Hartmut Engelmann

Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12.

Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll‐like receptor (TLR) expression. TLR1–TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDCbut not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL‐8, IP‐10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN‐α and IL‐12 including bioactive IL‐12u2004p70. Previous incubation of PDC with IL‐3 decreased the amount of CpG‐induced IFN‐α and shifted the cytokine response in favor of IL‐12. CpG ODN‐activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN‐stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL‐12u2004p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activatedT cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell‐controlled restriction to foreign antigens is maintained.

Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1 activation by endogenous membrane-anchored TNF.

Several members of the tumour necrosis factor receptor (TNF‐R) superfamily can induce cell death. For TNF‐R1, Fas/APO‐1, DR3, DR6, TRAIL‐R1 and TRAIL‐R2, a conserved ‘death domain’ in the intracellular region couples these receptors to activation of caspases. However, it is not yet known how TNF receptor family members lacking a death domain, such as TNF‐R2, CD40, LT‐βR, CD27 or CD30, execute their death‐inducing capability. Here we demonstrate in different cellular systems that cytotoxic effects induced by TNF‐R2, CD40 and CD30 are mediated by endogenous production of TNF and autotropic or paratropic activation of TNF‐R1. In addition, stimulation of TNF‐R2 and CD40 synergistically enhances TNF‐R1‐induced cytotoxicity. These findings describe a novel pro‐apoptotic mechanism induced by some members of the TNF‐R family.

Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor.

The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu‐p55‐TNF‐R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C‐terminally truncated mutants of the receptor, expressed in rodent cells. The ‘wild‐type’ receptor signalled for a cytocidal effect when cross‐linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4 beta‐phorbol‐12‐myristate‐13‐acetate (PMA). Receptor mutants with 53%, 83% and 96% C‐terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF‐R are mechanistically distinct.

TRAF6 is a critical mediator of signal transduction by the viral oncogene latent membrane protein 1

The oncogenic latent membrane protein 1 (LMP1) of the Epstein–Barr virus recruits tumor necrosis factor‐receptor (TNFR)‐associated factors (TRAFs), the TNFR‐associated death domain protein (TRADD) and JAK3 to induce intracellular signaling pathways. LMP1 serves as the prototype of a TRADD‐binding receptor that transforms cells but does not induce apoptosis. Here we show that TRAF6 critically mediates LMP1 signaling to p38 mitogen‐activated protein kinase (MAPK) via a MAPK kinase 6‐dependent pathway. In addition, NF‐κB but not c‐Jun N‐terminal kinase 1 (JNK1) induction by LMP1 involves TRAF6. The PxQxT motif of the LMP1 C‐terminal activator region 1 (CTAR1) and tyrosine 384 of CTAR2 together are essential for full p38 MAPK activation and for TRAF6 recruitment to the LMP1 signaling complex. Dominant‐negative TRADD blocks p38 MAPK activation by LMP1. The data suggest that entry of TRAF6 into the LMP1 complex is mediated by TRADD and TRAF2. In TRAF6‐knockout fibroblasts, significant induction of p38 MAPK by LMP1 is dependent on the ectopic expression of TRAF6. We describe a novel role of TRAF6 as an essential signaling mediator of a transforming oncogene, downstream of TRADD and TRAF2.

Replacement therapy for a homozygous protein C deficiency-state using a concentrate of human protein C and S

A severe congenital deficiency of protein C was diagnosed in a 10‐month‐old girl who had been suffering from skin necrosis since the age of 7 months. The patient was treated initially with fresh frozen plasma, 10 ml per kg body weight, every 24 h. Following treatment, the mean plasma level of protein C was 0.1 U/ml after 30 min and less than 0.02 U/ml after 24 h. The child was then treated with a concentrate of human protein C and S, 100 U protein C per kg body weight, given every 48 h for a period of 9 months. The mean plasma level of protein C was 0.93 U/ml 30 min after administration of the concentrate and 0.13 and 0.08 U/ml after 24 and 48 h, respectively. The mean post‐transfusional in vivo recovery of protein C was 44% and the half life was 8.3 h. The mean plasma level of ‘free’protein S increased from 1.1 to 2.2 U/ml after administration of the concentrate. There was no increase in ‘bound’protein S. The in vivo recovery of ‘free’protein S was 49% and the half life was about 17 h. Since the start of this replacement therapy using a human protein C and S concentrate, the patient has not developed any thromboembolic complications. These results indicate the therapeutic value of human protein C and S concentrate in the treatment of severe protein C deficiency.

