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Kaposi sarcoma-associated herpesvirus on in vitro colony formation of hematopoietic progenitor cells
Leukemia Research 29 (2005) 951–953
Short communication
Suppressive effect of human herpesvirus-8/Kaposi sarcoma-associated herpesvirus on in vitro colony formation of hematopoietic progenitor cells Mario Luppi ∗ , Raffaella Trovato, Patrizia Barozzi, Federica Gibellini, Leonardo Potenza, Giovanni Riva, Giuseppe Torelli Department of Oncology and Haematology, University of Modena and Reggio Emilia, Policlinico, Via del Pozzo 71, 41100 Modena, Italy Received 20 January 2005; accepted 26 January 2005 Available online 2 March 2005
We and others have described the occurrence of bone marrow (BM) failure and peripheral blood cytopenia as a result of primary infection and/or reactivation with the human herpesvirus-8 (HHV-8)/Kaposi sarcoma-associated herpesvirus both in solid organ and in BM/peripheral blood stem cell transplant patients [1,2]. Susceptibility of human fetalderived mesenchymal stem cells to HHV-8 infection in culture has also been demonstrated, raising the possibility that these cells may have a role in the development of HHV-8 related pathology of the marrow, through the horizontal transmission of the virus to the non-adherent hemopoietic progenitor cells [3]. This would, however, imply that the virus may exert a myelosuppressive effect on such cells, which has not yet been investigated. To address this issue, we examined the effect of HHV-8 on in vitro colony formation of hemopoietic progenitor cells in methylcellulose semi-solid media as well as in liquid cultures of BM mononuclear cells. HHV-8 particles were purified from the BCBL-1 lymphoma cell line supernatants, after treatment with tetradecanoyl phorbol acetate, and concentrated as described [4]. The viral titer was determined as reported [5]. An amount of 107 viral genome equivalents/ml of BCBL-1 culture supernatant was obtained [4]. One milliliter of viral preparation (which equals 40 ml of supernatant) was incubated with 1,5 million of Ficoll-Hypaque (Lymphoprep; Nycomed Pharma, Oslo, Norway) separated BM mononuclear cells, with the addition of Polybrene (2 g/ml) (Sigma–Aldrich, St Louis, MO), for 24 h. In the first set of experiments 80,000 BM cells were dispersed in methylcellulose culture (Stem Cell Technology Ins., Vancouver, CA), immediately after virus adsorption and after being maintained in liquid culture for one week, ∗
Corresponding author. Tel.: +39 059 4224641; fax: +39 059 4224549. E-mail address: [email protected] (M. Luppi).
0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.01.012
as described [6] (Fig. 1A). This latter approach may more closely resemble in vivo infection, as it allows secondary infection. After 24 h culture, the mean value of granulocytemacrophage-colony forming unit (CFU-GM) and erythroid 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 R&D Systems, Minneapolis, MN) and 10 U/ml erythropoietin (Gibco-BRL, Grand Island, NY) for one month (Fig. 1B). In HHV-8 infected BM culture the highest reduction in cell count (of about 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 some human cytomegalovirus clinical isolates, showing variable inhibition of colony formation, ranging from 36 ± 8% to 58 ± 8% for CFU-GM, and from 31 ± 17% to 44 ± 4% for BFU-E [7]. However, different clinical HHV-8 isolates should be tested to assess the myelosuppressive effect of HHV-8, more precisely. Earlier reports, before the discovery of HHV-8, have described hematologic manifestations in homosexual men with Kaposi sarcoma and AIDS patients,
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M. Luppi et al. / Leukemia Research 29 (2005) 951–953
