Jürgen Müllberg

ULBPs, Novel MHC Class I–Related Molecules, Bind to CMV Glycoprotein UL16 and Stimulate NK Cytotoxicity through the NKG2D Receptor

The human cytomegalovirus glycoprotein, UL16, binds to two members of a novel family of molecules, the ULBPs, and to the MHC class I homolog, MICB. The ULBPs are GPI-linked glycoproteins belonging to the extended MHC class I family but are only distantly related to MICB. The ULBP and MICB molecules are ligands for the activating receptor, NKG2D/DAP10, and this interaction is blocked by a soluble form of UL16. The ULBPs stimulate cytokine and chemokine production from NK cells, and expression of ULBPs in NK cell-resistant target cells confers susceptibility to NK cell cytotoxicity. Masking of NK cell recognition of ULBP or MIC antigens by UL16 provides a potential mechanism by which human cytomegalovirus-infected cells might evade attack by the immune system.

ULBP1, 2, 3: novel MHC class I‐related molecules that bind to human cytomegalovirus glycoprotein UL16, activate NK cells

New members of the extended MHC class I‐like family were identified based on their ability to bind human cytomegalovirus glycoprotein UL16 and/or their mutual homology. Soluble UL16 binding prteins (ULBP) competed with each other for binding to NK cells. Treatment of human and mouse NK cells with ULBP led to increased production of cytokines/chemokines, proliferation, cytotoxic activity and up‐regulation of activation‐associated surface molecules. The presence of ULBP during the stimulation phase of the CTL assay caused increased cytotoxic activity. Addition of soluble recombinant UL16 protein inhibited the biological activities mediated by ULBP, suggesting the existence of a novel mechanism utilized by CMV to evade elimination by the host immune system.

Structure-function analysis of human interleukin-6 : evidence for the involvement of the carboxy-terminus in function

C‐terminally deleted analogs of human interleukin‐6 (IL‐6) have been constructed at the cDNA level, and after cell‐free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL‐6 resulted in 58% helix, 14% β‐structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL‐6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% β‐structure, and 18% turn and coil.

Differential shedding of the two subunits of the interleukin‐6 receptor

cDNAs coding for the two receptor subunits of the interleukin‐6 receptor have been stably expressed in Madine Darby canine kidney (MDCK) cells. The fate of the IL‐6 binding protein (IL‐6R) and of the signal transducing protein gp130 was studied independently. Both proteins were proteolytically cleaved from cells metabolically labeled with [35S]methionine/cysteine leading to the release of soluble receptor proteins of 55 kDa and 100 kDa, respectively. In contrast to the shedding of the IL‐6R gp 130 was inefficiently released from the cells and the process was not significantly stimulated by the phorbolester PMA. In addition we show that the soluble forms of the IL‐6R and gp 130 released by transfected cells can form a ternary complexe with interleukin‐6 indicating that such complexes also may occur in vivo. gp 130; Interleukin‐6; Interleukin‐6‐receptor; Protein kinase C; Shedding

Protein kinase C activity is rate limiting for shedding of the interleukin-6 receptor

An analysis of the mechanism of generation of the soluble interleukin-6 receptor (IL-6R) has been performed. The membrane-bound receptor is proteolytically cleaved to release a soluble receptor form which retained its ligand binding capacity. Furthermore, the soluble IL-6R is unique in its ability to induce a biological signal in complex with the ligand interleukin-6 (IL-6) on cells which by themselves do not bind IL-6. Shedding of the IL-6R is strongly activated by PMA and can be inhibited by the protein kinase inhibitor staurosporine. The generation of the IL-6R is not dependent on protein synthesis. The inactive PMA analogue 4-alpha-phorbol-12,13-didecanoate fails to induce shedding of the IL-6R. Transfection of a protein kinase C expression plasmid into IL-6R expressing cells leads to enhanced shedding of the receptor. These experiments clearly show that protein kinase C regulates shedding of the IL-6R.

IL-6 Receptor Independent Stimulation of Human gp130 by Viral IL-6

The genome of human herpes virus 8, which is associated with Kaposi’s sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.

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.

Further evidence for a common mechanism for shedding of cell surface proteins

Pro‐TNFα, Steel factor, type II IL‐1R and IL‐2Rα were expressed in COS‐7 cells and the generation of their soluble forms was examined. The release of all four proteins was strongly stimulated by the phorbol ester PMA and completely blocked by a hydroxamate‐based inhibitor of metalloproteases. COS‐7 cell membranes were found to cleave various synthetic pro‐TNFα peptides with the same specificity as a partially purified TNFα converting enzyme purified from human monocytic cells, suggesting that the same enzyme may be responsible for at least some of the COS‐7 cell shedding activity.

THE MEMBRANE DISTAL HALF OF GP130 IS RESPONSIBLE FOR THE FORMATION OF A TERNARY COMPLEX WITH IL-6 AND THE IL-6 RECEPTOR

Gp130 is the signal transducing subunit of the interleukin‐6 receptor. Signaling is initiated by the complex formation of gp130 with IL‐6 bound to the IL‐6 receptor (IL‐6R). We have subdivided the extracellular domain of gp130 in two parts and expressed the mutant proteins as soluble IgG fusion proteins in COS‐7 cells. By studying the formation of the ternary complex we show that the membrane distal half of gp130 which contains a cytokine receptor domain is responsible for the interaction with the IL‐6/IL‐6R complex. Interestingly this is the same region which is believed to be involved in specific recognition of the related cytokines LIF, OM, and probably also of CNTF and IL‐11.

TIMP-1 protein expression is stimulated by IL-1β and IL-6 in primary rat hepatocytes

Degradation of extracellular matrix proteins is performed by metalloproteinases which are inhibited by tissue inhibitors of metalloproteinases (TIMP). We expressed the murine TIMP‐1 protein in E. coli and prepared a polyclonal antiserum against the recombinant protein. Using this antiserum we studied the biosynthesis and glycosylation of murine TIMP‐1 protein in COS‐7 cells transfected with a TIMP‐1 expression plasmid by metabolic labeling and indirect immunofluorescence studies. In primary rat hepatocytes we show for the first time that TIMP‐1 protein expression is up‐regulated upon stimulation with IL‐1β and IL‐6. Since TIMP‐1 is induced during the acute phase reaction it could possibly be involved in the pathogenesis of liver fibrosis.