Determination of haptoglobin expression in IL-6 treated HepG2 cells by ELISA and by RNA hybridization--evaluation of a quantitative method to measure IL-6.
Interleukin-6 (IL-6) is known to be an important modulator of acute phase (AP) protein expression in hepatocytes both in vivo and in vitro. In the present study the inducing activity of IL-6 on the expression of the AP protein haptoglobin (HP) by the human hepatoma cell line HepG2, has been evaluated. HP mRNA inducibility was analysed by Northern and slot-blot hybridization, while HP protein was detected by means of an ELISA procedure. A dose-response relationship from 0.3 to 4.8 ng/ml of a human recombinant IL-6 preparation derived from a Chinese hamster ovary (CHO) cell line was observed after 48 h of treatment. Comparable results were obtained by analysing both HP mRNA expression and HP protein secretion. Detectable induction of HP protein secretion was observed with as little as 25 pg/ml of IL-6. The effect of IL-6 was potentiated by dexamethasone, while an inhibition on HP mRNA inducibility could be prevented by lowering the foetal calf serum (FCS) concentration to 1%. Preliminary data indicate that neither IL-1 beta nor TNF-alpha were able to induce significantly HP mRNA expression and protein secretion. The activity ratio between two IL-6 preparations (from CHO and E. coli cells) obtained with a conventional IL-6 bioassay (i.e., T1165 cell growth assay) was comparable to that obtained in the induction of HP expression. The nominal specific activity of the CHO-derived IL-6 was two to three times higher with both responses.
Boe
A
A
Istituto di Ricerca Cesare Serono, Ardea, Rome, Italy.
Canosi
U
U
Donini
S
S
Mastrangeli
R
R
Ythier
A
A
Crescenzi
O S
OS
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005069:Evaluation Studies as Topic; D006242:Haptoglobins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis* / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D006242:Haptoglobins; D015850:Interleukin-6; D012333:RNA, Messenger