Rainer Zawatzky

IDENTIFICATION OF A NOVEL DENDRITIC CELL-LIKE SUBSET OF CD64(+)/CD16(+) BLOOD MONOCYTES

Human monocytes (Mo) consist of a major subset of Fcγ‐receptor I (CD64)‐positive typical low accessory phagocytes, and a minor CD64– DC‐like subset with high T cell‐accessory and IFN‐α‐releasing activity. Both populations also differentially express CD16 (Fcγ‐receptor III). Double labeling with anti‐CD64 and anti‐CD16 mAb, as performed here, identified four different subsets. The CD64– subset consists of CD64– / 16+ cells with high antigen‐presenting cell (APC) function and macrophage‐like phenotype, and a CD64– / 16– subset of less active APC but which exhibits a higher mixed lymphocyte reaction (MLR) stimulating and IFN‐α‐producing capacity, possibly resembling plasmacytoid dendritic cell type II (DC2) blood precursors. As well as the majority of CD64+ cells that appeared CD64+ / 16– and represent typical low‐accessory, CD14high Mo, we could identify and describe a novel minor subset of CD64+ / 16+ cells which is unique in combining typical DC and Mo characteristics in the same cell. These are high IL‐12 production, high accessory capacity for antigen‐ or allogen‐activated lymphocytes, and high expression of HLA‐DR, CD86, and CD11c.

Coactivator function of RIP140 for nf{kappa}b/rela-dependent cytokine gene expression

Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor kappaB (NFkappaB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that RIP140 suppresses the expression of metabolic gene networks, but we have found that genetic as well as acute deficiency of RIP140 leads to the inhibition of the proinflammatory program in macrophages. The ability of RIP140 to function as a coactivator for cytokine gene promoter activity relies on direct protein-protein interactions with the NFkappaB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP). RIP140-dependent control of proinflammatory gene expression via RelA/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.

Differential Antiviral Response of Endothelial Cells after Infection with Pathogenic and Nonpathogenic Hantaviruses

ABSTRACT Hantaviruses represent important human pathogens and can induce hemorrhagic fever with renal syndrome (HFRS), which is characterized by endothelial dysfunction. Both pathogenic and nonpathogenic hantaviruses replicate without causing any apparent cytopathic effect, suggesting that immunopathological mechanisms play an important role in pathogenesis. We compared the antiviral responses triggered by Hantaan virus (HTNV), a pathogenic hantavirus associated with HFRS, and Tula virus (TULV), a rather nonpathogenic hantavirus, in human umbilical vein endothelial cells (HUVECs). Both HTNV- and TULV-infected cells showed increased levels of molecules involved in antigen presentation. However, TULV-infected HUVECs upregulated HLA class I molecules more rapidly. Interestingly, HTNV clearly induced the production of beta interferon (IFN-β), whereas expression of this cytokine was barely detectable in the supernatant or in extracts from TULV-infected HUVECs. Nevertheless, the upregulation of HLA class I on both TULV- and HTNV-infected cells could be blocked by neutralizing anti-IFN-β antibodies. Most strikingly, the antiviral MxA protein, which interferes with hantavirus replication, was already induced 16 h after infection with TULV. In contrast, HTNV-infected HUVECs showed no expression of MxA until 48 h postinfection. In accordance with the kinetics of MxA expression, TULV replicated only inefficiently in HUVECs, whereas HTNV-infected cells produced high titers of virus particles that decreased after 48 h postinfection. Both hantavirus species, however, could replicate equally well in Vero E6 cells, which lack an IFN-induced MxA response. Thus, delayed induction of antiviral MxA in endothelial cells after infection with HTNV could allow viral dissemination and contribute to the pathogenesis leading to HFRS.

Experimental infection of inbred mice with herpes simplex virus type 1. I. Investigation of humoral and cellular immunity and of interferon induction.

Considerable differences exist in the lethality of herpes simplex virus type 1 (HSV-1) after intraperitoneal (i.p.) infection in different inbred strains of mice. In this study humoral and cellular immunity and interferon production were compared in resistant and susceptible mice. The serum IgG response, as determined by an enzyme-linked immunosorbent assay (ELISA), was the same in different strains of mice. There was also no difference in neutralizing antibodies between resistant C57BL/6 and susceptible DBA/2 mice. The production of macrophage migration inhibitory factor, obtained from spleen cells of mice 7 days after infection with different doses of HSV-1, was the same in resistant and susceptible mice. Serum interferon could be detected 8 h after i.p. injection of 10(7) p.f.u. of HSV-1, but not at lower virus doses. At 8 h, high interferon titres [less than 1000 reference research units (iu)/ml] were observed in the serum of C57BL/6 mice but low titres (about 100 iu/ml) were found in the serum of DBA/2 mice. At 24 h, the titres were low in both strains of mice. Interferon production was also measured in vitro in spleen cell cultures exposed to inactivated HSV-1. These studies also showed high interferon production in spleen cell cultures of resistant mice, whereas low titres were produced by spleen cells of susceptible mice. Thus, our study has failed to reveal any differences in humoral or cellular immunity in mice resistant or susceptible to HSV but a difference in HSV-induced interferon production.

