Activation of Toll‐like receptor 9 inhibits lipopolysaccharide‐induced receptor activator of nuclear factor kappa‐ B ligand expression in rat B lymphocytes

B lymphocytes express multiple TLRs that regulate their cytokine production. We investigated the effect of TLR4 and TLR9 activation on receptor activator of NF‐κB ligand (RANKL) expression by rat spleen B cells. Splenocytes or purified spleen B cells from Rowett rats were cultured with TLR4 ligand Escherichia coli LPS and/or TLR9 ligand CpG‐oligodeoxynucleotide (CpG‐ODN) for 2 days. RANKL mRNA expression and the percentage of RANKL‐positive B cells were increased in rat splenocytes challenged by E. coli LPS alone. The increases were less pronounced when cells were treated with both CpG‐ODN and E. coli LPS. Microarray analysis showed that expressions of multiple cyclin‐dependent kinase (CDK) pathway‐related genes were up‐regulated only in cells treated with both E. coli LPS and CpG‐ODN. This study suggests that CpG‐ODN inhibits LPS‐induced RANKL expression in rat B cells via regulation of the CDK pathway.     

ROS/Epac1-mediated Rap1/NF-kappaB activation is required for the expression of BAFF in Raw264.7 murine macrophages

B-cell activating factor (BAFF) plays a role for the maturation and the maintenance of B cells. Lipopolysaccharide (LPS) activates toll-like receptor 4 (TLR4)-dependent signal transduction, which resulted in BAFF expression through nuclear factor kappa B (NF-κB) activation. Here, we investigated whether BAFF expression could be regulated by p65 phosphorylation through the production of reactive oxygen species (ROS) or cyclic AMP (cAMP) in Raw264.7 murine macrophages. mBAFF expression was reduced by ROS scavengers and it was increased by dibutyl-cAMP, a cAMP analogue. mBAFF expression and mBAFF promoter activity were increased by co-transfection of p65 but they were reduced by p65-small interference (si) RNA. Serine (Ser) 276 phosphorylation of p65 was increased by LPS-mediated PKA activation or by the treatment with forskolin, adenylate cyclase activator and dibutyl-cAMP. In contrast, p65 phosphorylation at Ser276 was decreased by ROS scavengers. H₂O₂ increased intracellular cAMP concentration, significantly. While no increase in p65 phosphorylation at Ser276 was detected by the treatment with H₂O₂, CREB and p65 phosphorylation at Ser133 and Ser536 was observed, respectively. It implicates that p65 phosphorylation at Ser276 is independent of ROS-induced cAMP production. As another cAMP effector protein was cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor, NF-κB was activated by the treatment with 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT) that is an activator to Epac. Epac1-mediated Rap1 was activated by the treatment with H₂O₂ but it was inhibited by ROS scavengers. CPT induced p65 phosphorylation at both Ser276 and Ser536. CPT also increased not only mBAFF expression but mBAFF promoter activity. Data demonstrate that TLR4-mediated mBAFF expression was resulted from the crosstalk of p65 phosphorylation at Ser536 and Ser276 through ROS- and/or cAMP-mediated signal transduction. It suggests for the first time that ROS/Epac1-mediated Rap1/NF-κB pathway could be required for BAFF expression.     

Negative Regulation of Toll-like Receptor-4 Signaling through the Binding of Glycosylphosphatidylinositol-anchored Glycoprotein,CD14, with the Sialic Acid-binding Lectin,CD33

When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.     

Pathway of Toll-like receptor 7/B cell activating factor/B cell activating factor receptor plays a role in immune thrombocytopenia in vivo

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by anti-platelet autoantibody-mediated platelet destruction. Antigen-presenting cell (APC) dysfunction is considered to play crucial roles in ITP. However, how APC affects autoreactive B cells in ITP is still unknown. Using a mouse model of immune thrombocytopenia, we demonstrated an increase in levels of TLR7 in splenic mononuclear cells (SMCs). Using both TLR7 agonist and TLR7 silencing lentivirus, we found stimulation of TLR7 decreased platelet counts and increased levels of platelet-associated IgG (PAIgG) in ITP mice, which correlates TLR7 with platelet destruction by autoantibodies. Levels of serum BAFF increased significantly in ITP mice and stimulation of TLR7 promoted secretion of BAFF. Among the three BAFF receptors, only BAFF receptor (BAFF-R) increased in ITP mice. However, activation of TLR7 showed no effect on the expression of BAFF receptors. These findings indicate that upregulation of TLR7 may augment BAFF secretion by APC and through ligation of BAFF-R promote autoreactive B cell survival and thus anti-platelet autoantibody production. The pathway of TLR7/BAFF/BAFF-R provides us with an explanation of how activation of APC affects autoantibody production by B cells in ITP and thus might provide a reasonable therapeutic strategy for ITP.     

