Spontaneous apoptosis and highly active antiretroviral therapy (HAART)

0 2000 Editions

Dossier: 1999-2000

Spontaneous

scientitiques

Biomed & Pharmacother 2000 ; 54 : 16-20 et medicales Elsevier SAS. All rights reserved

Special AIDS issue

apoptosis and highly active antiretroviral (HAART)

therapy

T.N. Dieye1s2,J.P. Van VooreS, M.L. Delforge”, C. Liesnard”, M. Devleeschouwe?, C.M. Farber** I Centre National de Transfusion Sanguine du S~n&ul, Dakar; Erasme Hospital, 4 Department of Microbiology, Institute

z Immunodeficiency unit, Erasme Hospital, i Luhoratoy of Virology, of Pharmaq Universite Libre de Bruxelles, Brussels, Belgium

Summary -Increased programmed cell death (PCD) or apoptosis has been detected in the T cells of HIV-infected subjects; it is held partially responsible for the continuous loss of CD4+ T cells during the natural course of HIV infection. Highly active antiretroviral therapy (HAART) decreases the viral load and leads to an increase of CD4+ count in vivo. In this study we evaluated PCD in total peripheral blood mononuclear cells, CD8+ and CD4+ lymphocytes before and four weeks after initiation of HAART. Seven HIV-l-infected patients were investigated. Viral load was assessed by RT-polymerase chain reaction and PCD by flow cytometry using apoptosis by 7 amino actinomycin D (7AAD) and propidium iodide (PI). After four weeks of HAART, CD4+ T and CD8+ T cell levels were stable, and plasma HIVRNA copies were significantly decreased. In four of the patients (4/7), HIV-RNA levels were reduced to undetectable levels (fewer than 400 copies per milliliter). A statistically significant reduction of apoptosis among CD4+ cells was observed (P < 0.03). though neither in the CD8+ T cell population nor in peripheral blood mononuclear c,ells (PBMCS). These results demonstrate the beneficial effect of HAART on apoptosis of CD4+ ceils in the early treatment stage. 0 2000 Editions scientiliques et medicales Elsevier SAS 7AAD

I apoptosis

/ CD4+

cell / CDS+

cell / HAART

I HIV

I PBMC

The natural history of HIV disease involves CD4+ lymphocyte depletion; it has been shown that apoptosis or programmed-cell death (PCD) is a mechanism of CD4+ loss in HIV infection [l]. Mellors et al. [2] have shown that the importance of viraemia, measured as the HIV- 1 RNA level, is the best available surrogate marker of HIV-l disease progression and should help guide future therapies. Guidelines recommend that highly active antiretroviral treatment (HAART) should include the administration of at least one protease inhibitor and two reverse transcriptase inhibitors, and that it should be initiated when the plasma viral load is higher than 5,000 or 10,000 copies/ml [3]. HAART has been shown to reduce viral load and increase CD4+ cell number in HIV-infected patients [4]. CD4+ lymphocyte proliferation in response to in-vitro antigen stimulation increases after combination antiretroviral therapy [5]. In addition,

/ PCD

these regimens dramatically delay the progression of HIV infection, increasing survival and quality of life [6]. It is also known that CD4+ lymphocyte PCD is high in AIDS [ 11.Routine laboratory assessment of response to antiretroviral therapy is usually restricted to measurement of CD4+ lymphocytes and the plasma HIV RNA load. Flow cytometry allows not only lymphocyte subsets identification but also other determinations, including that of PCD in the same run. We wanted to know if the evaluation of circulating CD4+ and CDS+ lymphocyte PCD in the context of HAART could also have a clinical relevance. METHODS Patients Our protocol was approved by the local ethics committee and an informed consent was obtained from all patients. Seven

*Correspondence: CM. Farber, Unite de Traitement des Immunodeliciences (UTI), HGpital Erasme, ULB. 808 route de Lennik, 1070, Bruxelles, Belgium.

HIV-

1 infected,

previously

untreated

patients

were

studied before and four weeks after initiation of HAART. All patients were male, and mean age was 43 + 11 years. These

were

unselected

sequential

patients

who

presented

Apoptosis and HAART at the clinic a viral load superior to 10,000 copies of HIV- 1 RNA per milliliter. None of them had reached stage C of HIV-l disease. HAART consisted of the administration of two nucleoside analogues and one or two protease inhibitors. Five HIV-l negative healthy controls, mean age 40 f 15 years, were tested for PCD.

17

Plasma HIV- 1 RNA was quantified using a nested RT-PCR performed with GAG primers (Amplicor, Roche Diagnostic Systems Inc, Branchburg, NJ). Samples for viral load measurement and cell testing were collected simultaneously.

