Immunologic characteristics of healthy and HIV-1-infected Ethiopians

(1)

UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

Messele, T.

Publication date

2000

Link to publication

Citation for published version (APA):

Messele, T. (2000). Immunologic characteristics of healthy and HIV-1-infected Ethiopians.

General rights

It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulations

If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

(2)

CHAPTERR 6

Reducedd CCR5 expression in Ethiopian commercial sex

workerss with CCR2b-64l polymorphism

(3)

4 4

i i

(4)

Effectt of CCR2b-64l genotype on CCR5 expression 85

Reducedd CCR5 expression in Ethiopian commercial sex

workerss with CCR2b-64l polymorphism

Tsehayneshh Messele*, Mathias Aklilu\ Tsigereda Birru*, Margreet Brouwer*,

Arnaudd L. Fontanet*#, Tobias F. Rinke de Wit* , H. Schuitemaker* and Dörte

Hamann*. .

'Ethio-Netherlandss AIDS Research Project (ENARP) at the Ethiopian Health and Nutritionn Research Institute (EHNRI), Addis Ababa, Ethiopia.

^CLB,, Department of Clinical Viro-lmmunology and Laboratory for Experimental andd Clinical Immunology, Academic Medical Center, University of Amsterdam, the Netherlands. .

#

Municipall Health Service, Division of Health and Environment, Amsterdam, The Netherlands s

Abstract t

AA cross-sectional study was conducted in HIV-1-infected and -non-infectedd Ethiopian commercial sex workers (CSW) to determine expression levelss of CCR5 and CXCR4 and to study the association of CCR5 expression withh CCR2b polymorphism (CCR2b-64l). Expression of CCR5 was found to be

upregulatedd in CD45RA+CD4+ and total CD4+ T cells of HIV-- infected subjects

comparedd to the uninfected group, whereas the expression level of CXCR4 in

CD45RO+CD4++ and total CD4+ T cells was reduced in the HIV-1-positive CSW.

Thee overall frequency of the CCR2b-64l polymorphism was 55% in the CSW. Theree was no significant difference in the percentage of the CCR2b-64l genotypee between the HIV-1-infected (64.7%) and -non-infected (47.8%) subjects.. Remarkably, within the HIV-1-negative CSW a significantly reduced

CCR55 expression on total CD4+ T cells was observed in individuals with

CCR2b-64ll genotype compared to CCR2b wild type. This association was not presentt in HIV-1-infected CSW. Despite reduced CCR5 expression in vivo, in-vitrovitro susceptibility and p24 production after infection with different HIV-1 biologicall isolates were not different between HIV-1-negative CSW with CCR2b wild-typee and mutant genotype. The results of this study suggest that CCR2b-64II might mediate its disease progression delaying effect by affecting CCR5 expression. .

(5)

866 Chapter 6

Severall chemokine receptors CCR5, CXCR4, CCR2 and CCR3 have been shownn to serve as co-receptors for HIV-1 entry into target cells. HIV-1 variants thatt are restricted to primary cells use CCR5, whereas T-cell line tropic viruses

alternativelyy or additionally use CXCR41*3. CCR2b and CCR3 are used rarely4.

Polymorphismss in the HIV-1 co-receptors genes have been associated withh altered cellular susceptibility and disease progression. Although it is not absolute,, the A32 base pair deletion in the CCR5 gene was reported to confer

HIV-11 infection resistance5"8. In addition, individuals heterozygous for this

deletionn have been shown to have slower disease progression compared to

individualss with the wild-type genotype9'10. The CCR5 A32 deletion is very rare

inn Africans11. In contrast, a polymorphism in the CCR2b gene in which a valine

iss switched into isoleucine at position 64 of the transmembrane domain

(CCR2b-64l)) seems to be common in Africans12. This allelic variant of CCR2b

hass been associated with delayed disease progres-sion12"16. The mechanism

byy which this mutation protects against disease progression is not clear. However,, it has been reported that CCR2b-64l is linked with a point mutation in thee CCR5 regulatory region and a possible effect on the expression level of

CCR55 has been suggested16.

Wee studied CCR5 and CXCR4 expression in 64 female Ethiopian

commerciall sex workers (32 HIV" and 32 HIV+) and CCR5 expression in relation

too CCR2b genotype in 40 of these women (23 HIV" and 17 HIV+) after informed

consentt to participate in the study.