Anesthetic propofol reduces endotoxic inflammation by inhibiting reactive oxygen species-regulated Akt/IKKβ/NF-κB signaling

Background

Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages.

Methodology/Principal Findings

Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages.

Conclusions/Significance

These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.     

Expression of the adaptor protein hematopoietic Src homology 2 is up-regulated in response to stimuli that promote survival and differentiation of B cells

Analysis of hematopoietic Src homology 2 (HSH2) protein expression in mouse immune cells demonstrated that it is expressed at low levels in resting B cells but not T cells or macrophages. However, HSH2 expression is up-regulated within 6-12 h in response to multiple stimuli that promote activation, differentiation, and survival of splenic B cells. HSH2 expression is increased in response to anti-CD40 mAb, the TLR ligands LPS and CpG DNA, and B lymphocyte stimulator (BLyS), a key regulator of peripheral B cell survival and homeostasis. Stimulation of B cells with anti-CD40 mAb, LPS, CpG DNA, or BLyS has previously been shown to induce activation of NF-kappaB. In agreement with this finding, up-regulation of HSH2 expression in response to these stimuli is blocked by inhibitors of NF-kappaB activation and is potentiated by stimulation with PMA, suggesting that HSH2 expression is dependent on NF-kappaB activation. In contrast to CD40, BAFF receptor, TLR4, and TLR9 mediated signaling, stimulation of splenic B cells via the BCR was not observed to induce expression of HSH2 unless the cells had been stimulated previously through CD40. Finally, HSH2 expression is down-regulated in splenic B cells in response to stimulation with IL-21, which has been shown to induce apoptosis, even in the presence of anti-CD40 mAb, LPS, or CpG DNA. IL-21 stimulation also results in down-regulation of antiapoptotic proteins such as Bcl-x(L) and up-regulation of proapoptotic proteins like Bim. Therefore, HSH2 expression is coordinately up-regulated with known antiapoptotic molecules and directly correlates with B cell survival.     

Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production

Chronic infection, such as Helicobacter pylori infection, has been associated with the development of gastric cancer (GC). Pathogen-associated molecular patterns can trigger inflammatory responses via Toll-like receptors (TLRs) in GC. Here we showed that Toll-like receptor 4 (TLR4) was highly expressed in GC cells and was associated with the aggressiveness of GC. The binding of lipopolysaccharide (LPS) to TLR4 on GC cells enhanced proliferation without affecting apoptosis. Higher level of reactive oxygen species (ROS) was induced after activation of TLR4 signaling in GC. Using oxidase inhibitors and antioxidants, we found that mitochondrial ROS (mROS) was major source of TLR4-stimulated ROS generation. This elevated mROS production can be inhibited by diphenylene iodonium (DPI), and the blocking of the mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential, which were plausible reason for decreased cell viability. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of Akt phosphorylation and NF-κB p65 nuclear translocation. Altogether, these results reveal a novel pathway linking innate immune signaling to GC cell proliferation, implicate mROS as an important component of cell survival signals and further establish mitochondria as hubs for GC therapies.     

Augmented Osteogenic Responses in Human Aortic Valve Cells Exposed to oxLDL and TLR4 Agonist: A Mechanistic Role of Notch1 and NF-κB Interaction

