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Antimicrobial Agents and Chemotherapy, February 2000, p. 405-407, Vol. 44, No. 2
CEA, Service de Neurovirologie, DSV/DRM,
CRSSA, IPSC, Fontenay aux Roses,1
SPI-BIO, Massy,2 Laboratoire
d'Immunodifférenciation, Université Denis Diderot,
Paris,4 and Laboratoires
Mayoly-Spindler, Chatou,3 France
Received 5 May 1999/Returned for modification 10 August
1999/Accepted 18 October 1999
Amphotericin B derivatives, such as MS-8209, have been evaluated as
a therapeutic approach to human immunodeficiency virus (HIV) infection.
We show that MS-8209, like amphotericin B, increases tumor necrosis
factor alpha (TNF- Macrophages and related cells play a
key role in pathological events, particularly in inflammatory processes
associated with human immunodeficiency virus (HIV) disease. They are a
major target cell for HIV, and proinflammatory monokines such as tumor
necrosis factor alpha (TNF- We obtained human macrophages by 7-day differentiation of freshly
isolated monocytes. Monocytes were separated from peripheral blood
mononuclear cells using countercurrent centrifugal elutriation. Cells
were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and 1% triantibiotic
mixture (penicillin, neomycin, and streptomycin). Cell culture medium was endotoxin free, as shown by the Limulus amebocyte lysate
test. To study the effects of MS-8209 on TNF- MS-8209 dose dependently increased TNF-
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Effects of MS-8209, an Amphotericin B Derivative, on Tumor
Necrosis Factor Alpha Synthesis and Human Immunodeficiency Virus
Replication in Macrophages
ABSTRACT
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Abstract
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References
) mRNA expression and TNF- production and
consequently HIV replication in human macrophages. These effects
confirm the pharmacological risk associated with the administration of
amphotericin B or its derivatives to HIV-infected patients.
TEXT
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Abstract
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) increase HIV type 1 (HIV-1) gene
expression and replication, via intracellular activation and autocrine
and paracrine pathways (5, 14, 19). MS-8209 is a
water-soluble derivative of amphotericin B (AmB) of lower cellular and
animal toxicity and greater solubility than the parent compound
(13). MS-8209 exhibits antiretroviral activity in
mitogen-activated CD4+ T lymphocytes infected in vitro with
HIV-1 (4, 11). MS-8209 exerts its antiviral action by
inhibiting HIV entry into cells after CD4-gp120 interactions
(13). AmB provokes marked overexpression of TNF- in
murine and human macrophages (10, 15, 17). In view of the
deleterious effects of TNF- on HIV replication, it was therefore of
interest to investigate the possible effects of MS-8209 on TNF-
synthesis and HIV replication in macrophages.
synthesis,
monocyte-derived macrophages (MDM) were exposed to various
concentrations of MS-8209 (0, 5, and 10 µM) for 24 h, during
which TNF- was measured in cell supernatants by enzyme-linked
immunosorbent assay (ELISA), and its mRNA was quantified by reverse
transcriptase PCR (RT-PCR) (1). To investigate the effects
of MS-8209 on HIV replication, 1 million MDM were infected in vitro
with 10,000 50% tissue culture infective doses of either the reference
macrophage-tropic HIV-1/Ba-L strain or the primary HIV-1-DAS isolate.
TNF- and viral replication were measured throughout the culture in
cell supernatants by ELISA and by dosing RT activity, respectively, as
previously described (7). In these last experiments, cell
culture medium and MS-8209 (0, 1, 5, and 10 µM) were renewed twice a week.
synthesis and TNF- mRNA
expression during the first 24 h of MDM treatment (Fig. 1). In long-term experiments, a
dose-dependent increase in TNF- synthesis was also observed in
response to MS-8209. HIV-1/Ba-L and -DAS replicated efficiently in our
MDM cultures (7). The significant increase in HIV-1
replication induced by 5 and 10 µM MS-8209 was correlated with the
overexpression of TNF- (Fig. 2) but
unlike the increase in TNF- synthesis was not dose dependent, suggesting that it was only a consequence of TNF- and not of its
concentration. Similar results were observed with cells isolated from a
second blood donor and another AmB derivative (MS-1191 [data not
shown]). Furthermore, we have previously described the increase in
HIV-1/Ba-L replication associated with the overexpression of TNF- in
in vitro-infected MDM treated with dapsone (8). Increased
mortality was suspected for HIV-infected patients receiving dapsone as
prophylaxis for Pneumocystis carinii pneumonia and toxoplasmic encephalitis (16).
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FIG. 1.
TNF- mRNA expression and TNF- production in
uninfected MDM treated for 24 h with MS-8209. RNAs were quantified
using a noncompetitive RT-PCR (1), and the cytokine was
detected in cell culture supernatants by ELISA (Immunotech, Luminy,
France). Results are expressed as the means ± standard deviations
of three different culture wells. This experiment was performed using
cells from one given donor, and identical results were observed with
cells isolated from a second donor. Data were analyzed using an
unpaired t test (Statview microcomputer software; Abacus
Concept Inc., Berkeley, Calif.). Differences were considered to be
significant at P < 0.01 (**) or P < 0.05 (*). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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FIG. 2.