First,, we determined the expression pattern of the HIV-1 co-receptors in

thee two study groups. Naive and memory as well as total CD4+ T cells

expressingg CCR5 and CXCR4 were quantified in whole blood or isolated PBMC byy three colour flowcytometry using combinations of PercP-conjugated CD4 Moabb with FITC-conjugated CD45RA or CD45RO Moabs (Becton Dickinson, Sann Jose, CA) and PE-conjugated CCR5 (2D7) or CXCR4 (12G5) Moabs (Pharmingen,, La Jolla, CA). A minimum of 1500 CD4 cells were analayzed by

settingg a live gate around the CD4+ T cells using a FACScan flowcytometer with

thee Cellquest software.

Tablee 1. Median (95 percentile ranges) CCR5 and CXCR4 expression on total

ass well as naive (CD45RA+) and memory (CD45RO+) CD4+ T cells in

HIV-infectedd and -non-infected subjects

Co-receptorrr-celll HIV (n=32) HIV+ (n=32)

subset t %% CCR5+ cells within: Totall CD4+T cells 28(14-54) 36(17-58)T* CD45RA+CD4++ T cells 9(3-34) 14( 4-49) T* CD45RO+CD4++ T cells 35(19-83) 38(16-90) %% CXCR4+ cells within: Totall CD4* T cells 98(66-100) 87(55-98)1* CD45RA+CD4++ T cells 86(52-99) 92(38-98) CD45RO*CD4++ T cells 71 (35-94) 55(36-93)1*

(6)

Expressionn of CCR5 was found to be upregulated in CD4+CD45RA+ (naïve) and

totall CD4+ T cells of HIV-1 -infected CSW compared to HIV-1-negative CSW

{Tablee 1). The expression of CCR5 on the CD45RO+CD4+ (memory) T cells was

nott different between the two groups. The opposite was observed for CXCR4 in

thatt it's expression in both CD4+CD45RO+ and total CD4+ T cells was

significantlyy reduced in the HIV1positive group when compared to the HIV1 -negativee group supporting previous observations on the opposite regulation of

CCR55 and CXCR4 expression after cellular activation17. In contrast to previous

studiess reported from western countries where CCR5 is mainly upregulated on

memoryy cells17,18, we observed a significant increase of CCR5- expressing cells

withinn the naive but not the memory CD4+ T-cell population of HIV-1-infected

CSW.. Recently, it has been shown that CCR5 expression is already increased

inn memory subsets of healthy HIV-1-negative Ethiopians19 and our result may

suggestt that during HIV-1 infection the expression of CCR5 on the memory subsett is not further upregulated. It is remarkable that CCR5 was found to be

upregulatedd on naïve (CD45RA+) CD4+ T cells after HIV-1 infection. Previously,

itt has been shown that cytokines can induce activation and proliferation of naïve

TT cells without a switch to a memory phenotype20,21. Elevated cytokine levels

duee to chronic immune stimulation during HIV-1 infection could therefore lead to

upregulationn of CCR5 on phenotypically naïve CD4+ T cells.

Inn order to determine the frequency of CCR2b-64l polymorphism in the HIV-1-infectedd and -non-infected CSW and to analyze its effect on CCR5 expression,, CCR2b genotyping was performed on genomic DNA isolated from cryopreservedd PBMC (Qiagen blood kit, Westburg, Germany) using BsaBI

RFLP-PCRR analysis as described previously13. There was no difference in the

frequencyy of CCR2b-64l polymorphism between the HIV-1-infected and -non-infectedd groups (Table 2).

Tablee 2. Frequency of CCR2b-64l mutation in HIV-1-infected and- non-infected

CSW W Genotype e CCR2-64I/+ + CCR2-64I/64I I CCR2-64/+and64l/64l l Totall frequency of CCR2-64/+and64l/64l l H I V T C S WW HIV1+CSW 39.1(9/23)) 58.8(10/17) 8.7(2/23)) 5.9(1/17) 47.8%(11/23)) 64.7%(11/17) 55%% (22/40) PP value NS S NS S NS S

Thee expression of CCR5 was significantly lower on CD4+ T cells of the HIV1

-negativee CSW with the CCR2b-64l genotype (Fig. 1). Although not significant, thee tendency for lower expression of CCR5 was also observed for both

CD45RA** and CD45RO+ CD4+ T cells of the HIV-1-negative CSW (data not

shown).. In the HIV-1-infected CSW, the CCR5 expression in all subsets tested wass not different between wild-type and CCR2b-64l mutants.

