Suppressive effect of human herpesvirus-8/Kaposi sarcoma-associated herpesvirus on in vitro colony formation of hematopoietic progenitor cells

Abstract

0 d esvirus both in solid organ and in BM/peripheral blood stem ell transplant patients [1,2]. Susceptibility of human fetalerived mesenchymal stem cells to HHV-8 infection in culure has also been demonstrated, raising the possibility that hese cells may have a role in the development of HHV-8 reated pathology of the marrow, through the horizontal transission of the virus to the non-adherent hemopoietic progentor cells [3]. This would, however, imply that the virus may xert a myelosuppressive effect on such cells, which has not et been investigated. To address this issue, we examined the ffect of HHV-8 on in vitro colony formation of hemopoietic rogenitor cells in methylcellulose semi-solid media as well s in liquid cultures of BM mononuclear cells. HHV-8 particles were purified from the BCBL-1 lymhoma cell line supernatants, after treatment with tetradeanoyl phorbol acetate, and concentrated as described [4]. he viral titer was determined as reported [5]. An amount of burst forming-unit (BFU-E) numbers generated by HHV-8 infected BM resulted 33 and 34% , respectively lower than that obtained in control BM (Fig. 1A). At day 7, the myeloid colonies in viral infected cultures were still decreased of about 30%, while erythroid colonies drastically decreased of about 55% in infected BM compared with control uninfected cultures (Fig. 1A). HHV-8 orf-K1 gene sequences were detected in all the ten myeloid and erythroid pooled colonies tested by polymerase chain reaction, as described [1](data not shown). In another set of experiments, 1 million HHV-8 infected and uninfected BM cells were cultured in suspension with recombinant cytokines (50 ng/ml stem cell factor, 10 ng/ml granulocyte/monocyte-colony stimulation factor, 10 ng/ml interleukin 3 (all from RD Nycomed Pharma, slo, Norway) separated BM mononuclear cells, with the 40%) was observed at the first two weeks post-infection, compared with uninfected culture (Fig. 1B). In conclusion, inhibition of colony formation appears at least one mechanism to explain how HHV-8 may suppress hematopoieis. This effect is comparable to that observed with s a t f s H e h ddition of Polybrene (2 g/ml) (Sigma–Aldrich, St Louis, O), for 24 h. In the first set of experiments 80,000 BM cells ere dispersed in methylcellulose culture (Stem Cell Techology Ins., Vancouver, CA), immediately after virus adsorpion and after being maintained in liquid culture for one week,