RT activity and TNF- in culture supernatants of MDM
treated with 1, 5, and 10 µM MS-8209. MDM were treated throughout the
culture and infected either with the reference macrophage-tropic
HIV-1/Ba-L strain or with the primary macrophage-tropic HIV-1-DAS
isolate (10,000 50% tissue culture infective doses). HIV replication
was assessed throughout the culture by the assay of RT activity using
the RetroSys kit (Innovagen, Lund, Sweden). TNF- was measured in
cell culture supernatants of uninfected MDM by ELISA. Each point is the
mean ± standard deviation of results from three different culture
wells. This experiment was performed using cells from one given cell
donor, and identical results were observed with cells from a second
blood donor. Data were analyzed using an unpaired t test.
Differences were considered to be significant at P < 0.01 (**) or P < 0.05 (*).
These results obtained with AmB derivatives are consistent with
previously published data showing an increase in TNF- synthesis after exposure of murine and human macrophages to AmB (10, 15, 17) and TNF--induced enhancement of HIV replication
(19). Like AmB, its derivatives may have contrasting effects
on the cell population studied, i.e., macrophages or T cells
(4). In vivo use of AmB derivatives could therefore be
unsuccessful in HIV infection. More particularly, the enhancement of
HIV replication in MDM could account for the relative inefficiency of
MS-8209 in simian immunodeficiency virus (SIV) mac251-infected
macaques, especially since our SIVmac251 isolate infects
cells of the macrophage lineage (unpublished data). Moreover, this
TNF- overexpression could explain the deleterious or beneficial
effects of AmB observed for other infections. Elevated intracranial
pressure is a sometimes-fatal adverse reaction in HIV-infected patients
treated with AmB for cryptococcal meningitis (18), and
TNF- amplifies contraction of cerebral arteries (9). Low
intracellular catalase levels exacerbate AmB-related toxicity and
interfere with AmB's efficacy in mice infected with the bacterium
Listeria monocytogenes (2). As TNF- increases
the synthesis of superoxide dismutase, which produces the substrate of
catalase, i.e., hydrogen peroxide, high hydrogen peroxide levels may
exceed catalase's capacity and have effects similar to those of low
catalase levels.
It is likely that the effects of AmB derivatives are mediated by
NF-
B, a transcription factor of the NF-B-Rel family which regulates the expression of numerous cellular genes, particularly those
involved in the inflammatory response, such as TNF- (3), and of HIV provirus through its binding sites in the HIV long terminal
repeat promoter (6). Even though the role of other IB
proteins remains unclear, NF-B is activated by TNF- and a
pathogenic agent such as HIV using this activation pathway. Its
specific inhibitor subunit IB is phosphorylated, the cytoplasmic NF-B-IB complex is then disrupted, and NF-B is translocated into the nucleus, where it can bind to its specific binding sites. By
enhancing HIV replication and TNF- synthesis in cells of macrophage lineage, AmB derivatives could induce neurological disorders in HIV-infected patients. The neuropathogenesis of HIV infection is
governed by HIV replication in mononuclear phagocytes, the production
of viral and cellular components mediating inflammation and
neurotoxicity, and neuronal injury and death. TNF- participates in
inflammation and induces neuronal apoptosis through the platelet activating factor (12).
Although the precise mode of action of AmB and its derivatives is not well understood, our study confirms that they interfere with the macrophage activation state and its ability to produce proinflammatory cytokines and should be used carefully in management of HIV-infected patients.
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ACKNOWLEDGMENTS |
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We acknowledge the Centre de Transfusion Sanguine des Armées (CTSA; Clamart, France) and the Service de Cytaphérèse de l'Hôpital Saint-Louis (Paris, France).
This work was supported by the Agence Nationale de Recherches sur le SIDA (ANRS; Paris, France), the Institut de Formation Supérieure Biomédicale (IFSBM; Villejuif, France), the Association pour la Recherche en Neurovirologie (ARN; Griselles, France), the Association Claude Bernard (Paris, France), the Association Naturalia-Biologia (Paris, France), and the Ministère de l'Education Nationale, de la Recherche et de la Technologie (Action Concertée 10, Paris, France).
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FOOTNOTES |
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* Corresponding author. Mailing address: Service de Neurovirologie, CEA/DSV/DRM, 60-68, avenue de la Division Leclerc, B.P. 6, 92265, Fontenay aux Roses Cedex, France. Phone: 33 (0)1 46 54 87 69. Fax: 33 (0)1 46 54 77 26. E-mail: clayette{at}dsvidf.cea.fr.
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Antimicrobial Agents and Chemotherapy, February 2000, p. 405-407, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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