JAC Advance Access originally published online on July 2, 2007
Journal of Antimicrobial Chemotherapy 2007 60(3):685-689; doi:10.1093/jac/dkm230
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Factors impacting the expression of membrane-bound proteins in lymphocytes from HIV-positive subjects
Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GF, UK
* Correspondence address. Pharmacology Research Laboratories, 70 Pembroke Place, Liverpool L69 3GF, UK. Tel: +44-151-7948211; Fax: +44-151-7945656; E-mail: rjchand{at}liv.ac.uk
Received 28 March 2007; returned 27 May 2007; revised 31 May 2007; accepted 31 May 2007
 |
Abstract |
|---|
 Top Abstract IntroductionPatients, materials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Background: The cellular transport proteins ABCB1, ABCC1 and ABCG2 have been implicated in the efflux of some antiretroviral drugs, thus decreasing their intracellular concentrations. Decreased drug accumulation in lymphocytes may allow viral replication and the subsequent emergence of viral resistance leading to treatment failure. Expression of HIV co-receptors on the surface of lymphocytes may influence viral tropism and therefore viral pathogenicity and disease progression. Here, we describe the relationship between expression of transport proteins and chemokine receptors in lymphocytes isolated from HIV-infected individuals and also investigate their relationship with demographic, therapeutic and virological factors.
Methods: Peripheral blood mononuclear cells (PBMC) isolated from HIV-positive individuals were co-stained for expression of CD4 and ABCB1, ABCC1, ABCG2, CXCR4 and CCR5. The influence of gender, ethnicity, treatment status, viral load and CD4 count was assessed on expression of each protein as well as correlations between expression of the proteins by univariate and multivariate analyses.
Results: Expression of ABCB1 was independently associated with gender (n = 98) and expression of ABCG2 and CXCR4. Gender also correlated with expression of ABCC1 and CXCR4 in univariate analysis with lower expression being detected in females compared with males.
Conclusions: Here we confirm that the previously reported correlation between ABCB1 and CXCR4 observed in PBMC isolated from healthy volunteers is also found in HIV-positive individuals. The influence of gender on the expression of drug efflux proteins could be a determinant of intracellular drug concentrations in vivo.
Keywords: drug transport , gender , chemokine receptors , HIV
|
Introduction |
|---|
|
TopAbstractIntroductionPatients, materials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
ABCB1 (P-glycoprotein), ABCC1 (multidrug resistance associated protein) and ABCG2 (breast cancer resistance protein) are all members of the ATP-binding cassette transporter superfamily and each protein has been implicated in the transport of one or more antiretroviral compounds.1 These transporters are expressed in many tissues including lymphocytes, and their activity may serve to lower intracellular accumulation of compounds, including the HIV protease inhibitors (PIs)2 at their site of action. This creates a sanctuary site for viral replication, thus accelerating the emergence of resistant virus and decreasing efficacy. Expression of ABCB1 has been reported to be influenced by several factors including polymorphisms within the ABCB1 gene (the frequency of which may differ between ethnic groups),3 gender4 and various external stimuli. However, relatively few studies have sought to determine demographic predictors of expression of other transport proteins.
The chemokine receptors CXCR4 and CCR5 act as co-receptors for HIV entry into CD4+ cells and their expression has been linked to susceptibility to HIV infection and disease progression following infection. CCR5 is preferentially utilized by HIV in early infection. However, a switch to CXCR4 usage has been temporally linked to rapid CD4+ cell decline and onset of AIDS.5 Expression of these co-receptors is highly variable and may be affected by numerous factors including genetic polymorphisms,6 gender7 and HIV treatment status.8 We have previously observed a correlation between ABCB1 and CXCR4 expression on peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals.9 No such correlation has yet been reported in HIV-positive individuals, although there is evidence to suggest that expression of both transport proteins1 and chemokine receptors8 may be influenced by HIV infection (both directly and indirectly via immune modulation) and by constituents of antiretroviral therapy. It is therefore important to determine whether the relationship persists in PBMC isolated from these patients, in which the activity of transporters and the availability of HIV co-receptors for viral entry play an important role in viral pathogenesis and treatment. Further, as a role for both ABCC1 and ABCG2 has been suggested in the transport of antiretroviral drugs, relationships between expression of these proteins and chemokine receptors may also be of interest.