0.1% (PBA). Two hundred pL (5. lo5 cells) were incubated in the dark for 15 min at + 4 “C with either 5 pL of conjugated monoclonal anti-CD4-FITC (12.5 l.tg/mL) or 5 pL of anti-CD&FITC (12.5 pg/mL) (Becton Dickinson). After two washes in PBA, cells were resuspended in a solution of 20 ug/mL of 7AAD in PBA for 20 min at + 4” C. The latter reaction was stopped by centrifugation, cells were resuspended in a solution of 20 pg/mL of actinomycin D (AD) in PBA and fixed for 20 min in the same buffer containing 1% paraformaldehyde (Sigma, St. Louis, MO). The samples were immediately acquired on a FACScan in the staining solution. Seven AAD emission was detected in the ‘red channel’, FL3 (650 nm < wavelength < 850 nm) on the FACScan flow cytometer (Becton Dickinson).

Immunophenotyping by flow cytometry

Propidium iodide(PI) staining

Quantitative HIV-l RNA load

Flow cytometry analysis was performed on peripheral whole blood in standard conditions using a FACScan flow cytometer connected to Lysis II software (Becton Dickinson, Erembodegem, Belgium). Peridinin chlorophyll protein-conjugated anti-CD3 antibody (6.25 pg/mL), fluorescein isothiocyanate-conjugated anti-CD4 (12.5 pg/mL) and phycoerythrin-conjugated anti-CD8 (12.5 pg/mL) monoclonal antibodies were used (Becton Dickinson). Phenotyping was performed according to the manufacturer’s recommendations.

Cell preparation and culture Peripheral blood mononuclear cells (PBMC) were isolated from heparinized whole blood by Ficoll-Hypaque (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) gradient density and washed three times in Hank’s Balanced Salt Solution (HBSS) (Gibco, BRL, Life Technologies, Paisley, Scotland). Cultures were performed (1 mL/well) in 24 flat-bottom well plates, lo6 cells per mL in RPM1 1640 supplemented with 10% (vol/vol) heat-inactivated foetal calf serum (Gibco BRL). The plates were incubated at 37” C in a water-saturated atmosphere containing 5% CO,. PCD was evaluated after 24 hours of incubation.

Quantification of apoptosis 7 aminoactinomycin D (7AAD) staining This technique identifies cells in the late stages of PCD since it relies on increased cell membrane permeability. We adapted the technique of Schmid et al. [7] as follows: cells recovered after 24 hours’ culture were centrifuged at 200 g for 10 min and resuspended in PBS-BSA 1%-NaN,

This technique identifies apoptotic cells irrespective of cellular membrane permeability, thus cells in early as well as late stages of PCD. Apoptosis was quantified by staining nuclei with PI. Fluorescence was analysed with the FACScan as originally described by Nicoletti et al. [8] and adapted by Gougeon et al. [9]. Briefly, following 24 hours of incubation, the cells were collected and centrifuged at 200 g for 10 min. The pellet was resuspended in a hypotonic solution of PI (50 pg of PI diluted in 0.1% Triton X-100 [Sigma]). Cells were incubated overnight at + 4” C and analysed using the FACScan to determine the PI fluorescence. Apoptotic nuclei appear as broad hypodiploid DNA peaks that are easily distinguished from the narrow peak of nuclei with normal (diploid) DNA content in the red fluorescence channel (FL2, bandpass 585 nm, bandwidth 24 nm).

Statistical analysis Comparisons were performed using paired t tests or Mann-Whitney tests. Correlations were calculated by linear regression analysis.

RESULTS Effects of HAART on HIV-l burden in peripheral blood Results obtained after HAART are summarized in table I. A significant reduction in plasma HIV-l RNA (> 2 log) was observed in all patients after four weeks of HAART except for patient P4. In four patients, plasma HIV- 1 RNA were reduced to undetectable levels (fewer than 400 collies per mL).

18 Table

T.N. Dieye et al

I. Effects

of highly

active antiretroviral

1 Therapy

therapy

(HAART)

2

before/after

on HIV-I

Studysubjects 4

3

before/after

viral load and T lymphocyte

before/after

bgfore/after

5 before/after

210,197/661

20,629/9,648

78,362/533

subsets

6 before/after

7 before/after

HIV-l RNA (copies/mL)

64 1,087/<

T Lymphocytes CD3% /mm’ CD4% /mm3 CD8% /mm’

