Processing of TNF-receptors to soluble receptor forms in myeloid cells

University dissertation from Research Department 2, University Hospital, S-221 85 LUND, SWEDEN

Abstract: TNF (Tumor Necrosis Factor) is a pro-inflammatory cytokine which binds to two cell surface receptors, TNF-R55 and TNF-R75. The receptors undergo proteolytic cleavage and release a fragment (soluble TNF-receptors or TNF-Binding Protein), TNF-R55-BP and TNF-R75-BP. Soluble TNF-receptors are able to bind and inactivate TNF. The release of soluble TNF-receptors was measured with ELISA. TNF binding capacity to cells was measured with iodinated TNF. PMA induced rapid release of TNF-R55-BP and TNF-R75-BP in myeloid cell lines. TNF induced release of soluble TNF receptors after long term incubation. Both PMA- and TNF-induced release of soluble TNF-receptors was inhibited by addition of the pH increasing agents NH4Cl and monensin. Short time incubation with NH4Cl and monensin did not affect PMA-induced downregulation, indicating an intracellular processing site of TNF-receptors to soluble receptor forms. Addition of cAMP resulted in release of TNF-R75-BP. cAMP and PMA had additive effects regarding the production of TNF-R75-BP. Thus, PKA and PKC may regulate the production of soluble TNF-receptors independently of each other. Addition of serineprotease and metalloprotease inhibitors resulted in decreased release of soluble TNF-receptors in myeloid cell lines. In addition, PMA-induced downregulation was inhibited by the metalloprotease inhibitor 1,10-phenanthroline. The effect was reversed by addition of Zn, indicating that Zn-dependent metalloproteases are involved in the release of soluble TNF-receptors. Neutrophil granulocytes released soluble TNF-receptors upon contact with a fibrinogen coated surface. The release was not dependent on integrins. However, TNF-receptors was downregulated by crosslinking integrins in suspension. Incubation of neutrophils on an endothelium which had been prestimulated with IL-1 resulted in adherence and release of soluble TNF-receptors. Both adherence and release of soluble TNF-receptors was blocked by addition of antibodies to integrins. In addition, recombinant TNF-R55-BP was able to inhibit the production of TNF from endothelial cells. Contact with an endothelium resulted in a release of soluble TNF-receptors in monocyte-like cells (THP-1). The release was not dependent on adhesion molecules on the endothelial cells.

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