In this study, we investigated the expression of ABCB1, ABCC1 and ABCG2 as well as the HIV co-receptors CXCR4 and CCR5 in a cross-sectional cohort of HIV-positive patients with differing demographic, virological and treatment backgrounds.
|
Patients, materials and methods |
|---|
|
TopAbstractIntroductionPatients, materials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Patient details
Patients were recruited from the Royal Liverpool University Hospital. Each participant gave informed, written consent and the study was approved by the local research Ethics Committee.
CXCR4- and CCR5-specific antibodies were provided by the AIDS Reagent Project of the National Institute of Biological Standards and Controls (South Mimms, UK). IgG2a negative control and goat anti-mouse IgG2a:RPE were obtained from Serotech Ltd (Oxford, UK). UIC2 antibody was purchased from Immunotech (Marseilles, France). ABCC1-specific antibody QCRL1 was purchased from Calbiochem (Nottingham, UK) and FITC-conjugated anti-CD4 and Hanks' balanced salt solution were supplied by Sigma Chemical Co. Ltd (Poole, UK). CellFIX was purchased from Becton Dickinson (Oxford, UK). Methanol was obtained from Fisher Scientific (Loughborough, UK) and Ficoll was obtained from Amersham Biosciences (Bucks, UK).
PBMC were isolated from whole blood (20 mL) obtained from each patient (n = 98) by density gradient centrifugation over Ficoll-Hypaque and stained for expression of ABCB1, ABCC1, ABCG2, CXCR4 and CCR5 as previously described9,10 using rPE-labelled antibodies. Cells were co-stained for ABCB1 and CXCR4 with CD4 using an FITC-conjugated antibody, and CD4+ and CD4– populations were gated by their positions on the FL1 fluorescence scale following compensation for spectral overlap.
For total PBMC and CD4+ populations, FL2 (rPE) fluorescence was plotted against the number of events and the data registered on a logarithmic scale prior to calculation of the median fluorescence. Surface expression in each population was then determined by subtracting the median fluorescence of isotype control antibody from that of test antibody. Expression data are presented in relative fluorescence units (RFU; arbitrary units) for each protein.
All data were tested for normality using the Shapiro–Wilk test. Categorical data were analysed by Mann–Whitney U-test and continuous data by Spearman's rank correlation. Data with a P < 0.1 by univariate analysis was log transformed and tested for significance by multiple linear regression using best subset selection (StatsDirect).
|
Results |
|---|
|
TopAbstractIntroductionPatients, materials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Of the HIV-positive participants (n = 98; 67 male, 31 female), 34 had undetectable viral load (<50 copies/mL), those with detectable viral load had a median of 3.69 log copies/mL (range 1.75–5.81 log) and the cohort had a median CD4 count of 347 cells/mm3 (range 8–1318). Patients were on a range of antiretroviral regimens, with 17 on a PI-containing and 42 a non-PI-containing regimen. Thirty-nine patients were treatment naive. Of the patients included in the ethnicity analysis, 72 were Caucasian and 19 Black African.
Expression of ABCB1 on CD4+ cells correlated with that measured on total PBMC (
= 0.45, P = 0.0001, n = 69) as did CXCR4 ( = 0.41, P = 0.0007, n = 67). Univariate and multivariate analyses of expression data in PBMC are presented in Table 1 and Figure 1. Univariate analysis showed a gender effect on ABCB1 expression (mean expression in males 1.02 ± 0.45, n = 62, compared with 0.76 ± 0.42 RFU in females, n = 30, P = 0.001; Figure 1a). Expression of ABCB1 correlated with expression of ABCC1 ( = 0.25, P = 0.02, n = 89; Figure 1b), ABCG2 ( = 0.24, P = 0.03, n = 78, Figure 1c) and CXCR4 ( = 0.54, P < 0.0001, n = 90; Figure 1d). Multivariate analysis showed gender (P = 0.02) and expression of ABCG2 (P = 0.005) and CXCR4 (P = 0.01) to be independent predictors of ABCB1 expression (Table 1).
