Kiyoshi Yasukawa

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 gp130 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 gp130 released by transfected cells can form a ternary complex with interleukin-6 indicating that such complexes also may occur in vivo.

Peripheral blood monocytes are common precursor cells of dendritic cells (DCs) and macrophages. We have searched for factors with the potential to regulate the differentiation of monocytes to DCs and macrophages. When CD14+ monocytes are cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) 4, the CD14+CD1a- population, which consists of macrophages, was found in the serum-containing cultures but not in the serum-free cultures. Addition of IL-6 receptor-neutralizing monoclonal antibody (mAb) or gp130-neutralizing mAb to the serum-containing cultures resulted in a decreased population of CD14+CD1a- cells. An increase in the CD14+CD1a- population with reduction in CD14-CD1a+ DCs was observed with the addition of IL-6 to cultures, whereas IL-11, leukaemia inhibitory factor, oncostatin M or macrophage colony-stimulating factor did not affect the differentiation of monocytes in the presence of GM-CSF plus IL-4. This effect of IL-6 was blocked by tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), IL-1beta, CD40 ligand (CD40L) and transforming growth factor beta1 (TGF-beta1). Among these factors, TNF-alpha was most potent in interfering with the action of IL-6. These results suggest that IL-6 inhibits the differentiation of monocytes to DCs by promoting their differentiation toward macrophages, which is modulated by factors such as TNF-alpha, LPS, IL-1beta, CD40L and TGF-beta1.

Interleukin‐6 (IL‐6) exerts its action via a receptor complex composed of a ligand‐binding sub‐unit (gp80) and a signal transducer (gp130) which both belong to the hematopoietic receptor super‐family. Very little is known about the biosynthesis and the biological half‐lives of proteins of this superfamily. Therefore, we studied the biosynthesis and maturation of the interleukin‐6 receptor and its signaling subunit gp130 by pulse chase experiments in stably transfected Madin‐Darby canine kidney cells. We found that both proteins are synthesized as precursors with apparent molecular masses of 67 kDa and 130 kDa, respectively. These receptor forms are processed within 45–60 min into mature proteins of 82 kDa and 150 kDa containing complex‐type oligosaccharides. The signal transducer gp130 shows a similar maturation in human hepatoma cells HepG2. The IL‐6 receptor appears at the cell surface 45 min after completion of its synthesis in the endoplasmic reticulum. In both cell types studied, gp80 and gp130 are rapidly turned over with half‐lives of 2–3 h. These half‐lives were unaffected by the presence of the ligand IL‐6.