Karl-Josef Kallen

Cellular Cholesterol Depletion Triggers Shedding of the Human Interleukin-6 Receptor by ADAM10 and ADAM17 (TACE)

Interleukin-6 (IL-6) activates cells by binding to the membrane-bound IL-6 receptor (IL-6R) and subsequent formation of a glycoprotein 130 homodimer. Cells that express glycoprotein 130, but not the IL-6R, can be activated by IL-6 and the soluble IL-6R which is generated by shedding from the cell surface or by alternative splicing. Here we show that cholesterol depletion of cells with methyl-β-cyclodextrin increases IL-6R shedding independent of protein kinase C activation and thus differs from phorbol ester-induced shedding. Contrary to cholesterol depletion, cholesterol enrichment did not increase IL-6R shedding. Shedding of the IL-6R because of cholesterol depletion is highly dependent on the metalloproteinase ADAM17 (tumor necrosis factor-α-converting enzyme), and the related ADAM10, which is identified here for the first time as an enzyme involved in constitutive and induced shedding of the human IL-6R. When combined with protein kinase C inhibition by staurosporine or rottlerin, breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide also increased IL-6R shedding. The effect of cholesterol depletion was confirmed in human THP-1 and Hep3B cells and in primary human peripheral blood monocytes, which naturally express the IL-6R. For decades, high cholesterol levels have been considered harmful. This study indicates that low cholesterol levels may play a role in shedding of the membrane-bound IL-6R and thereby in the immunopathogenesis of human diseases.

The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma and TNF-alpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10.

The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-γ and TNF-α, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-γ and TNF-α synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.

The role of transsignalling via the agonistic soluble IL-6 receptor in human diseases.

The activation of cells that do not express the membrane bound interleukin-6 6 receptor (IL-6R) by IL-6 and the soluble IL-6 receptor (sIL-6R) is termed transsignalling. Transsignalling may be an pathogenetic factor in human diseases as diverse as multiple myeloma (MM), Castlemans disease, prostate carcinoma, Crohns disease, systemic sclerosis, Stills disease, osteoporosis and cardiovascular diseases. IL-6 and sIL-6R may directly or indirectly enhance their own production on endothelial or bone marrow stromal cells. Positive feedback autocrine loops thus created in affected organs may either cause or maintain disease progression. In autoimmune or vasculitic disease, the ability of the IL-6/sIL-6R complex to inhibit apoptosis of autoreactive T-cells may be central to the development of tissue specific autoimmunity. The anti-apoptotic effect of the IL-6/sIL-6R complex may be involved in tumour genesis and resistance to chemotherapy. Only in rare cases, where counterregulation has failed, there is a notable systemic effect of IL-6/sIL-6R. Appropriate animal models are necessary to establish the pathogenetic role of the IL-6/sIL-6R complex. A specific treatment option for diseases influenced by the sIL-6R could be based on gp130-Fc, a soluble gp130 (sgp130) linked to the Fc-fragment of IgG1. gp130-Fc has shown efficacy in vivo in animal models of Crohns disease.

Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection

Despite substantial improvements, influenza vaccine production—and availability—remain suboptimal. Influenza vaccines based on mRNA may offer a solution as sequence-matched, clinical-grade material could be produced reliably and rapidly in a scalable process, allowing quick response to the emergence of pandemic strains. Here we show that mRNA vaccines induce balanced, long-lived and protective immunity to influenza A virus infections in even very young and very old mice and that the vaccine remains protective upon thermal stress. This vaccine format elicits B and T cell–dependent protection and targets multiple antigens, including the highly conserved viral nucleoprotein, indicating its usefulness as a cross-protective vaccine. In ferrets and pigs, mRNA vaccines induce immunological correlates of protection and protective effects similar to those of a licensed influenza vaccine in pigs. Thus, mRNA vaccines could address substantial medical need in the area of influenza prophylaxis and the broader realm of anti-infective vaccinology.

Depletion of cellular cholesterol and lipid rafts increases shedding of CD30.

CD30, a lymphoid activation marker, is shed into the cell environment after endoproteolytic cleavage of its ectodomain. Soluble (s)CD30 is able to suppress the Th1-type immune response. Because high serum levels of sCD30 and cholesterol-lowering drugs seem to be beneficial in some Th1-type autoimmune diseases, we focused on a link between CD30 shedding and the amount of cellular cholesterol. Cholesterol depletion of human Hodgkin lymphoma- and non-Hodgkin lymphoma-derived cell lines by methyl-β-cyclodextrin led to a down-regulation of membrane-bound CD30 and increased release of sCD30. Additionally, the cholesterol-interfering drugs lovastatin, cholesterol oxidase, and filipin increased CD30 shedding. Both the down-regulation of membrane-anchored CD30 and the release of sCD30 were dependent on metalloproteinases. Using specific inhibitors, we detected TNF-α converting enzyme (TACE) as the leading enzyme responsible for cholesterol-dependent CD30 shedding. A Triton X-100-based method for lipid raft isolation revealed that CD30 was partially present in lipid rafts, whereas TACE was localized in the nonraft fractions. Disintegration of lipid rafts by cholesterol depletion might therefore lead to dynamic interactions of CD30 with TACE, resulting in enhanced shedding of CD30. Our results suggest a possible role of cholesterol-dependent shedding of CD30 in the pathogenesis of immune diseases.

IL-6 type cytokine receptor complexes: hexamer, tetramer or both?