|
|
There was a gender effect on ABCC1 (mean expression in males 0.44 ± 0.52 RFU, n = 59, compared with 0.24 ± 0.3 in females, n = 29, P = 0.02; Figure 1e), ABCC1 expression correlated with ABCB1 expression (see above) and CXCR4 expression (
= 0.26, P = 0.01, n = 86; Figure 1f). However, none of these variables were independent predictors of ABCC1 expression in multivariate analysis. ABCG2 expression correlated with ABCB1 expression (see above). There was a significant gender effect on CXCR4 expression (mean expression in males 0.47 ± 0.22 RFU, n = 62, compared with 0.38 ± 0.22 in females, n = 30, P = 0.04; Figure 1g) and a correlation with ABCB1 and ABCC1 expression (see above). ABCB1 expression was an independent predictor of CXCR4 expression following multivariate analysis (P = 0.01).
|
Discussion |
|---|
|
TopAbstractIntroductionPatients, materials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
We have previously demonstrated a positive correlation between expression of ABCB1 and CXCR4 on PBMC isolated from healthy volunteers and have also shown that these two proteins are co-localized within the cell membrane.9 In this study, we have confirmed these findings in PBMC isolated from HIV-positive individuals. We also found ABCB1 expression to correlate with ABCG2. This raises the possibility that the emergence of CXCR4-tropic strains of HIV, observed in patients with high lymphocyte CXCR4 expression,11 may be exacerbated by decreased antiretroviral efficacy as a consequence of concomitantly high transporter expression. These strains have been shown to be more lytic to CD4+ cells in vitro.12
The pharmacokinetics of some HIV drugs (e.g. saquinavir) has been found to vary according to gender with higher exposure seen in women.13 It is unclear what mechanism is responsible for this difference or whether there is an impact on response to therapy. We found gender to be associated with the expression of ABCB1, with males having higher expression than females; similar correlations were not observed for other transporters by multivariate analysis. Taken together with previous reports of hepatic ABCB1 expression being higher in males than females,14 it could be argued that our finding of decreased ABCB1 expression in PBMC from female subjects is an indicator of altered expression in other tissues which may in part explain the higher drug exposure of saquinavir13 and indinavir15 in females. However, it is important to note that the pharmacokinetics of PIs is driven by first-pass metabolism by cytochrome P450 enzymes as well as drug transporters in the gut and liver, and it is currently unknown whether transporter expression on the PBMC is a valid surrogate for transporters at these sites. Paine et al.16 have reported no difference in ABCB1 expression in the proximal small intestine between healthy male and female subjects, although hepatic expression has been reported to be higher in males than in females.14 However, there are no data available regarding HIV-positive patients.
We have previously reported that ex vivo lymphocyte ABCB1 expression inversely correlates with the in vitro antiviral EC50 of saquinavir9 and it is possible that any gender-related differences in expression at the lymphocyte may be an additional consideration over and above any gender effects on first-pass metabolism. Studies as to whether treatment outcome varies between genders are equivocal with some studies observing a higher proportion of women than men achieving viral suppression following highly active antiretroviral therapy17,18 and others finding no difference in virological or immunological success between the genders.19,20
In summary, we present data confirming that the ABCB1/CXCR4 correlation observed in PBMC isolated from healthy volunteers is also present in cells isolated from HIV-positive individuals. Expression of ABCB1 is also influenced by gender in HIV-positive individuals.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionPatients, materials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
B. C. is supported by the British Society for Antimicrobial Chemotherapy and Roche. M. D. was supported by the Royal Liverpool & Broadgreen University Hospitals NHS Trust R & D support scheme.