(7)

888 Chapter 6 1000 r 800 -m -m cc cc o o Ü Ü ?600 [ 400 -g 2 0 0 O O I I II * * HIV--p = 0 . 0 22 —11 ,, HIV+ + I II "J | I I

--CCR2bb wild type CCR2b mutant CCR2b wild type CCR2b mutant

Fig.. 1. Percentage of CCR5 expressing CD4+ T cells of HIV-infected and -non-infectedd CSW with CCR2 wild and mutant genotype. Each dot represents a single subject.. Bars indicate the means and the standard deviations.

Too study whether the difference in CCR5 expression between CCR2b-64l and CCR2bb groups is also reflected in in-vitro susceptibility to HIV-1 infection, we

comparedd TCID50/ml and p24 production of PBMC from HIV-1-negative CSW

withh CCR2b-64l mutant and wild-type genotype. The PBMC were prestimulated withh PHA for three days and infected by four different HIV-1 primary isolates. Susceptibilityy was determined by measurement of p24 production over a two weekss period. The difference in virus titer expressed as 50% tissue culture

infectiouss dose (TCID50) per milliliter of supernatant was used as a measure of

in-vitroin-vitro susceptibility. As shown in Fig. 2 A and B, the observed difference in CCR55 expression between the CCR2b and CCR2b-64l groups was not

associatedd with a difference in in-vitro susceptibility as measured by TCID50 and

(8)

Effectt of CCR2b-64l genotype on CCR5 expression 89 B B E E S3 3 Q Q U U CCR2bwildd CCR2b mutant 0 0 0 0 100 0 --1 —:—' ' ii 1 ff ' ' CCR22 mutanl

Fig.. 2. In-vitro susceptibility of PBMC from HIV non-infected CSW with CCR2b

wild-typee and CCR2b-64l mutant genotype to infection by 4 HIV-1 biological

cloness expressed as A) mean log(TCID5o/ml), calculated as the average

log(TCID50/ml)) of the four HIV-1 clones and B) mean P24 production, calculated

ass the average P24 production of the four HIV-1 clones. Each dot represents PBMCC from a single subject. Bars indicate the means and the standard deviations. .

(9)

900 Chapter 6

Thee expression pattern of the HIV-1 co-receptors on the surface of T

cellss is believed to have an influence on susceptibility of CD4+ T cells to HIV-1

infection,, viral tropism and rate of disease progression22,23. Primary isolates

usee CCR5 as a co-receptor whereas CXCR4 is used by SI viruses that appear

laterr in the infection period1"3. In Ethiopia, the circulating virus is subtype C,

whichh has been shown to use mainly CCR5 as co-receptor. Furthermore, the appearancee of SI viruses later in the course of the infection was found to be

raree for subtype C viruses24. Therefore, CCR5 plays a crucial role in HIV-1

infectionn with subtype C. The results of our study show that the expression of

CCR55 on CD4+ T cells is significantly reduced in HIV- uninfected Ethiopian

CSWW with the CCR2b-64l genotype. Despite the difference in CCR5 expressionn levels, no association with reduced in-vitro susceptibility was found supportingg previous reports on the lack of an association of CCR2b-64l

polymorphismm and HIV-1 trans-mission14,23. These data are in agreement with

ourr previous observation that CCR5 expression level is not a limiting factor for HIV-11 infection in Ethiopians (Messele et a/., submitted for publication). It is remarkablee that in Caucasians where CCR5 expression levels have been

shownn to correlate with in-vitro susceptibility to HIV-118, the CCR2b-64l

genotypee was associated with very low in-vitro susceptibility (H. Blaak, personal communication).. CCR5 expression was not different in HIV-1-infected Ethiopiann CSW with either a CCR2b-64l or wild-type genotype. This may be a reflectionn of the increased state of cellular activation in HIV-1-infected individuals,, resulting in upregulation of CCR5 which was also observed in this study,, possibly masking the difference in the expression levels of CCR5 linked too the CCR2b-64l genotype. Although several reports have confirmed that CCR2b-64ll is associated with a delay in progression to AIDS, the mechanism of thiss delaying effect is not clear. CCR2b is not a major co-receptor and is rarely usedd by primary HIV-1 strains. In addition, CCR2b and CCR2b-64l function

equallyy well as HIV-1 co-receptors25. Therefore, it has been suggested that it

mayy exert its protective effect indirectly through interaction with other chemokinee receptors. The CCR5 and CCR2 chemokine genes are located at closee proximity on chromosome 3 separated by 2 0 K B . Recently, it was reportedd that another polymorphism in the CCR5 promotor region

