive and memory CD4 T cells, we show that B cells activated by LPS and by poly-I:C can provide co-stimulatory signal to both naive and memory CD4 T cells. By contrast, zymosan particles, which are known to activate macrophages in a variety of assays, do not activate B cells to become co-stimulatory, but do induce this activity in macrophages. These data demonstrate that a variety of infectious agents or their constituents can induce accessory cells to become co-stimulatory for CD4 T cells. They are interpreted in light of a proposed role for two classes of recognition in the induction of the immune responses, specific recognition of antigens and non-specific recognition of infectious agents. These data support the contention that the immune system uses this mechanism to discriminate infectious non-self from non-infectious self.     

Toll-like receptor 4-mediated signaling regulates IL-7-driven proliferation and differentiation of B-cell precursors

Lipopolysaccharide （LPS） is known to be a potent activator of mature B cells by signaling through Toll-like receptor 4 （TLR4）. Its impact on early B-cell development, however, is not well defined. When comparing to C3H/HeN mice, TLR4-mutant C3H/HeJ mice showed an increase in the number of pro-B and pre-B cells in the bone marrow. When cultured in the presence of IL-7, the proliferation of pro-B and large pre-B cells was significantly inhibited by LPS, possibly due to reduced IL-7 receptor-a （IL-7Ra） expression. Meanwhile, the generation of IgM＋/IgD＋ B cells was greatly enhanced in IL-7 cultures of pro-B and pre-B cells. Consistent with these results, treatment with LPS facilitated the progression of adoptively transferred B220＋IgM-IgD- precursors into IgD＋ cells. Overall, these data suggest that LPS has a profound influence on early B-cell development, which may contribute to the deregulated B-cell development under physiological and pathological conditions such as bacterial infections.     

Genes expressed during the IFN gamma-induced maturation of pre-B cells.

Interferon-gamma (IFN gamma) exerts diverse responses in B cell development ranging from growth arrest and apoptosis to proliferation and differentiation. IFN gamma stimulates murine 70Z/3 pre-B cells to express surface immunoglobulin (Ig) and this system serves as a useful model for the pre-B to immature B cell transition in B cell development. To analyze this developmental transition, we used a PCR-based subtractive hybridization in combination with miniarray screening to identify differentially-expressed genes in IFN gamma-stimulated compared with unstimulated 70Z/3 pre-B cells. The majority (44%) of the differentially-expressed genes obtained were known IFN gamma-inducible. These included multiple isolates from each of three multi-gene families, including two guanylate-binding protein (47 and 67kDa GBP) families of GTPases and the hematopoietic IFN gamma-inducible nuclear protein family (HIN-200). These multiple isolates of genes comprised the majority of the total isolated and sequenced clones. Other known IFN gamma-induced genes in this group included Ig kappa light chain and Ly-6, as well as genes with functions in antigen processing, cellular regulation, and cytoskeletal organization. Another 36% of the genes identified were previously known, but not known to be IFN gamma-inducible (e.g. pre-B cell enhancing factor, PBEF). The remaining 20% of the IFN gamma-induced isolates did not match entries in Genbank, and thus, may represent novel genes involved in IFN gamma responses and/or in the pre-B to immature B cell transition. Overall, the majority of the individual genes isolated were either not known to be IFN gamma responsive or were not previously known.     

Heparin alters the expression of different forms of immunoglobulin mu heavy chains and their associated proteins by pre-B cell lines and normal Ly-1 (CD5+) B cells.

Studies presented here show that heparin alters immunoglobulin expression by murine pre-B cell lines and normal Ly-1 (CD5+) B cells. Previous studies have shown that pre-B cell lines 70Z/3 and NFS-5.3 express mu heavy chains in the cytoplasm and a small amount on the cell surface. Both these cytoplasmic and surface mu are disulfide-linked to omega (lambda 5) surrogate light chains and are noncovalently associated with iota (Vpre-B) variable region-like proteins. We show that culturing 70Z/3 with heparin reduces the amount of the membrane-form mu (micron) on the cell surface. Culturing NFS-5.3 with heparin similarly decreases the membrane-form mu; however, it increases the surface level of a pentameric mu molecule containing secreted-form mu (microS) heavy chains, disulfide-linked omega (lambda 5) chains, and noncovalently associated proteins. Culturing peritoneal B cells with heparin also increases the production of the secreted-form microS, detectable in this case by the secretion of classical pentameric IgM. Similarly, injecting heparin intraperitoneally increases IgM secretion by peritoneal Ly-1 B cells. Thus heparin could influence pre-B cell and B cell differentiation and function.     

川芎嗪抑制LPS诱导的人Ⅱ型肺泡上皮细胞炎性反应

目的体外探讨川芎嗪(TMP)对脂多糖(LPS)诱导的人Ⅱ型肺泡上皮细胞(HAECⅡ)炎性反应的保护作用及机制。方法体外培养HAECⅡ(A549细胞来源),LPS刺激建立炎性模型,分别加入TMP和前B细胞克隆增强因子(PBEF)抑制剂FK866进行干预。q-PCR和Western blot检测肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)和PBEF的mRNA和蛋白表达;通过Western blot检测细胞核和细胞质内磷酸化P65蛋白来反映核因子κB(NF-κB)的激活。结果 LPS刺激A549细胞后,TNF-α、IL-1β、IL-8和PBEF mRNA及蛋白表达均较对照组明显增高(P0.001),伴随着细胞核和细胞质磷酸化的P65蛋白增高(P0.001);TMP干预后,上述炎性因子mRNA和蛋白表达下降,磷酸化P65蛋白表达降低(P0.05);FK866干预后,TNF-α、IL-1β和IL-8表达以及磷酸化P65蛋白降低(P0.01)。结论 TMP可能通过降低PBEF的表达,从而抑制NF-κB的激活,减轻肺泡上皮细胞炎性反应。     