Abstract The typical protein fold of most cytokines is a bundle of four antiparallel helices. This ‘four-helical bundle fold’ seems to be unique to cytokines and has not been detected in other proteins. Cytokine receptors, however, can be classified as a subfamily of the immunoglobulin superfamily. Cytokines using the same receptor subunits are grouped into cytokine families. The interleukin-6 (IL-6) type cytokine family comprises six members. IL-6 type cytokines may interact with three receptor subunits instead of the usual two subunits. A tetramer would be the simplest model to describe such a receptor complex, but present orthodoxy describes the active complexes of IL-6 and ciliary neurotrophic factor (CNTF) as hexamers. Here, we summarize the structural and biochemical information on IL-6 type cytokines and discuss interactions between cytokine and individual receptor subunits at alternative positions. Contradictory results regarding the stoichiometry and assembly of signaling receptor complexes are rationalized by a new, unique model. The model stipulates that a ligand-induced transition from an active tetrameric to an inactive hexameric complex serves as a molecular switch that turns off cytokine signals in the presence of supraoptimal cytokine concentrations.

Receptor Recognition Sites of Cytokines Are Organized as Exchangeable Modules TRANSFER OF THE LEUKEMIA INHIBITORY FACTOR RECEPTOR-BINDING SITE FROM CILIARY NEUROTROPHIC FACTOR TO INTERLEUKIN-6

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are “4–helical bundle” cytokines of the IL–6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.

Dynamics of the gp130 cytokine complex: A model for assembly on the cellular membrane

Cytokines of the interleukin‐6 (IL‐6)‐type family all bind to the glycoprotein gp130 on the cell surface and require interaction with two gp130 or one gp130 and another related signal transducing receptor subunit. In addition, some cytokines of this family, such as IL‐6, interleukin‐11, ciliary neurotrophic factor, neuropoietin, cardiotrophin‐1, and cardiotrophin‐1‐like‐cytokine, interact with specific ligand binding receptor proteins. High‐ and low‐affinity binding sites have been determined for these cytokines. So far, however, the stoichiometry of the signaling receptor complexes has remained unclear, because the formation of the cytokine/cytokine‐receptor complexes has been analyzed with soluble receptor components in solution, which do not necessarily reflect the situation on the cellular membrane. Consequently, the binding affinities measured in solution have been orders of magnitude below the values obtained with whole cells. We have expressed two gp130 extracellular domains in the context of a Fc‐fusion protein, which fixes the receptors within one dimension and thereby restricts the flexibility of the proteins in a fashion similar to that within the plasma membrane. We measured binding of IL‐6 and interleukin‐b receptor (IL‐6R) by means of fluorescence‐correlation spectroscopy. For the first time we have succeeded in recapitulating in a cell‐free condition the binding affinities and dynamics of IL‐6 and IL‐6R to the gp130 receptor proteins, which have been determined on whole cells. Our results demonstrate that a dimer of gp130 first binds one IL‐6/IL‐6R complex and only at higher ligand concentrations does it bind a second IL‐6/IL‐6R complex. This view contrasts with the current perception of IL‐6 receptor activation and reveals an alternative receptor activation mechanism.

Highly potent mRNA based cancer vaccines represent an attractive platform for combination therapies supporting an improved therapeutic effect

Direct vaccination with mRNA encoding tumor antigens is a novel and promising approach in cancer immunotherapy. CureVacs mRNA vaccines contain free and protamine‐complexed mRNA. Such two‐component mRNA vaccines support both antigen expression and immune stimulation. These self‐adjuvanting RNA vaccines, administered intradermally without any additional adjuvant, induce a comprehensive balanced immune response, comprising antigen specific CD4+ T cells, CD8+ T cells and B cells. The balanced immune response results in a strong anti‐tumor effect and complete protection against antigen positive tumor cells. This tumor inhibition elicited by mRNA vaccines is a result of the concerted action of different players. After just two intradermal vaccinations, we observe multiple changes at the tumor site, including the up‐regulation of many genes connected to T and natural killer cell activation, as well as genes responsible for improved infiltration of immune cells into the tumor via chemotaxis. The two‐component mRNA vaccines induce a very fast and boostable immune response. Therefore, the vaccination schedules can be adjusted to suit the clinical situation. Moreover, by combining the mRNA vaccines with therapies in clinical use (chemotherapy or anti‐CTLA‐4 antibody therapy), an even more effective anti‐tumor response can be elicited. The first clinical data obtained from two separate Phase I/IIa trials conducted in PCA (prostate cancer) and NSCLC (non‐small cell lung carcinoma) patients have shown that the two‐component mRNA vaccines are safe, well tolerated and highly immunogenic in humans. Copyright

A novel, disruptive vaccination technology: Self-adjuvanted RNActive® vaccines

Nucleotide based vaccines represent an enticing, novel approach to vaccination. We have developed a novel immunization technology, RNActive® vaccines, that have two important characteristics: mRNA molecules are used whose protein expression capacity has been enhanced by 4 to 5 orders of magnitude by modifications of the nucleotide sequence with the naturally occurring nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) that do not affect the primary amino acid sequence. Second, they are complexed with protamine and thus activate the immune system by involvement of toll-like receptor (TLR) 7. Essentially, this bestows self-adjuvant activity on RNActive® vaccines. RNActive® vaccines induce strong, balanced immune responses comprising humoral and cellular responses, effector and memory responses as well as activation of important subpopulations of immune cells, such as Th1 and Th2 cells. Pre-germinal center and germinal center B cells were detected in human patients upon vaccination. RNActive® vaccines successfully protect against lethal challenges with a variety of different influenza strains in preclinical models. Anti-tumor activity was observed preclinically under therapeutic as well as prophylactic conditions. Initial clinical experiences suggest that the preclinical immunogenicity of RNActive® could be successfully translated to humans.