(CCR5-5963T)) is tightly linked to the CCR2b-64l genotype16 in all races including

Africans27.. In contrast to a report by Marianni et a/.28, we observed a reduced

CCR55 expression in HIV-1- negative individuals heterozygous or homozygous forr CCR2b-64l. Although the reduced expression of CCR5 was not observed in HIV-1-infectedd individuals with CCR2b-64l mutation, the protective effect of CCR2b-64ll may still be mediated by affecting CCR5 expression levels. Reducedd in vivo, CCR5 expression could influence the viral set point shortly afterr primary HIV-1 infection. During chronic HIV-1 infection, CCR5 expression mightt be upregulated due to persisent immune activation masking the effect of CCR2b-64ll genotype. The lack of association between the difference in the in-vivovivo expression of CCR5 and in-vitro susceptibility and p24 production could be explainedd by the prestimulation of PBMC with PHA. This may lead to an upregulationn of CCR5 to levels above a certain threshold in CCR2b-64l and wild-typee individuals.

(10)

Ourr observation of reduced in-vivo CCR5 expression in CCR2b-64l HIV-1-negativee individuals together with a recent report that the delaying effect off this polymorphism is more pronounced during infection by NSI HIV-1

variants133 suggests that the slower disease progression associated with this

mutationn could be mediated by reduced CCR5 expression. Finally, our data showw that the CCR2-64I mutation is sufficiently present in Ethiopians and the usee of this mutation for prognostic genotyping in this population needs to be assessed. .

Acknowledgements s

Thiss study is part of the Ethio-Netherlands AIDS Research Program (ENARP),, a collaborative effort of the Ethiopian Health and Nutrition Research Institutee (EHNRI), the Amsterdam Municipal Health Service (GG&GD), the CLB andd the Academic Medical Center of the University of Amsterdam (AMC). The Netherlandss Ministry of Foreign Affairs and the Ethiopian Ministry of Health (MOH)) financially support ENARP as a bilateral project. We thank Ronald van Rijj for stimulating discussion and critical reading of the manuscript.

References s

1.. Feng Y, Broder CC, Kennedy PE, et al. HIV-1 entry cofactor: Functional cDNA cloning of a seven-transmembranee domain, G-protein coupled receptor. Science 1996;272:872-888. 2.. Deng H, Liu R, Ellmeier W, ef al. Identification of a major co-receptor for primary isolates of

HIV-1.. Nature 1996; 381:661-666.

3.. Deng HK, Unutmaz D, KewalRamani VN and Littman DR. Expression cloning of new receptorss by simian and human immunodeficiency viruses. Nature 1997;388:296.

4.. Doranz BJ, Rucker J, Yi Y, ef at.: A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokinee receptors CKR-5, CKR-, and CKR-2b as fusin cofacors. Cell 1996;851149-1158. .

5.. O'Brien TJ, Winkler C, Dean M, et al. HIV-1 infection in a man homozygous for CCR5A32. Lancett 1997;349:1219.

6.. Theodora I, Meyer L, Magrierowska M, et al. HIV-1 infection in an individual homozygous forr CCR5A32. Lancet 1997; 1219-1220.

7.. Zimmerman PA, Buckler-White A, Alkhatib G, ef al. Inherited resistance to HIV-1 confer-red byy inactivating mutation in CC chemokine receptor 5:Studies in populations with contrasting clinicall phenotypes, defined racial background, and quantified risk. Mol Med 1997;3:23-36. 8.. Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1 infection and

progressionn to AIDS by a deletion allele of the CKR5 structural gene. Nature 1996; 382 722-725. .

9.. De Roda Husman AM, Koot M, Cornelissen M, ef al. Association between CCR5 genotype andd the clinical course of HIV-1 infection. Ann Intern Med 1997;127:882-890.

10.. Huang Y, Paxton WA, Wolinsky SM, ef al. The role of mutant CCR5 allele in HIV-1 transmissionn and disease progression. Nature Medicine 1996;2:1240-1243.

