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HEMATOPOIESIS
From the National Heart, Lung and Blood Institute,
National Institutes of Health, Bethesda, MD, and the Division of
Infectious Diseases, Georgetown University Medical Center,
Washington, DC.
Highly active retroviral therapy has been associated with a decline
in the frequency of cytopenia in patients with human immunodeficiency virus (HIV) infection. This may result from lower hematologic toxicity
of newer antiviral drugs and their increased efficacy against HIV-1.
Protease inhibitors, in addition to their effects on HIV replication,
appear to affect various cellular functions. Recently, it was
reported that ritonavir inhibited caspase-1 expression in
normal CD4+ cells. It was hypothesized that protease
inhibitors may improve hematopoietic function owing to their direct
effects on the bone marrow progenitor cells. When ritonavir was added
to methylcellulose cultures of bone marrow cells from HIV-infected
patients and normal controls, colony formation increased 2.4-fold
(n = 5) in control cultures and 4-fold (n = 5) in cultures of cells
from HIV-infected patients. In the presence of ritonavir, cultures of
CD34+ cells showed markedly decreased apoptosis in
comparison with untreated cultures (45% decrease in apoptotic cell
number; n = 6). A synthetic inhibitor of caspase 1 (Ac-Tyr-Val-Ala-Asp-aldehyde [single-letter amino acid codes]), which
inhibits activation of several caspases including CPP32 and interleukin
1 Advanced HIV infection is associated with
cytopenias that are, in part, related to bone marrow (BM)
failure. Although direct infection of progenitor cells may be possible
under some circumstances in vitro,1 CD34+
cells from human immunodeficiency virus (HIV)-infected patients rarely
demonstrate evidence of viral infection.2,3 Although the
stem cell compartment appears to be relatively well preserved early in
the disease in asymptomatic HIV-infected individuals, in patients with
low CD4 counts or with opportunistic infections, there is a marked
deficiency in primitive progenitors as measured by reduction in
long-term colony-initiating cell (LTCIC) numbers.4 Depletion of progenitor cells may be related a number of reasons including infection with HIV or apoptosis, related to changes in
cytokine expresssion or other immunological factors. Data from our
laboratory failed to demonstrate any evidence of infection of the most
immature progenitors, the LTCICs, after exposure to HIV-1 or HIV-2
isolates.5 On the other hand, hematopoietic progenitor
cells derived from BM of HIV-infected patients have been reported to
undergo apoptosis at an increased rate.6 Apoptosis of
hematopoietic progenitor cells, through the Fas-ligand
(Fas-L)/Fas-receptor (Fas-R) pathway, is a major mechanism by which
activated T cells kill virus-infected cells.7 It is likely
that Fas-L and other cytokine products of activated T cells contribute
to the hematopoietic inhibition of HIV-1 infection.8,9
Infection with HIV or exposure of BM accessory cells to gp120 increases
production of tumor necrosis factor- HIV protease inhibitors have led to increased CD4+ cell
numbers and decreased viral loads, resulting in decreased mortality rate and clinical improvement of AIDS patients. Previously, we demonstrated that HIV protease inhibitors blocked apoptosis and caspase-1 expression in culture of CD4+ cells from normal,
uninfected patients and HIV-infected persons; HIV-infected patients
receiving protease inhibitor combinations also demonstrated decreases
in CD4+ cell apoptosis and caspase-1
content.18 On the basis of these findings, we hypothesized
that a similar mechanism may also operate in early hematopoietic cells.
In this study, we attempted to determine if ritonavir, a protease
inhibitor, could block apoptosis, decrease ICE expression, and increase
hematopoietic colony formation in BM cells derived from HIV-infected
patients and normal controls.
