Antibodies to human IL-10 neutralize ebvIL-10-mediated cytokine suppression but have no effect on cmvIL-10 activity

Abstract

Interleukin-10 is a pivotal determinant of virus clearance or persistence. Two human herpesviruses, Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) are unique among persistent viruses because they not only trigger production of host IL-10, but both viruses also encode homologs of IL-10 that are expressed during infection. Because anti-human IL-10 antibodies have diagnostic value and therapeutic potential for many chronic infections, cross-reactivity with ebvIL-10 and cmvIL-10 was evaluated in this study. Six of seven anti-hIL-10 antibodies tested recognized ebvIL-10 and neutralized its immunosuppressive activity. In contrast, cmvIL-10 was neither recognized nor neutralized by any anti-human IL-10 antibody. These findings demonstrate that IL-10-neutralizing treatments in HCMV- or EBV-infected patients may require consideration of the contribution of viral IL-10 to disease pathology.

Fig. 1

Cross-reactivity of anti-hIL-10 antibodies by Western blot. Purified recombinant hIL-10, IFNγ, cmvIL-10, or ebvIL-10 (R&D Systems) were diluted to 10 μg/ml in PBS and analyzed by SDS-PAGE, transferred to a PVDF membrane, blocked, and then probed with the indicated antibodies at a dilution of 1:100 in PBS plus 5% milk. All anti-hIL-10 monoclonal antibodies are referred to by the clone name and were obtained from Santa Cruz Biotechnology. HCMV and EBV goat polyclonal antisera were obtained from R&D Systems and the HCMV monoclonal antibody was kindly provided by Dr. Gavin Wilkinson. Appropriate alkaline phosphatase conjugated secondary antibodies were used for detection at a dilution of 1:1000. Promega Western Blue Substrate was used for colorimetric detection of bands. Ponceau S staining was performed to confirm the presence of each cytokine on the membrane prior to Western blotting. Results are representative of three separate experiments.

Fig. 2

ELISA detection by anti-human IL-10 antibodies. Purified hIL-10, cmvIL-10, or ebvIL-10 proteins were diluted to 2.5 μg/ml in PBS and used to coat a microtiter plate overnight at 4°C. After blocking, the indicated antibodies were added (4 μg/ml), followed by horseradish peroxidase-conjugated secondary antibody (1:1000) and substrate reagent. An antibody to NF-κB family protein c-rel served as a negative control. Optical density was read at 450 nm following the addition of 1 M sulfuric acid stop solution to each well. Error bars represent standard error. Statistical analysis was performed using a two-tailed Student' t-test, and * indicates P< 0.01. Results are representative of four separate experiments.

Fig. 3

Effect of anti-human IL-10 antibodies onTNFα production. THP-1 monocytes were seeded into 96-well culture dishes at 2 × 10⁴ cells per well and treated with 1 ng/ml LPS in the presence or absence of hIL-10, cmvIL-10, or ebvIL-10 (10ng/ml). After 24 h, supernatants were collected and TNFα levels determined by ELISA (R&D Systems) to determine total cytokine suppression for each cytokine. The indicated antibodies (5 μg/ml) were included just prior to the addition of human or viral IL-10 to neutralize CSIF activity. Results are expressed as percent neutralization, or the extent to which antibodies relieved TNFα suppression caused by the cytokines alone. Error bars represent standard error. Statistical analysis was performed using a two-tailed Student' t-test, and * indicates P<0.01. Results are representative of four separate experiments.