Fig. 1. Influence of KSHV on the number of either colony-forming BM cells in methylcellulose cultures or of BM cells in liquid cultures. (A) BM cells were dispersed, immediately after virus adsorption, in 2 ml of methylcellulose medium (Stem Cell Technology Ins., Vancouver, CA) containing Iscove’s modified Dulbecco’s medium (Gibco-BRL, Grand Island, NY), supplemented with 20% fetal bovine serum (FBS) (Gibco-BRL), and with recombinant cytokines: 50 ng/ml stem cell factor, 10 ng/ml granulocyte/monocyte-colony stimulation factor, 10 ng/ml interleukin 3 (all from R&D Systems, Minneapolis, MN), and 10 U/ml erythropoietin (Gibco-BRL). KSHV infected and uninfected BM cells were resuspended in 2.5 ml of long-term culture medium (Dexter original medium), as described (7), and maintained in culture. At day 7 cells were removed from liquid culture and the progenitor number was determined by methylcellulose culture. After 14 days the number of granulocyte-macrophage-colony forming units (CFU-GM) and erythroid burst forming-units (BFU-E) was counted either in KSHV infected or in uninfected control BM cell cultures, plated in duplicate. Bars represent the number of colony-forming cells per 2 × 105 cells in terms of the mean percent ± S.D. of uninfected cultures grown in parallel. Filled bars represent myeloid colonies and empty bars represent erythroid colonies. Results are representative of four independent experiments, each performed on the BM cells obtained from four healthy individuals, respectively. (B) BM cells were cultured in suspension in Iscove’s modified Dulbecco’s medium (Gibco-BRL), supplemented with 20% fetal bovine serum (FBS) (Gibco-BRL), and recombinant cytokines. Cell numbers of infected and uninfected BM cells were assessed every week, by light microscope. Bars represent the number of BM cells in infected cultures in terms of the mean percent ± S.D. of uninfected cultures grown in parallel. Results are representative of three independent experiments.
including alterations in marrow cellularity and peripheral cytopenia [8]. Our findings advocate a possible pathogenetic role of HHV-8 also in human immunodeficiency virus associated cytopenias.
ica Gibellini, performed the experiments. Leonardo Potenza, Giovanni Riva, and Giuseppe Torelli contributed to data analyisis.
References Acknowledgements Grant support was provided by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C., Milan, Italy). Contributions. Mario Luppi, designed the study and wrote the paper. Raffaella Trovato, Patrizia Barozzi, and Feder-
[1] Luppi M, Barozzi P, Schulz TF, Setti G, Staskus K, Trovato R, et al. Bone marrow failure associated with human herpesvirus-8 infection after transplantation. New Engl J Med 2000;343:1378– 85. [2] Cuzzola M, Irrora G, Iacopino O, Cuzzocrea A, Messina G, Console G, et al. Bone marrow failure associated with herpesvirus 8 infec-
M. Luppi et al. / Leukemia Research 29 (2005) 951–953 tion in a patient undergoing autologous peripheral blood stem cell transplantation. Clin Infect Dis 2003;37:e102–6. [3] Parsons CH, Szomju B, Kedes DH. Susceptibility of human fetal mesenchymal stem cells to Kaposi’s sarcoma-associated herpesvirus. Blood 2004;104:2736–8. [4] Renne R, Blackbourn D, Whitby D, Levy J, Ganem D. Limited transmission of Kaposi’s Sarcoma-associated herpesvirus in cultured cells. J Virol 1998;72:5182–8. [5] Lagunoff M, Bechtel J, Venetsanakos E, Roy AM, Abbey N, Herndier B, et al. De Novo Infection and Serial Transmission of Kaposi’s Sarcoma-associated herpesvirus in cultured endothelial cells. J Virol 2002;76:2440–8.
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[6] Dominici M, Luppi M, Campione D, Lanza F, Barozzi P, Milani R, et al. PCR with degenerate primers for highly conserved DNA polymerase gene of the herpesvirus family shows neither human herpesvirus 8 nor a related variant in bone marrow stromal cells from multiple myeloma patients. Int J Cancer 2000;86: 76–82. [7] Simmons P, Kaushansky K, Torok-Storb B. Mechanisms of cytomegalovirus-mediated myelosuppression: perturbation of stromal cell function versus direct infection of myeloid cells. Proc Natl Acad Sci USA 1990;87:1386–90. [8] Spivak JL. Hematologic abnormalities in the acquired immune deficiency syndrome. Am J Med 1984;77:224–8.