Patient selection
Cell preparation
Flow cytometric analysis Phycoerythrin (PE)-conjugated monoclonal antibody (mAb) directed against human CD34+ antigen (Becton Dickinson, Mountain View, CA) was used for phenotypic analysis of BM progenitor cells. Samples were analyzed by means of an Epics Elite flow cytometer (Coulter, Hialeah, FL).Intracellular staining Intracellular staining for ICE expression was performed with the PharMingen Intracellular Staining Kit (Franklin Lakes, NJ).19 BMMCs or peripheral blood mononuclear cells (PBMCs) were stained with PE-conjugated CD34+ antibody, fixed, and permeabilized and stained with anti-ICE antibody and fluorescein isothiocyanate-labeled (FITC)-conjugated antirabbit antibody. Permeabilized isotypic controls were stained with secondary antibodies. Previous data17,18 demonstrated correlation of intracellular staining for ICE with immunoblot.Separation of CD34+ cells After washing with phosphate-buffered saline (PBS) (Life Technologies) supplemented with 2% human albumin, cells were applied to an affinity column containing biotin-coated beads, and the CD34+ cell fraction was eluted with PBS. An aliquot of eluted cells was stained with PE-conjugated anti-CD34+ mAb (Becton Dickinson) to assess purity; usually, 70% to 90% of separated cells were positive for CD34 antigen. For preparations of higher purity, cells were further fractionated: column-purified cells were stained with FITC anti-CD34 mAb, washed with PBS, and sorted by flow cytometry (Epics V; Coulter); by this combined method, the purity of cells was 97% to 99%.Hematopoietic cell culture Numbers of hematopoietic colony-forming cells were measured in methylcellulose colony cultures under standard conditions. Freshly isolated BMMCs were plated in methylcellulose (Stem Cell Technology, Vancouver, BC, Canada) in the presence of 50 ng/mL interleukin-3 (IL-3; Genzyme, Boston, MA), 20 ng/mL granulocyte-macrophage colony stimulating factor (Boehringer, Indianapolis, IN), 50 ng/mL stem cell factor (Amgen, Thousand Oaks, CA), and 2 U/mL erythropoietin (Amgen). Total BMMCs were plated at a density of 1 × 105 in 1 mL of medium in 35-mm dishes. CD34+ cells were plated at a density of 1 × 103 cells/0.5 mL methylcellulose in 48-well 11-mm plates. Colonies on replicate plates were counted, and the average number of colonies per 105 cells was calculated. When appropriate, 100 µg/mL of ICE inhibitor (Ac-Tyr-Val-Ala-Asp-aldehyde) (Calbiochem, Cambridge, MA) was added to culture of BM obtained from HIV-infected patients (this concentration was previously found to be optimal17 for increasing colony formation in normal donors). This ICE inhibitor has demonstrated specificity for inhibition of caspase 1, 8, and 3.20Long-term BM culture for the determination of LTCIC numbers was
performed. We used 10 × 106 BMMCs to initiate stromal
culture. Allogeneic stroma was grown to confluence. Culture medium
consisted of stem cell medium (Stem Cell Technology)
supplemented with 1 × 10 Apoptosis assays Apoptotic cells were quantitated by means of the annexin assay.21 BMMCs were prepared as described above. After culture, cells were washed with PBS and stained with annexin and propidium iodide as previously described.17,21 Samples were analyzed by means of flow cytometry. Gates were drawn to exclude dead, propidium-iodide-staining cells, and the number of apoptotic cells staining with annexin were quantitated.Statistical analysis Statistical significance was calculated by means of a nonparametric Wilcoxon 2-sample or 1-sample test.
Effect of ritonavir on hematopoietic colony formation in short-term methylcellulose colony assay and long-term BM culture First, we determined the effect of ritonavir on colony formation measured in short-term culture. When ritonavir was added to methylcellulose cultures of BM cells from HIV-infected patients and normal controls, colony formation was augmented; in cultures from uninfected normal controls, the number of colonies was increased 2.4-fold (n = 5; P < .01) (Figure 1) at concentrations of 5 nm; in cultures of cells from HIV-infected patients there was a 4-fold increase (n = 3) (P < .05). Both myeloid and erythroid colonies were equally increased.
To assess the effect of ritonavir on the most primitive progenitor
cells, we also placed BM of normal donors with ritonavir (5 nm) in
long-term culture, and determined progenitor number by replating
experiments performed after 5 weeks. There was a 2-fold increase in the
yield of secondary colonies in cultures containing ritonavir (Table
1).