Viral IFN-Regulatory Factors Inhibit Activation-Induced Cell Death Via Two Positive Regulatory IFN-Regulatory Factor 1-Dependent Domains in the CD95 Ligand Promoter

The CD95 (also called APO-1/Fas) system plays a major role in the induction of apoptosis in lymphoid and nonlymphoid tissues. The CD95 ligand (CD95L) is induced in response to a variety of signals, including IFN-γ and TCR/CD3 stimulation. Here we report the identification of two positive regulatory IFN-regulatory factor-dependent domains (PRIDDs) in the CD95L promoter and its 5′ untranslated region, respectively. EMSAs demonstrate specific binding of IFN-γ-induced IFN-regulatory factor 1 (IRF-1) to the PRIDD sequences. Ectopic IRF-1 expression induces CD95L promoter activity. Furthermore, we demonstrate that PRIDDs play an important role in TCR/CD3-mediated CD95L induction. Most interestingly, viral IRFs of human herpes virus 8 (HHV8) totally abolish IRF-1-mediated and strongly reduce TCR/CD3-mediated CD95L induction. We demonstrate here for the first time that viral IRFs inhibit activation-induced cell death. Thus, these results demonstrate an important mechanism of HHV8 to modulate the immune response by down-regulation of CD95L expression. Inhibition of CD95-dependent T cell function might contribute to the immune escape of HHV8.

Post-Translational Control of IL-1β via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53

Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1β) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1β production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1β processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1β regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1β was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1β that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1β is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1β regulation which ultimately inhibits the secretion of IL-1β in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1β towards cervical cancer could be discerned. Hence, attenuation of IL-1β by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy.

Modification of tumor cells by a low dose of Newcastle disease virus. III. Potentiation of tumor-specific cytolytic T cell activity via induction of interferon-alpha/beta.

Abstract To investigate possibilities of augmenting tumor-specific immune responses against the highly metastatic murine lymphoma ESb, we tested the effects of the interferon inducer newcastle disease virus (NDV) or of interferon- α β as costimulator in mixed lymphocyte-tumor cell cultures (MLTC) on the tumor-specific cytolytic T cell (CTL) response. Both approaches, namely stimulation of ESb immune spleen cells with NDV-modified stimulator cells or with ESb stimulator cells and exogenous IFN- α β , led to a selective potentiation of tumor-specific CTL activity. The potent activation of tumor-specific CTL precursor (CTLP) required the simultaneous presence of the specific ESb tumor antigen—possibly to mediate a signal via the corresponding T cell receptor—and costimulators—possibly to mediate second activation signals. Increased CTL activity required only very low amounts of NDV or IFN- α β . The generation of CTL activity in the MLTC cultures could be blocked by antisera to IFN- α β , not, however by control sera. Similar effects were observed in vivo , suggesting that IFN- α β not only caused an increase in CTL activity, but was essential for the generation of CTL activity. The reduction of the generation of CTL by antiserum to IFN- α β could be overcome by excess interferon, especially when using ESb-NDV as stimulator Cells.

An X-linked locus influences the amount of circulating interferon induced in the mouse by herpes simplex virus type 1.

Production of circulating interferon (IFN) was measured in inbred mouse strains following intravenous injection of herpes simplex virus type 1 (HSV-1). IFN titres reached maximal levels 2 to 3 h after injection of virus and a 10-fold difference was found between C57BL/6 mice and BALB/c, as high and low producers respectively. Mendelian analysis revealed that HSV-induced IFN production is governed by several loci, one of which is X-linked. The strain distribution pattern obtained from results in recombinant inbred lines and the results obtained in the congenic B6-C-H-28c-If-1(1) strain furthermore indicated an absence of close linkage to If-1. It is concluded that the levels of HSV-induced early IFN production are influenced by several autosomal loci and one X-linked locus.

Staphylococcus aureus-Induced Plasmacytoid Dendritic Cell Activation Is Based on an IgG-Mediated Memory Response

Type I IFNs represent a major antimicrobial defense mechanism due to their property of enhancing immune responses by priming both innate and adaptive immune cells. Plasmacytoid dendritic cells (pDC) are the major source of type I IFN in the human body and represent innate immune cells involved in first-line defense against invading pathogens. Although pDC activation has been extensively studied upon stimulation with synthetic TLR ligands, viruses, and intracellular bacteria, there is only scarce information on extracellular bacteria. In this study we show that the triggering of human pDC-derived IFN-α secretion by Staphylococcus aureus is independent of TLR2 and specific for coagulase-positive staphylococci. Specificity of the pDC response to S. aureus is independent of the bacterial virulence factors protein A and α-toxin but is mediated by Ag-specific IgG and CD32. S. aureus-induced pDC activation can be blocked by inhibitory DNA oligonucleotides and chloroquine, suggesting that engagement of TLR7/9 by bacterial nucleic acids after CD32-mediated uptake of these compounds may play a central role in this process. Altogether, we propose that in marked contrast to nonselective TLR2-dependent activation of most innate immune cells, pDC activation by S. aureus represents an Ag-specific memory response since it requires the presence of class-switched immunoglobulins.

Experimental Infection of Inbred Mice with Herpes Simplex Virus. III. Comparison between Newborn and Adult C57BL/6 Mice

We have previously shown that adult C57BL/6 mice are relatively resistant to intraperitoneal (i.p.) infection with herpes simplex virus type 1 (HSV-1). Here we show that newborn mice of the strain C57BL/6 are highly susceptible to i.p. infection with HSV-1. Newborn C57BL/6 mice, in contrast to adult mice, did not develop natural killer cell activity in the peritoneal cavity 24 h after i.p injection of HSV-1, and showed only minimal titres of interferon in the peritoneal fluid after 4 h. After 24 h the peritoneal fluid of newborn mice contained high amounts of interferon and high titres of HSV-1. In contrast, the virus titres in the peritoneal cavity of adult mice were significantly lower. It is suggested that the early titres of interferon at the infection site that are observed in adult, resistant C57BL/6 mice but not in susceptible, newborn mice play a decisive role in resistance.