Interleukin-6 and dexamethasone modulate in vitro asymmetric antibody synthesis and UDP-Glc glycoprotein glycosyltransferase activity.
The increased production of asymmetric IgG protective antibodies is one of the mechanisms proposed to explain a successful semiallogeneic pregnancy. We have previously demonstrated that IL-6 was able to enhance the synthesis of these antibodies by a murine hybridoma, while the glucocorticoid dexamethasone (DEXA) diminished it. In order to investigate the mechanism of asymmetric antibody synthesis, we investigate the role of UDP-Glc glycoprotein glucosyltransferase (GT), an endoplasmic reticulum enzyme involved in the quality control and folding of glycoproteins. Either recombinant murine rmIL-6 (0-10-40-160-320-640 ng/ml) or DEXA (0.15 microM) were added to a mouse hybridoma culture and incubated for 24 and 72 h in the first case, and for 4h in the presence of DEXA. Anti-DNP asymmetric antibodies were determined in the culture supernatants by ELISA. After harvesting, hybridoma cells were sonicated and GT activity was analysed in isolated microsomal fractions by measuring UDP((14)C)-Glc incorporation into urea-denatured thyroglobulin (urea-Tg). In the present paper, we showed that IL-6, mainly at 40 ng/ml and t=24h, was able to upregulate both in vitro GT activity (+74%) and asymmetric molecule synthesis (+227%). Lower increases were obtained employing 10 and 160 ng/ml. On the other hand, DEXA, at 0.15 microM and t=4h, showed a mild inhibition of enzyme activity (-10%) and a diminished proportion of asymmetric IgG (-49%). A direct relationship between GT activity and proportion of the asymmetric antibody synthesised was found in our hybridoma cells employing IL-6 and DEXA in different conditions, as was indicated by a correlation analysis. These results suggest that GT might be involved in the synthesis of asymmetric antibodies.
Miranda
S
S
Instituto de Estudios en Inmunidad Humoral (IDEHU), CONICET-UBA, Junín 956, 4P-1113 Buenos Aires, Argentina. smiranda@ffyb.uba.ar
Canellada
A
A
Gentile
T
T
Margni
R
R
D000818:Animals; D003907:Dexamethasone / Q000494:pharmacology*; D005964:Glucosyltransferases / Q000378:metabolism*; D006825:Hybridomas; D007074:Immunoglobulin G / Q000096:biosynthesis*; D015850:Interleukin-6 / Q000494:pharmacology*; D051379:Mice; D015854:Up-Regulation; D014530:Uridine Diphosphate / Q000378:metabolism
D007074:Immunoglobulin G; D015850:Interleukin-6; D014530:Uridine Diphosphate; D003907:Dexamethasone; D005964:Glucosyltransferases; C072423:mannosylglycoprotein 1,3-glucosyltransferase