Effect of a caspase inhibitor on colony formation of PBMCs obtained from HIV-infected patients We previously demonstrated that CD34+ cells demonstrated up-regulated expression of capsase-1 during in vitro culture and that the level of caspase-1 expression correlated with apoptosis of these cells.17 In addition, a caspase-1 inhibitor also increased colony growth in normal BM culture. We now determined whether addition of a synthetic caspase inhibitor can also affect colony formation in BM obtained from HIV-infected patients. A caspase-1 inhibitor, in concentrations similar to those reported to increase normal BM growth, increased colony growth of BM progenitor cells obtained from HIV-infected patients17 (Figure 2).
Effect of ritonavir on apoptosis and ICE expression of CD34+ cells CD34+ cells obtained from HIV-infected BM reportedly show increased apoptosis.6 Because of the increase in colony formation in both long-term and short-term cultures performed in the presence of ritonavir, we studied the effect of ritonavir on apoptosis and ICE expression of purified CD34+ cells. We also measured expression of caspase 1 to determine whether the caspase-mediated transduction cascade was involved in the effect of ritonavir. When CD34+ cells were cultured in the presence of ritonavir, there was a 45% decrease in the number of apoptotic cells as determined in the annexin assay (n = 6; P < .05) (Figure 3). This effect was dose-related up to the concentration of 5 nm; higher concentrations of ritonavir resulted in toxicity and drastically decreased cell viability (data not shown). Fas-R expression was unaffected by the addition of ritonavir to CD34+ cells (data not shown); the expression of caspase 1, as measured by double-staining with CD34 mAb and intracellular staining for ICE, was decreased after 2 days of culture (n = 5; P < .05) (Figure 3).
The HIV protease inhibitors decrease viral replication and
result in reduced viremia and slowed progression to AIDS. Clinically, these agents have significantly improved the quality of life of patients with HIV-1 infection and have led to remarkably decreased mortality due to AIDS. Following the introduction of treatment with
protease inhibitor combinations, symptomatic cytopenias and the
transfusion requirements in HIV-infected patients have concurrently fallen. It is likely that decreased viremia in patients successfully treated with antiviral combination therapy also results in diminished levels of cytokines with inhibitory properties on the hematopoietic system, such as IFN- We hypothesized that protease inhibitors must have a direct
effect on hematopoietic colony formation by inhibiting apoptosis and,
more specifically, caspase expression in CD34+ cells. In
our study, we have shown that ritonavir increased colony formation of
normal BM progenitors and progenitor cells derived from HIV-1-infected
persons. This positive effect was paralleled by decreased apoptosis and
caspase expression in the CD34+ cell compartment. In
agreement with these results, we have previously reported that normal
unmanipulated CD34+ cells expressed caspase-1 mRNA, though
we did not detect significant expression of ICE protein in these
cells.17 However, most CD34+ cells expressed
ICE protein in tissue culture and underwent apoptosis that could be
blocked by addition of a synthetic caspase inhibitor. IFN- The beneficial effect of caspase blockade on normal marrow may be related to inhibitory cytokines produced constitutively in the BM microenvironment. It is important to note that we measured ICE (caspase 1) expression, other caspases may also have been involved.16 We recently demonstrated that caspase 1 is most important in modulating CD34+ cell apoptosis.24 In summary, ritonavir increased colony growth of BM obtained either from HIV-infected patients with advanced disease or from normal individuals. Apoptosis and ICE expression in purified CD34+ were also decreased during treatment in vitro. Protease inhibitors may improve hematopoiesis in HIV-infected patient by a mechanism that is independent of their effect on HIV viremia. Whether this class of drugs could be used in other marrow failure states needs further research.
Submitted May 18, 1999; accepted June 7, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Elaine M. Sloand, National Heart, Lung, and Blood Institute, National Institutes of Health, 31 Center Dr, MSC 2490, Bldg 31, Rm 4A11, Bethesda, MD 20892-2490.
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© 2000 by The American Society of Hematology.