|
References |
|---|
|
TopAbstractIntroductionPatients, materials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
1 Owen A, Chandler B, Back DJ. The implications of P-glycoprotein in HIV: friend or foe? Fundam Clin Pharmacol (2005) 19:283–96.[CrossRef][Web of Science][Medline]
2 Janneh O, Owen A, Chandler B, et al. Modulation of the intracellular accumulation of saquinavir in peripheral blood mononuclear cells by inhibitors of MRP1, MRP2, P-gp and BCRP. AIDS (2005) 19:2097–102.[Web of Science][Medline]
3 Kim RB, Leake BF, Choo EF, et al. Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther (2001) 70:189–99.[CrossRef][Web of Science][Medline]
4 Salphati L, Benet LZ. Modulation of P-glycoprotein expression by cytochrome P450 3A inducers in male and female rat livers. Biochem Pharmacol (1998) 55:387–95.[CrossRef][Web of Science][Medline]
5 Schols D. HIV co-receptor inhibitors as novel class of anti-HIV drugs. Antiviral Res (2006) 71:216–26.[CrossRef][Web of Science][Medline]
6 Paxton WA, Kang S, Koup RA. The HIV type 1 coreceptor CCR5 and its role in viral transmission and disease progression. AIDS Res Hum Retroviruses (1998) 14(Suppl. 1):S89–S92.[Web of Science][Medline]
7
Portales P, Clot J, Corbeau P. Sex differences in HIV-1 viral load due to sex difference in CCR5 expression. Ann Intern Med (2001) 134:81–2.
8 Giovannetti A, Pierdominici M, Mazzetta F, et al. T cell responses to highly active antiretroviral therapy defined by chemokine receptors expression, cytokine production, T cell receptor repertoire and anti-HIV T-lymphocyte activity. Clin Exp Immunol (2001) 124:21–31.[CrossRef][Web of Science][Medline]
9
Owen A, Chandler B, Bray PG, et al. Functional correlation of P-glycoprotein expression and genotype with expression of the human immunodeficiency virus type 1 coreceptor CXCR4. J Virol (2004) 78:12022–9.
10 Minderman H, Suvannasankha A, O'Loughlin KL, et al. Flow cytometric analysis of breast cancer resistance protein expression and function. Cytometry (2002) 48:59–65.[CrossRef][Web of Science][Medline]
11 Manetti R, Cosmi L, Galli G, et al. Enhanced expression of the CXCR4 co-receptor in HIV-1-infected individuals correlates with the emergence of syncytia-inducing strains. Cytokines Cell Mol Ther (2000) 6:19–24.[CrossRef][Web of Science][Medline]
12 Glushakova S, Yi Y, Grivel JC, et al. Preferential coreceptor utilization and cytopathicity by dual-tropic HIV-1 in human lymphoid tissue ex vivo. J Clin Invest (1999) 104:R7–R11.[Medline]
13
Ribera E, Lopez RM, Diaz M, et al. Steady-state pharmacokinetics of a double-boosting regimen of saquinavir soft gel plus lopinavir plus minidose ritonavir in human immunodeficiency virus-infected adults. Antimicrob Agents Chemother (2004) 48:4256–62.
14
Schuetz EG, Furuya KN, Schuetz JD. Interindividual variation in expression of P-glycoprotein in normal human liver and secondary hepatic neoplasms. J Pharmacol Exp Ther (1995) 275:1011–8.
15 Brendel K, Legrand M, Taburet AM, et al. Population pharmacokinetic analysis of indinavir in HIV-infected patient treated with a stable antiretroviral therapy. Fundam Clin Pharmacol (2005) 19:373–83.[CrossRef][Web of Science][Medline]
16
Paine MF, Ludington SS, Chen ML, et al. Do men and women differ in proximal small intestinal CYP3A or P-glycoprotein expression? Drug Metab Dispos (2005) 33:426–33.
17 Fletcher CV, Jiang H, Brundage RC, et al. Sex-based differences in saquinavir pharmacology and virologic response in AIDS Clinical Trials Group Study 359. J Infect Dis (2004) 189:1176–84.[CrossRef][Web of Science][Medline]
18 Hoen B, Fournier I, Lacabaratz C, et al. Structured treatment interruptions in primary HIV-1 infection: the ANRS 100 PRIMSTOP trial. J Acquir Immune Defic Syndr (2005) 40:307–16.[CrossRef][Web of Science][Medline]
19 Nicastri E, Angeletti C, Palmisano L, et al. Gender differences in clinical progression of HIV-1-infected individuals during long-term highly active antiretroviral therapy. AIDS (2005) 19:577–83.[Web of Science][Medline]
20 Mocroft A, Gill MJ, Davidson W, et al. Are there gender differences in starting protease inhibitors, HAART, and disease progression despite equal access to care? J Acquir Immune Defic Syndr (2000) 24:475–82.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||