Short communication
Suppressive effect of human herpesvirus-8/Kaposi sarcoma-associated herpesvirus on in vitro colony formation of hematopoietic progenitor cells Mario Luppi ∗ , Raffaella Trovato, Patrizia Barozzi, Federica Gibellini, Leonardo Potenza, Giovanni Riva, Giuseppe Torelli Department of Oncology and Haematology, University of Modena and Reggio Emilia, Policlinico, Via del Pozzo 71, 41100 Modena, Italy Received 20 January 2005; accepted 26 January 2005 Available online 2 March 2005
We and others have described the occurrence of bone marrow (BM) failure and peripheral blood cytopenia as a result of primary infection and/or reactivation with the human herpesvirus-8 (HHV-8)/Kaposi sarcoma-associated herpesvirus both in solid organ and in BM/peripheral blood stem cell transplant patients [1,2]. Susceptibility of human fetalderived mesenchymal stem cells to HHV-8 infection in culture has also been demonstrated, raising the possibility that these cells may have a role in the development of HHV-8 related pathology of the marrow, through the horizontal transmission of the virus to the non-adherent hemopoietic progenitor cells [3]. This would, however, imply that the virus may exert a myelosuppressive effect on such cells, which has not yet been investigated. To address this issue, we examined the effect of HHV-8 on in vitro colony formation of hemopoietic progenitor cells in methylcellulose semi-solid media as well as in liquid cultures of BM mononuclear cells. HHV-8 particles were purified from the BCBL-1 lymphoma cell line supernatants, after treatment with tetradecanoyl phorbol acetate, and concentrated as described [4]. The viral titer was determined as reported [5]. An amount of 107 viral genome equivalents/ml of BCBL-1 culture supernatant was obtained [4]. One milliliter of viral preparation (which equals 40 ml of supernatant) was incubated with 1,5 million of Ficoll-Hypaque (Lymphoprep; Nycomed Pharma, Oslo, Norway) separated BM mononuclear cells, with the addition of Polybrene (2 g/ml) (Sigma–Aldrich, St Louis, MO), for 24 h. In the first set of experiments 80,000 BM cells were dispersed in methylcellulose culture (Stem Cell Technology Ins., Vancouver, CA), immediately after virus adsorption and after being maintained in liquid culture for one week, ∗
Corresponding author. Tel.: +39 059 4224641; fax: +39 059 4224549. E-mail address: [email protected] (M. Luppi).
0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.01.012
as described [6] (Fig. 1A). This latter approach may more closely resemble in vivo infection, as it allows secondary infection. After 24 h culture, the mean value of granulocytemacrophage-colony forming unit (CFU-GM) and erythroid 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 R&D Systems, Minneapolis, MN) and 10 U/ml erythropoietin (Gibco-BRL, Grand Island, NY) for one month (Fig. 1B). In HHV-8 infected BM culture the highest reduction in cell count (of about 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 some human cytomegalovirus clinical isolates, showing variable inhibition of colony formation, ranging from 36 ± 8% to 58 ± 8% for CFU-GM, and from 31 ± 17% to 44 ± 4% for BFU-E [7]. However, different clinical HHV-8 isolates should be tested to assess the myelosuppressive effect of HHV-8, more precisely. Earlier reports, before the discovery of HHV-8, have described hematologic manifestations in homosexual men with Kaposi sarcoma and AIDS patients,
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M. Luppi et al. / Leukemia Research 29 (2005) 951–953