CD40 induces resistance to TNF‐mediated apoptosis in a fibroblast cell line

CD40, a member of the TNF receptor family, has been characterized as an important T‐B cell interaction molecule. In B cells it co‐stimulates isotype switching, proliferation, adhesion and is involved in cell death regulation. In addition to B cells, CD40 expression was found on transformed cells and carcinomas. However, little is known about its functions in these cell types. Recent studies show that CD40 mediates the production of pro‐inflammatory cytokines in non‐hematopoietic cells, inhibits proliferation or induces cell death. In some cell types the apoptotic program triggered by CD40 is only executed when protein synthesis is blocked, suggesting the existence of constitutively expressed resistance proteins. Here we demonstrate that CD40, similar to the 55‐kDa TNF receptor (p55TNFR), has a dual role in the regulation of apoptosis in such cells. In the fibroblast cell line SV80 both CD40 and the p55TNFR trigger apoptosis when protein synthesis is blocked with cycloheximide (CHX). Simultaneous activation of both receptors results in markedly enhanced cell death. However, CD40 activation more than 4u2009h prior to a challenge with TNF/CHX paradoxically conferred resistance to TNF‐induced cell death. Protection correlated with NF‐κB induction and up‐regulation of the anti‐apoptotic zinc finger protein A20. Overexpression of A20 in turn rendered SV80 cells resistant to TNF cytotoxicity. In conclusion, our data provide evidence that CD40 may regulate cell death in non‐hematopoietic cells in a dual fashion: the decision upon apoptosis or survival of a CD40‐activated cell seems to depend on its ability to up‐regulate resistance factors.

Circulating plasma receptors for tumour necrosis factor in Malawian children with severe falciparum malaria

Tumour necrosis factor (TNF) concentrations are increased in the plasma during a malarial illness, and are highest in patients with severe or fatal disease. We have studied the plasma concentrations of two soluble receptors (sTNF-R1 and sTNF-R2), which act as binding proteins for TNF, in children with falciparum malaria. In 52 Malawian children with malaria, plasma concentrations of both sTNF-R1 (mean (S.D.) 4759(2552) pg/ml) and sTNF-R2 (59077(37102) pg/ml) were greatly increased when compared with levels of convalescence (sTNF-R1 718(68), and sTNF-R2 8015(7021) pg/ml), and in controls without malaria (486(1353) and 4380(2168)). Concentrations of both receptors correlated with plasma levels of TNF measured by immunoradiometric assay, but not with those of another cytokine, IL-6. The mean plasma concentrations of both immunoreactive TNF and soluble TNF receptors were greater in patients with cerebral malaria than those with uncomplicated malaria. Despite high levels of immunoreactive TNF in the plasma of patients acutely ill with malaria, no bioactive TNF could be detected in these patients by the WEHI cell bioassay. Soluble TNF receptors are present in greatly increased concentrations in the plasma of patients with malaria and may play a role in mediating or modulating the pathogenetic effects of the cytokine.

Purification of TNF Binding Proteins

The finding that the two tumor necrosis factor receptors (TNFR) exist in soluble form in various body fluids not only has substantiated the paradigm of naturally existing soluble cytokine receptors but also has represented a milestone on the road to the biochemical and biological characterization of the two TNFRs. This chapter gives a simple, basic protocol for the purification of the two soluble TNFRs. The protocols found here may be easily adapted for the purification of various other soluble cytokine receptors. The purified proteins may be used in biological experiments or for the generation of specific research tools such as polyclonal or monoclonal antibodies.