Aortic valve calcification causes the progression of calcific aortic valve disease (CAVD). Stimulation of aortic valve interstitial cells (AVICs) with lipopolysaccharide (LPS) up-regulates the expression of osteogenic mediators, and NF-κB plays a central role in mediating AVIC osteogenic responses to Toll-like receptor 4 (TLR4) stimulation. Diseased aortic valves exhibit greater levels of oxidized low-density lipoprotein (oxLDL). This study tested the hypothesis that oxLDL augments the osteogenic responses in human AVICs through modulation of NF-κB and Notch1 activation. AVICs isolated from normal human aortic valves were treated with LPS (0.1 µg/ml), oxLDL (20 µg/ml) or LPS plus oxLDL for 48 h. OxLDL alone increased cellular bone morphogenetic protein-2 (BMP-2) levels while it had no effect on alkaline phosphatase (ALP) levels. Cells exposed to LPS plus oxLDL produced higher levels of BMP-2 and ALP than cells exposed to LPS alone. Further, LPS plus oxLDL induced greater NF-κB activation, and inhibition of NF-κB markedly reduced the expression of BMP-2 and ALP in cells treated with LPS plus oxLDL. OxLDL also induced Notch1 activation and resulted in augmented Notch1 activation when it was combined with LPS. Inhibition of Notch1 cleavage attenuated NF-κB activation induced by LPS plus oxLDL, and inhibition of NF-κB suppressed the expression of BMP-2 and ALP induced by the synergistic effect of Jagged1 and LPS. These findings demonstrate that oxLDL up-regulates BMP-2 expression in human AVICs and synergizes with LPS to elicit augmented AVIC osteogenic responses. OxLDL exerts its effect through modulation of the Notch1-NF-κB signaling cascade. Thus, oxLDL may play a role in the mechanism underlying CAVD progression.     

The Toll-Like Receptor 4 Polymorphism Asp299Gly but Not Thr399Ile Influences TLR4 Signaling and Function

The common, co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms (SNPs), Asp299Gly and Thr399Ile, are associated with hyporesponsiveness to inhaled lipopolysaccharide (LPS) and increased susceptibility to Gram negative pathogens in humans. The purpose of this study was to identify the relative contributions of the Asp299Gly and the Thr399Ile variants in inhibiting the function of TLR4. 293/hMD2-CD14 cell line was transfected with lentiviral constructs containing human wild type (WT) TLR4-EGFP or TLR4-EGFP with Asp299Gly, Thr399Ile or Asp299Gly/Thr399Ile complementary DNA (cDNA). Multiple stable cell lines were established for each construct: three for WT TLR4, Asp299Gly, and Thr399Ile, and only two for Asp299Gly/Thr399Ile mutants and EGFP control. We did not observe a significant effect of polymorphisms on cell surface and intracellular TLR4 expression nor were there any significant differences in TLR4 and EGFP protein levels assessed by Western blotting and confocal microscopy among the multiple cell lines of each of the constructs. All cell lines had a dose-dependent responsiveness to LPS stimulation. However, compared to the WT TLR4, cells expressing TLR4 with Asp299Gly but not Thr399Ile polymorphism produced significantly less (P<0.05) IL-8 following LPS stimulation. Similarly, cells expressing TLR4 Asp299Gly but not Thr399Ile allele had significantly lower percentage of phosphorylated and total NF-κB P65 following LPS stimulation. While we could not do statistics on the Asp299Gly/Thr399Ile group, we observed a reduced responsiveness to LPS compared to WT TLR4. Taken together, we observed that the TLR4 Asp299Gly variant, but not the Thr399Ile variant, is responsible for impaired responsiveness of TLR4 to LPS and corresponding activation of NF-κB.     

Airway epithelial cells produce B cell-activating factor of TNF family by an IFN-beta-dependent mechanism

Activation of B cells in the airways is now believed to be of great importance in immunity to pathogens, and it participates in the pathogenesis of airway diseases. However, little is known about the mechanisms of local activation of B cells in airway mucosa. We investigated the expression of members of the B cell-activating TNF superfamily (B cell-activating factor of TNF family (BAFF) and a proliferation-inducing ligand (APRIL)) in resting and TLR ligand-treated BEAS-2B cells and primary human bronchial epithelial cells (PBEC). In unstimulated cells, expression of BAFF and APRIL was minimal. However, BAFF mRNA was significantly up-regulated by TLR3 ligand (dsRNA), but not by other TLR ligands, in both BEAS-2B cells (376-fold) and PBEC (224-fold). APRIL mRNA was up-regulated by dsRNA in PBEC (7-fold), but not in BEAS-2B cells. Membrane-bound BAFF protein was detectable after stimulation with dsRNA. Soluble BAFF protein was also induced by dsRNA (> 200 pg/ml). The biological activity of the epithelial cell-produced BAFF was verified using a B cell survival assay. BAFF was also strongly induced by IFN-beta, a cytokine induced by dsRNA. Induction of BAFF by dsRNA was dependent upon protein synthesis and IFN-alphabeta receptor-JAK-STAT signaling, as indicated by studies with cycloheximide, the JAK inhibitor I, and small interfering RNA against STAT1 and IFN-alphabeta receptor 2. These results suggest that BAFF is induced by dsRNA in airway epithelial cells and that the response results via an autocrine pathway involving IFN-beta. The production of BAFF and APRIL by epithelial cells may contribute to local accumulation, activation, class switch recombination, and Ig synthesis by B cells in the airways.     

Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts

Saikosaponin a (SSa), the major triterpenoid saponin derivatives from Radix bupleuri (RB), has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effects of SSa on lipopolysaccharide (LPS)-induced oxidative stress and inflammatory response in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with LPS in the presence or absence of SSa. The levels of TNF-α and IL-8 were detected by ELISA. The expression of COX-2 and iNOS, NF-κB and IκB protein were determined by Western blotting. To investigate the protective mechanisms of SSa, TLR4 expression was detected by Western blotting and membrane lipid rafts were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The results showed that SSa dose-dependently inhibited the production of ROS, TNF-α, IL-8, COX-2 and iNOS in LPS-stimulated HUVECs. Western blot analysis showed that SSa suppressed LPS-induced NF-κB activation. SSa did not affect the expression of TLR4 induced by LPS. However, translocation of TLR4 into lipid rafts and oligomerization of TLR4 induce by LPS was inhibited by SSa. Furthermore, SSa disrupted the formation of lipid rafts by depleting cholesterol. Moreover, SSa activated LXRα-ABCA1 signaling pathway, which could induce cholesterol efflux from lipid rafts. Knockdown of LXRα abrogated the anti-inflammatory effects of SSa. In conclusion, the effects of SSa is associated with activating LXRα-ABCA1 signaling pathway which results in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts and oligomerization of TLR4, thereby attenuating LPS mediated oxidative and inflammatory responses.     

MNK kinases regulate multiple TLR pathways and innate proinflammatory cytokines in macrophages

The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

B1 cells produce nitric oxide in response to a series of toll-like receptor ligands

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5⁺ IgM⁺ WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-κB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-β (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.     

In vitro and in vivo activation induces BAFF and APRIL expression in B cells

B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. BAFF and APRIL are produced by a variety of cell types such as macrophages/monocytes and dendritic cells. Our analysis shows that BAFF mRNA is also expressed in all B cell subsets isolated from bone marrow, spleen, and peritoneal cavity of BALB/c mice. APRIL expression is restricted to early stages of B cell development in the bone marrow and the peritoneal B1 subset. Stimulation of B2 and B1 cells with LPS or CpG-oligodeoxynucleotides induced MyD88-dependent plasma cell differentiation and intracellular expression of BAFF and APRIL. Furthermore, activation of B cells up-regulated membrane expression of BAFF. The finding that in vitro activation of B cells is inhibited by the antagonist transmembrane activator and calcium modulator ligand interactor Ig, indicates that BAFF and/or APRIL are released into the culture supernatants. It shows that B cell survival, proliferation, and differentiation are supported by an autocrine pathway. In vivo activation of B cells with a T-dependent Ag- induced BAFF expression in germinal center B cells. In (NZB x NZW)F(1) mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of BAFF. In (NZB x NZW)F(1) mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease.     

The radioprotective 105/MD-1 complex links TLR2 and TLR4/MD-2 in antibody response to microbial membranes

Low-affinity IgG3 Abs to microbial membranes are important for primary immune defense against microbes, but little is known about the importance of TLRs in their production. IgG3 levels were extremely low in mice lacking radioprotective 105 (RP105), a B cell surface molecule structurally related to TLRs. RP105(-/-) B cells proliferated poorly in response to not only the TLR4 ligand LPS but also TLR2 ligand lipoproteins, both of which mediate the immunostimulatory activity of microbial membranes. RP105(-/-) mice were severely impaired in hapten-specific Ab production against LPS or lipoproteins. CD138 (syndecan-1)-positive plasma cells were detected after lipid A injection in wild-type spleen but much less in RP105(-/-) spleen. RP105 ligation in vivo induced plasma cell differentiation. RP105 expression was approximately 3-fold higher on marginal zone B cells than on follicular and B1 cells and was down-regulated on germinal center cells. These results demonstrate that a signal via RP105 is uniquely important for regulating TLR-dependent Ab production to microbial membranes.