Enhancement of in vitro HSP 70 expression by placental IL-6

S. MIRANDA, A. CIOCCHINI, T. GENTILE, R. MARGNI

Punta del Este, Uruguay

Congreso; 5º Congreso Latinoamericano de Inmunología; 1999

We have recently demonstrated that mouse crude placental supernatants (PSs) induced an increase of asymmetrically glycosylated blocking antibodies synthesized by an hybridoma and IL-6 appeared to be involved in this effect. Taking into account that molecular chaperones are important in post-translational modifications and in monitoring the process of folding and assembly of intracellular proteins, in the present work we have investigated the participation of the cytoplasmatic hsp72/73 in the immunoregulatory mechanism triggered by placental factors. Two hybridomas secreting both symmetric and, asymmetric mAbs (112B4 and 112B2) were cultured (4xlO6 cells/ml) for 72 hr In the presence of 5% mouse AKR/J x AKR/J and AKR/J x BALB/c crude placental supernatants (76 +/- 4 mg of placental tissue/ml) obtained from primiparous and multiparous females, each n=5. Asymmetric and total anti-DNP Ab production were checked by ELISA with or without previous adsorption of the asymmetric molecules employing Sepharose-ConA. The presence of hsp72/73 was investigated in the respective cellular lysates by dot-blot employing anti-hsp72/73 mAb and a luminiscent detection system. To evaluate the capacity of the cells for inducing hsp72, hybridomas were heat shocked (42.5 ± 0.5°C). Cellular lysates obtained from hybridoma control culture flasks did not noticeable expressed hsp70 indicating that constitutive hsp73 was not detected in our working conditions. While lysates obtained from heat-shock hybridoma cultures and from multiparous PSs-hybridoma cultures showed evident signals, primiparous PSs-hybridoma cultures were negative. When exogenous rmIL-6 was added to 112B2 hybridoma cultures (0-50-100 pg/ml), positive signals corresponding to hsp70 in the cell lysates were observed. All PSs were analized by ELISA to determine IL-6 concentration. It was found that those PSs that were able to induce hsp72 synthesis contained up to 900 pg IL-6/ml. Finally, each hsp72 inducing-PSs were respectively incubated with goat anti IL-6 antibody (O-10-40 mg/ml) and then 112B2 cells were added and incubated When the presence of hsp72/73 was investigated in the cell lysates obtained from the hybridoma cultured in the presence of neutralizing antibody, no signal was obtained indicating that it did not have intrinsic inducing effect. The goat anti IL-6 (free and bound to placental lL-6) added in the neutralization assay was adsorpted with ProteinG-Sepharose. Free IL-6 was not detected by ELISA in these supernatants, indicating that the two concentration tested appeared to be enough for blocking all the placental IL-6 and neutralized the enhancement of the in vitro hsp70 expression by PSs. These results indicate that IL-6 is the unique placental factor able to induce in vitro hsp72 synthesis. Considering the involvement of this cytokine in the in vitro glycosylating effect, our hypothesis is that this molecular chaperone could be implicated in the increased synthesis of asymmetric antibodies anti-paternal antigens observed during gestation.