11.. Martinson JJ, Chapman NH, Rees DC, et al. Global distribution of the CCR5 gene 32-basepatrr deletion. Nature Genet 1997;16:221-222.

12.. Anzala AO, Ball TB, Rostron T ef al. Collaboration for HIV-1 research: CCR2-64I allele and genotypee association with delayed AIDS progression in African women Lancet 1998 351:1632-1633. .

(11)

922 Chapter 6

13.. van Rij RP, De Roda Husman AM, Brouwer M, et al. Role of CCR2 genotype in the clinical coursee of syncytium-inducing phenotype (SI) or non-SI human immunodeficiency virus type

11 infection and in the time to conversion to SI virus variants. J Infect Dis 1998:178:1806-1811. .

14.. Michael NL, Louie LG, Rohrbaugh AL, et al. The role of CCR5 and CCR2 polymorphisms in HIV-11 transmission and disease progression. Nature Med 1997;3:1160-1162.

15.. Smith MW, Dean M, Carrington M, et al. Contrasting genetic influence of CCR2 and CCR5 variantss on HIV-1 infection and disease progression. Hemophilia growth and deve-lopment studyy (HGDS), Multicenter AIDS cohort study (MACS), San Francisco City Cohort (SFCC), ALIVEE Study. Science 1997;277:959-65.

16.. Kostrikis L, Huang Y, Moore JP, et at. A chemokine receptor CCR2 allele delays HIV-1 diseasee progression and is associated with a CCR5 promotor mutation. Nat Med

1998;4:350-353. .

17.. Bleul CC, Wu L, Hoxie JA, et al:. The HIV-1 coreceptors CXCR4 and CCR5 are differentially expressedd and regulated on human T lymphocytes. 1997; 94:1925-1930.

18.. Blaak H, Ran LJ, Rientsma R, et al. Susceptibility of in-vitro stimulated PBMC to infection withh NSI HIV-1 is associated with levels of CCR5 expression and beta-chemokine production.. Virology. 2000;267:237-246.

19.. Kalinkovich A, Weisman Z, Leng Q, et al. Increased CCR5 expression with decreased 6 chemokinee secretion in Ethiopians: Relevance to AIDS in Africa. J Hum Virol 1999;2:283-289. .

20.. Unutmaz D, Pileri P.Abrignani S. Antigen-independent activation of naive and memory restingg T cells by cytokine combination. J Exp Med 1994;180:1159-1164.

2 1 .. Soares MV, Borthwick NJ, Maini MK, et al. IL-7 dependent extrathymic expansion of CD45RA++ T cells enables preservation of a naive repertoire. J Immunol 1998;161:5909-5917. .

22.. Wu I, Paxton LA, Kassam N, et al. CCR5 levels and expression pattern correlate with infectabilityy by macrophage-tropic HIV-1, in vitro. J Exp Med 1997;185:1681-1691.

23.. Mummidi S, Ahuja SS, Gonzalez E, et al. Genealogy of the CCR5 locus and chemokine systemm gene variants associated with altered rates of HIV-1 disease progression. Nature Medd 1998;4:786-793.

24.. Abebe A., Demissie D., Goudsmit J. et al:. HIV-1 subtype C syncytium- and non-syncytium-inducingg phenotypes and co-receptor usage among Ethiopian patients with AIDS. AIDS 1999;; 13: 1305-1311.

25.. Lee B, Doranz BJr Rna S, Yi Y, Mellado M, Frade JM, et al. Influence of the CCR2-V64I

polymorphismm on human immunodeficiency virus type 1 co-receptor activity and on chemokinee receptor function of CCR2b, CCR3, CCR5, and CXCR4. J Virol 1998; 72:7450-7458. .

26.. Raport CJ, Gosling J, Schweickart VL, Gray PW, Charo IF. Molecular cloning and functional characterizationn of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1beta, andd MIP-1alpha. J Biol Chem 1996;271:17161-17166.

27.. Martinson JJ, Hong L, Karanicolas R, et al. Global distribution of the CCR2-64I/CCR5-59653TT HIV-1 disease-protective haplotype. AIDS 2000;14:483-489.

28.. Mariani R, Wong S, Mulder LC, Wilkinson DA, Reinhart AL, LaRosa G, ef al. CCR2-64I polymorphismm is not associated with altered CCR5 expression or co-receptor function. J Viroll 1999 73:2450-2459.