Fig. 1. Influence of KSHV on the number of either colony-forming BM cells in methylcellulose cultures or of BM cells in liquid cultures. (A) BM cells were dispersed, immediately after virus adsorption, in 2 ml of methylcellulose medium (Stem Cell Technology Ins., Vancouver, CA) containing Iscove’s modified Dulbecco’s medium (Gibco-BRL, Grand Island, NY), supplemented with 20% fetal bovine serum (FBS) (Gibco-BRL), and with recombinant cytokines: 50 ng/ml stem cell factor, 10 ng/ml granulocyte/monocyte-colony stimulation factor, 10 ng/ml interleukin 3 (all from R&D Systems, Minneapolis, MN), and 10 U/ml erythropoietin (Gibco-BRL). KSHV infected and uninfected BM cells were resuspended in 2.5 ml of long-term culture medium (Dexter original medium), as described (7), and maintained in culture. At day 7 cells were removed from liquid culture and the progenitor number was determined by methylcellulose culture. After 14 days the number of granulocyte-macrophage-colony forming units (CFU-GM) and erythroid burst forming-units (BFU-E) was counted either in KSHV infected or in uninfected control BM cell cultures, plated in duplicate. Bars represent the number of colony-forming cells per 2 × 105 cells in terms of the mean percent ± S.D. of uninfected cultures grown in parallel. Filled bars represent myeloid colonies and empty bars represent erythroid colonies. Results are representative of four independent experiments, each performed on the BM cells obtained from four healthy individuals, respectively. (B) BM cells were cultured in suspension in Iscove’s modified Dulbecco’s medium (Gibco-BRL), supplemented with 20% fetal bovine serum (FBS) (Gibco-BRL), and recombinant cytokines. Cell numbers of infected and uninfected BM cells were assessed every week, by light microscope. Bars represent the number of BM cells in infected cultures in terms of the mean percent ± S.D. of uninfected cultures grown in parallel. Results are representative of three independent experiments.
including alterations in marrow cellularity and peripheral cytopenia [8]. Our findings advocate a possible pathogenetic role of HHV-8 also in human immunodeficiency virus associated cytopenias.
ica Gibellini, performed the experiments. Leonardo Potenza, Giovanni Riva, and Giuseppe Torelli contributed to data analyisis.
References Acknowledgements Grant support was provided by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C., Milan, Italy). Contributions. Mario Luppi, designed the study and wrote the paper. Raffaella Trovato, Patrizia Barozzi, and Feder-
[1] Luppi M, Barozzi P, Schulz TF, Setti G, Staskus K, Trovato R, et al. Bone marrow failure associated with human herpesvirus-8 infection after transplantation. New Engl J Med 2000;343:1378– 85. [2] Cuzzola M, Irrora G, Iacopino O, Cuzzocrea A, Messina G, Console G, et al. Bone marrow failure associated with herpesvirus 8 infec-
M. Luppi et al. / Leukemia Research 29 (2005) 951–953 tion in a patient undergoing autologous peripheral blood stem cell transplantation. Clin Infect Dis 2003;37:e102–6. [3] Parsons CH, Szomju B, Kedes DH. Susceptibility of human fetal mesenchymal stem cells to Kaposi’s sarcoma-associated herpesvirus. Blood 2004;104:2736–8. [4] Renne R, Blackbourn D, Whitby D, Levy J, Ganem D. Limited transmission of Kaposi’s Sarcoma-associated herpesvirus in cultured cells. J Virol 1998;72:5182–8. [5] Lagunoff M, Bechtel J, Venetsanakos E, Roy AM, Abbey N, Herndier B, et al. De Novo Infection and Serial Transmission of Kaposi’s Sarcoma-associated herpesvirus in cultured endothelial cells. J Virol 2002;76:2440–8.
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[6] Dominici M, Luppi M, Campione D, Lanza F, Barozzi P, Milani R, et al. PCR with degenerate primers for highly conserved DNA polymerase gene of the herpesvirus family shows neither human herpesvirus 8 nor a related variant in bone marrow stromal cells from multiple myeloma patients. Int J Cancer 2000;86: 76–82. [7] Simmons P, Kaushansky K, Torok-Storb B. Mechanisms of cytomegalovirus-mediated myelosuppression: perturbation of stromal cell function versus direct infection of myeloid cells. Proc Natl Acad Sci USA 1990;87:1386–90. [8] Spivak JL. Hematologic abnormalities in the acquired immune deficiency syndrome. Am J Med 1984;77:224–8.