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  A. D. Redd, A. Avalos, and M. Essex Infection of hematopoietic progenitor cells by HIV-1 subtype C, and its association with anemia in southern Africa Blood, November 1, 2007; 110(9): 3143 - 3149. [Abstract] [Full Text] [PDF]
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E. M. Sloand, L. Pfannes, G. Chen, S. Shah, E. E. Solomou, J. Barrett, and N. S. Young CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins Blood, March 15, 2007; 109(6): 2399 - 2405. [Abstract] [Full Text] [PDF]
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 D. T. Scadden, V. V. Muse, and R. P. Hasserjian Case records of the Massachusetts General Hospital. Case 30-2006. A 41-year-old man with dyspnea, fever, and lymphadenopathy. N. Engl. J. Med., September 28, 2006; 355(13): 1358 - 1368. [Full Text] [PDF]
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 P. S. Koka, C. M. R. Kitchen, and S. T. Reddy Targeting c-Mpl for Revival of Human Immunodeficiency Virus Type 1-Induced Hematopoietic Inhibition When CD34+ Progenitor Cells Are Re-Engrafted into a Fresh Stromal Microenvironment In Vivo J. Virol., October 15, 2004; 78(20): 11385 - 11392. [Abstract] [Full Text] [PDF]
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 P. Matarrese, L. Gambardella, A. Cassone, S. Vella, R. Cauda, and W. Malorni Mitochondrial Membrane Hyperpolarization Hijacks Activated T Lymphocytes Toward the Apoptotic-Prone Phenotype: Homeostatic Mechanisms of HIV Protease Inhibitors J. Immunol., June 15, 2003; 170(12): 6006 - 6015. [Abstract] [Full Text] [PDF]
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J. Estaquier, J.-D. Lelievre, F. Petit, T. Brunner, L. Moutouh-de Parseval, D. D. Richman, J. C. Ameisen, and J. Corbeil Effects of Antiretroviral Drugs on Human Immunodeficiency Virus Type 1-Induced CD4+ T-Cell Death J. Virol., May 13, 2002; 76(12): 5966 - 5973. [Abstract] [Full Text] [PDF]
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B. N. Phenix, J. J. Lum, Z. Nie, J. Sanchez-Dardon, and A. D. Badley Antiapoptotic mechanism of HIV protease inhibitors: preventing mitochondrial transmembrane potential loss Blood, August 15, 2001; 98(4): 1078 - 1085. [Abstract] [Full Text] [PDF]
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S. Chavan, S. Kodoth, R. Pahwa, and S. Pahwa The HIV protease inhibitor Indinavir inhibits cell-cycle progression in vitro in lymphocytes of HIV-infected and uninfected individuals Blood, July 15, 2001; 98(2): 383 - 389. [Abstract] [Full Text] [PDF]
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 A. M. Levine, D. T. Scadden, J. A. Zaia, and A. Krishnan Hematologic Aspects of HIV/AIDS Hematology, January 1, 2001; 2001(1): 463 - 478. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-converting enzyme (ICE or caspase 1), also
decreased the rate of apoptosis and enhanced colony formation by
progenitor cells derived from HIV-infected patients (3-fold; n = 5).
In ritonavir-treated samples derived from HIV-infected individuals, the
number of cells expressing ICE also decreased. In conclusion, HIV
protease inhibitors may, by blocking the caspase-dependent apoptotic
pathway, overcome inhibition of hematopoiesis seen in patients with HIV
infection, an effect unrelated to their antiviral activity.
(Blood. 2000;96:2735-2739)
(TNF-
(IFN-
6 mol/L hydrocortisone
succinate (Sigma, St Louis, MO) and was replaced weekly. After 3 weeks
of culture, stromal cells were briefly treated with trypsin, washed,
and placed in 48-well plates. After reestablishment of a confluent
cellular layer, the plates were irradiated (15 Gy of 250-kV
x-rays) and used for the measurement of LTCIC content in mononuclear
cell fractions derived from BM and PB of patients and healthy
volunteers. At least 2 (if cell numbers were low) but more often 3 or
more cell concentrations were applied to preestablished, irradiated
stromal feeder layers and cultured at 33°C for 5 weeks.
Media changes were performed weekly. After 5 weeks, the adherent cells
were harvested by treatment with trypsin (Life Technologies), washed,
and replated in duplicate in methylcellulose in order to estimate the
numbers of cells able to form secondary colonies.
indicates a 2-nmol/L
concentration of Ritonavir; 
, 5-nmol/L concentration; 
, 10-nmol/L
concentration; 
,
20-nmol/L concentration.