Human recombinant heat shock protein 70 affects the maturation pathways of dendritic cells in vitro and has an in vivo adjuvant activity

Heat shock proteins (HSPs) are potent inducers of an antigen-specific immunological response. A role of chaperon of immunogenic peptides and a direct effect on APC activation and function have been described. However, the signal transduction events involved in the activation of human APCs are poorly characterized. We investigated, using human monocyte-derived dendritic cells (DCs), the signal transduction pathways activated by a human recombinant HSP70 (r)HSP70 purified from eukaryotic cells. rHSP70 effectively induced a partial maturation of DCs in vitro and a significant increase in the titers of antigen-specific IgG when used as a vaccine adjuvant in vivo. rHSP70 did not desensitize human DCs to LPS stimulation and retained its adjuvant properties in C3H/HeJ mice, which are LPS-resistant as a result of a mutation in TLR-4, ruling out the potential interference of LPS contamination. Effects on DC maturation and in vivo functions correlate to the ability of rHSP70 to activate IkappaB-alpha/NF-kappaB and ERK1/2 pathways in human DCs. No activation of p38 was induced in the same experimental conditions. Our data suggest that the IkappaB-alpha/NF-kappaB pathway has a critical role in the partial maturation of DCs induced by rHSP70.     

Phosphatidylserine inhibits NFκB and p38 MAPK activation in human monocyte derived dendritic cells

Phosphatidylserine (PS) is an anionic phospholipid restricted to the inner surface of the plasma membrane. PS translocates to the cell surface during early apoptosis where it serves as a marker for rapid uptake by phagocytes. PS is also thought to regulate immune responses. Dendritic cells (DC) are the most potent antigen presenting cells. Previous studies demonstrated that PS inhibits the expression of MHC and co-stimulatory molecules, the secretion of IL-12p70, and the ability to activate T cells by human monocyte derived DCs. However, the cell signaling mechanisms by which PS regulated DCs are not well described. In the current study we tested the effects of PS on signal transduction pathways thought to regulate human myeloid DC maturation and IL-12p70 production. We showed that PS inhibited the activation of nuclear factor-κB (NFκB) in response to LPS by preventing IκBα phosphorylation and degradation. PS also increased the total IκBα levels in immature DCs and inhibited p38 mitogen activated protein kinase (MAPK) phosphorylation and activation. The findings suggest a possible mechanism for regulating the immunostimulatory function of DCs by PS.     

Cross-linking of surface IgM activates NF-kappa B in B lymphocyte.

In B lymphocytes, cross-linking of surface IgM activates changes in both the cell cycle and differentiation. In normal B cells and B cell tumors, many stimuli induce the activation of NF-kappa B and its translocation from the cytoplasm to the nucleus. In this study we sought to determine if cross-linking of surface IgM led to the activation of NF-kappa B. Our results show that activation of B cells by cross-linking anti-IgM antibodies activated NF-kappa B in the murine B lymphoid cell lines 70Z/3 and M12, and in the dense fraction of splenic cells. The activation of NF-kappa B required optimal doses of anti-IgM antibodies and took 5 to 10 min to reach maximal levels. Cross-linking of IgM has also been shown to activate protein kinases including protein kinase C (PKC). To test whether PKC activation was required for NF-kappa B translocation, we treated 70Z/3 cells for 18 h with phorbol 12-myristate 13-acetate, a procedure which depletes these cells of functional PKC. This treatment did not abrogate the nuclear translocation of NF-kappa B following anti-IgM cross-linking. These results indicate that the nuclear translocation of NF-kappa B is rapidly induced by surface IgM cross-linking and that this activation appears to use a pathway which does not require PKC.     

Signal transduction through mu kappa B-cell receptors expressed on pre-B cells is different from that through B-cell receptors on mature B cells.

We introduced kappa light chain genes into pre-B cells to increase the surface mu HC expression, and established transfectants expressing mature type of B-cell receptors (BCR) on pre-B-cell surfaces. Since the cytoplasmic conformations of the reconstituted BCR and intrinsic pre-B-cell receptor (pre-BCR) are identical, they would be connected with the identical signal transduction pathways in pre-B cells. By using the transfectants, we revealed that the reconstituted BCR on pre-B cells was functionally equivalent to BCR on mature B cells in terms of the induction of intracellular Ca++ mobilization. However, we found that the signal-transduction pathways through BCR on pre-B cells were quantitatively different from those mature B cells in two ways. First, cross-linkage of the reconstituted BCR on pre-B cells induced preferential tyrosine phosphorylation of p120 and p100, which was not observed when BCR on mature B cells was cross-linked. Second, BCR in pre-B cells was physically associated with a larger amount of phosphatidylinositol-3 kinase (PI-3K) than BCR in mature B cells in spite of the fact that both pre-B and B cells expressed a similar amount of PI-3K in cytoplasm. Signals through pre-BCR and BCR are known to cause distinct biological effects in B-cell development. The biochemical features in the downstream of reconstituted BCR on pre-B cells, which we revealed in this study, will be of help in understanding the mechanism of functional differences between pre-BCR and BCR.