91 3,640 4 160 85 3.400

90 1,800 21 420 64 1,280

92 2,760 11 330 80 2,400

94 3,190 8 280 84 2,940

85 1,280 23 345 65 975

82 1,390 26 440 58 980

85 1,275 5 105 78 1,060

84 760 6 54 73 660

73 470 12 78 54 350

89 680 12 91 65 490

85 970 21 238 61 690

91 1,017 26 294 63 710

81 1,620 3% 65 1,300

82 1,720 21 440 59 1,240

0.05

0.33

0.14

0.10

0.38

0.45

0.10

0.10

0.23

0.18

0.34

0.41

0.25

0.35

CD4:CD8

400

29,840/c

400

Effects on CD4+ and CDS+ lymphocyte

33,477/<

400

81,761/c

400

subsets

The mean CD4+ (225 + 111.106/L) before and (288 + 16 1.106/L) after four weeks of HAART did not vary significantly, but CD4+ counts of two patients, Pl and P7, increased (fourfold for Pl and 1.5-fold for P7) (table I). CD8+ counts remained stable, mean (1,453 & 1,071.106/L) before and (1,185 + 828.106/L) after four weeks of HAART. Comparison between total PBMC apoptosis before and after HAART PCD was higher in HIV-l carriers than in controls. Apoptosis of total PBMC did not vary significantly after one month of HAART. Results obtained by 7AAD and PI staining (data not shown) were similar and correlated (r = 0.39; P = 0.005). CD4+ T and CDS+ T cell apoptosis before and after HAART Dual staining with 7AAD and the relevant monoclonal antibody allows us to differentiate the PCD of CD4+ and CDS+ lymphocytes. CD4+ PCD was significantly decreased after one month of HAART (mean 38.2 + 13.7% vs 17.9&3.9%, P
60

g t 8 I.g s! 8

B L s s e

50

40

30

20

14

0

30 W

Figure 1. CD4+ lymphocyte and after treatment (7-AAD of PCD in P5.

PCD in individual patients, before staining). Note the dramatic increase

Apoptosis

19

and HAART

60 -,

P< 0.004

Figure 2. Percentage of apoptotic cells after 24 hours of incubation and after 7 AAD staining. Percentage of apoptotic PBMC, CD4+ and CDs+ lymphocyte subsets from HIV-I infected persons and from seronegative controls measured by 7 AAD staining. A oaired t test compares PCD beFore iday 0) and after tdav 30) HAART in HIV-I oatients. and a Mann-Whitney test compares PCD between HIV- 1 patients and control subjects.

77 NS

HIV-l CD4

ctrl

-___________ HIV-1

day 30

ral treatment (76% vs 17.9%). In this patient (P5), a Hodgkin’s lymphoma was diagnosed six weeks after the beginning of HAART. This patient also showed an increase in the percentage of CD8+ apoptotic cells over time (9.1% vs 2.2%). DISCUSSION We have observed a significant decrease of both CD4+ lymphocyte PCD and viral load after a short period (four weeks) of HAART in five out of seven patients. We saw no significant increase of CD4+ counts in the whole group; however. this might be due to our small sample size. Viral load remained high, with a reduction of PCD in one patient who was found to be noncompliant with therapy. Viral load became undetectable but CD4+ and CD8+ lymphocyte PCD increased dramatically in another case; a Hodgkin’s lymphoma was diagnosed a few weeks later. We used two different techniques to analyse PCD in total PBMC. Results obtained were not significantly different between the two although they identify different phenomena associated with PCD. The use of 7AAD allows simultaneous labeling of cell surface antigens and thus identification of CD4+ and CD8+ subsets; this is not the case with PI because cell membranes are lysed during processing. Previous studies using this tech-

HIV-1 PBMC

ctrl

CD8 jLI

ctrl

control

nique have demonstrated that susceptibility to PCD of CD4+ and CD8+ cells correlates positively with viral load and disease progression [ 1, 10, I 11. In this study, PCD remained higher in HIV- 1 carriers than in healthy uninfected controls, even in the four patients whose viral load became undetectable. A long-term follow-up and the use of ultrasensitive HIV- 1 RT PCR are needed to confirm these preliminary data. Badley et al. [ 121 have shown a reduction of PCD in lymphoid tissues and a reduction of Fas-susceptibility apoptosis in peripheral CD4+ T lymphocytes during HAART. The increase of PCD observed in our patient with Hodgkin’s lymphoma and the decrease of PCD in a noncompliant patient show that PCD and viral load do not always evolve in parallel. The role of associated conditions should also be taken into account. For example, we have previously studied this latter phenomenon in healthy HIV- 1 carriers, and shown an increase of PCD after recall tetanus immunization without a concomitant change in viral load [ 131. We think that the introduction of 7AAD PCD monitoring during antiretroviral treatment might be considered since it brings clinically relevant information complementary to viral load and lymphocyte phenotyping; it does not require more technical skills or equipment than routine mononuclear cell subsets identification.

20

T.N. Dieye

ACKNOWLEDGEMENTS TND

tration (AGCD).

is the recipient

of a scholarship

G&ne’rale de la Coope’ration This

work

from the Adrninisuu De’veloppement by the Fends National

was supported (FNRS).

pour la Recherche Scientijique

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