Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.     

TLR1/TLR2 heterodimers play an important role in the recognition of Borrelia spirochetes

After infection with Borrelia species, the risk for developing Lyme disease varies significantly between individuals. Recognition of Borrelia by the immune system is mediated by pattern recognition receptors (PRRs), such as TLRs. While TLR2 is the main recognition receptor for Borrelia spp., little is known about the role of TLR1 and TLR6, which both can form functionally active heterodimers with TLR2. Here we investigated the recognition of Borrelia by both murine and human TLR1 and TLR6. Peritoneal macrophages from TLR1- and TLR6- gene deficient mice were isolated and exposed to Borrelia. Human PBMCs were stimulated with Borrelia with or without specific TLR1 and TLR6 blocking using specific antibodies. Finally, the functional consequences of TLR polymorphisms on Borrelia-induced cytokine production were assessed. Splenocytes isolated from both TLR1-/- and TLR6-/- mice displayed a distorted Th1/Th2 cytokine balance after stimulation with B.burgdorferi, while no differences in pro-inflammatory cytokine production were observed. In contrast, blockade of TLR1 with specific neutralizing antibodies led to decreased cytokine production by human PBMCs after exposure to B.burgdorferi. Blockade of human TLR6 did not lead to suppression of cytokine production. When PBMCs from healthy individuals bearing polymorphisms in TLR1 were exposed to B.burgdorferi, a remarkably decreased in vitro cytokine production was observed in comparison to wild-type controls. TLR6 polymorphisms lead to a minor modified cytokine production. This study indicates a dominant role for TLR1/TLR2 heterodimers in the induction of the early inflammatory response by Borrelia spirochetes in humans.     

Cytokine production of stimulated whole blood cultures in rheumatoid arthritis patients receiving short-term infliximab therapy

Patients with rheumatoid arthritis (RA) treated with anti-tumor necrosis factor (TNF) strategies have an increased susceptibility to infections, especially those caused by intracellular pathogens. In this study we assessed the cytokine production capacity in patients with RA and we further investigated whether anti-TNF therapy modulates the production of pro-inflammatory cytokines involved in the resistance against infections. Whole blood cultures from 10 RA patients and 10 healthy controls were stimulated with heat-killed Candida albicans, Salmonella typhimurium, Staphyloccocus aureus, Aspergillus fumigatus or Mycobacterium tuberculosis and production of interleukin (IL)-1beta, IL-6, IL-10, interferon (IFN)-gamma and TNF-alpha was measured. Before anti-TNF therapy, whole blood cultures from RA patients released significantly less IFN-gamma than healthy controls after stimulation with all tested microorganisms. Short-term anti-TNF therapy did not have an inhibitory effect on the release of the cytokines tested. We conclude that cells of patients with RA have a strongly reduced production capacity of IFN-gamma after bacterial challenge. Although short-term therapy with anti-TNF agents did not further decrease the release of other proinflammatory cytokines, the combination of defective IFN-gamma production in basal conditions and TNF neutralization during anti-TNF therapy is likely to be responsible for the higher susceptibility to infections in patients with RA.     

Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium

A chimaeric gene has been constructed that expresses -D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3–4 days and then declined; if selection was imposed expression increased again after 10–14 days. The extent of transient expression after 4 days correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3–4 days after inoculation. The most surprising result was that the blue staining was concentrated in localized zones on the circumference of the disc; within these zones, essentially all the cells appeared to be expressing GUS. We suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of leaf explants, but that the frequency of stable integration is several orders of magnitude lower.     

Dectin-1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages

The beta-glucan receptor dectin-1 and Toll-like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin-1 amplifies TLR2-dependent induction of cytokines in mouse models. In the present study we hypothesized that dectin-1 has potent synergistic effects with both TLR2 and TLR4 in human PBMCs and macrophages. Human PBMCs and monocyte-derived macrophages were stimulated with curdlan, a linear beta-1,3-glucan-polymer derived from Alcaligenes faecalis with specific ligand affinity for dectin-1, in combination with the synthetic TLR2 ligand Pam3Cys and the ultrapure TLR4 ligand LPS. TNF-alpha and IL-10 production was measured in the supernatants with ELISA. Curdlan is a specific dectin-1 ligand without TLR2- or TLR4-stimulating properties. Human primary monocytes and macrophages express dectin-1 on the cell membrane. Stimulation of human PBMCs with curdlan in combination with Pam3Cys or LPS leads to synergistic increase in TNF-alpha production that was inhibited by GE2, a neutralizing dectin-1 antibody. Dectin-1-dependent synergy between curdlan and TLR agonists was also apparent in human monocyte-derived macrophages. Conclusively, dectin-1 synergizes with both TLR2 and TLR4 pathways for the production of TNF-alpha in human primary PBMCs and in monocyte-derived macrophages.     

An EGP-2/Ep-CAM-expressing transgenic rat model to evaluate antibody-mediated immunotherapy

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), also known as 17-1A or Ep-CAM, is a 38-kDa transmembrane antigen, commonly used for targeted immunotherapy of carcinomas. Although strongly expressed by most carcinomas, EGP-2 is also expressed in most simple epithelia. To evaluate treatment-associated effects and side-effects on tumor and normal tissue respectively, we generated an EGP-2-expressing transgenic Wistar rat. To express the cDNA of the EGP-2 in an epithelium-specific manner, the 5′ and 3′ distal flanking regions of the human keratin 18 (K18) gene were used. EGP-2 protein expression was observed in the liver and pancreas, whereas EGP-2 mRNA could also be detected in lung, intestine, stomach and kidney tissues. In this rat, EGP-2-positive tumors can be induced by injecting a rat-derived carcinoma cell line transfected with the GA733-2 cDNA encoding EGP-2. Transgenic rats were used to study specific in vivo localization of an i.v. anti-EGP-2 monoclonal antibody, MOC31, applied i.v. Immunohistochemical analyses showed the specific localization of MOC31 in s.c. induced EGP-2-positive tumors, as well as in the liver. In contrast, in EGP-2-transgenic rats, MOC31 did not bind to EGP-2-negative tumors, the pancreas, or other normal tissues in vivo. In conclusion, an EGP-2-transgenic rat model has been generated that serves as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities. Received: 9 March 1999 / Accepted: 6 May 1999     

BcR-induced apoptosis involves differential regulation of C16 and C24-ceramide formation and sphingolipid-dependent activation of the proteasome

In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C(16)- ceramide predominated, was observed 6 h after BcR triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BcR triggering. The formation of these very long-chain ceramide species, in which C(24)-ceramide predominated, required the activation of effector caspases. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which long-chain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta-lactone, an irreversible inhibitor of the proteasome, prevented BcR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BcR cross-linking-induced activation of caspases. The formation of C(16)-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C(24)-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and important insights into the significance of specific ceramide species in defined stages of apoptosis.