Biomrd & Phormucothrr 1998 : 52 : 436-39

@ Elsevier. Paris

Dossier “AIDS Sciences”

Binding of HIV-l to RBCs involves the Duffy Antigen Receptors for Chemokines (DARC)

A Lachgar’, G Jaureguiberry2, H Le Buenacl, B Bizzinil, JF Zagury’, J Rappaport”, D Zagury’

’ Laborutoiw dr Ph:<sio/ogie Crliulairr. Univrrsitl P et A4 Curie, 4, plocr Jussieu RC. Box IYK, 75005 I’mis: -‘EP 1790 CNRS, Laboratoire de Biologir Parusituirr, Mu.seum NHN. Paris, Frunce: ‘Allqheny Uuiwrsit>~ of Heolt!~ Sciences.

Philadrlphict, PA, [ISA

Summary - The Duffy Antigen Receptor for Chemokines (DARC) belongs to a family of erythrocyte chemokine receptors that bind C-X-C and C-C chemokines such as interleukin 8 (IL-g), monocyte chemoattractant protein I (MCP-I) and regulated-on- activation, normal T cell-expressed and -secreted (RANTES). but not macrophage inflammatory protein la (MIP-la) or MIP-lb. DARC has also been identified to a receptor for malaria parasites Plrunzodirtm vivux and Plasrmdiun~ !~mw[r:;i. In the present study, we show that HI\‘-1 binds to RBC% from Caucasian individuals via DARC making RBCs able to transmit HIV to peripheral blood mononuclear cells (PBMCs). Furthermore, bindin g of HIV-I particles to RBCs is inhibited by treating these cells with recombinant RANTES. but not with recombinant MIP-la prior to their incubation with HIV-I. This finding suggests that RBCs may function as a reservoir for HIV-I or as a receptor for the entry of HIV-I into CDJ-cell subsets as well a!, neurons or endothelial cells. 0 1998 Elsevier, Paris

DARC / RBC I Duffy / HIV-1 / RANTES I chemokines

INTRODUCTION

Duffy antigens appear to be multimeric erythro- cyte-membrane proteins composed of different su- bunits. A glycoprotein of 35-45 kD named GPD is the major subunit of the protein complex and it carries the antigenic determinants as defined by anti-Fy(a), anti-Fy(b) antibodies [I]. Recognition of Duffy antigen as a receptor on the red blood cell (RBC) for the malarial parasite, Plasmodium vivax and the simian Plasmodium knowlesi [2], as well as its identification to a receptor for che- moattractant cytokines, or chemokines have been reported by several authors [3]. Many red cell po- lymorphic forms that have been considered to be related to malaria [4] selection are found with high frequency in populations with endemic ma- laria. Several linea of evidence support the as- sumption that the Duffy blood group antigen is the erythrocyte receptor for the chemokines such as regulated-on-activation, normal T cell-expres- sed and -secreted (RANTES) [3, 51, interleukin 8 (IL-g) [5, 61 and melanoma growth stimulatory activity (MGSA) [3, 51. Among chemokines

RANTES does not bind at all to Duffy-negative erythrocytes [a]. A monoclonal antibody to the Duffy blood group antigen also blocked binding of IL-8 and RANTES to Duffy-positive erythro- cytes. Moreover both MGSA and IL-8 prevented of human erythrocytes from being invaded by Plasmodium knowlesi 12, 71.

Finding that the Duffy Antigen on Erythrocytes as a Chemokine receptor is also expressed by en- dothelial cells lining postcapillary venules [8] and splenic sinusoids [9] as well as by some subsets of neurons in the central nervous system suggests additional roles for Duffy Antigen Receptor for Chemokines (DARC) in the physiologic role of the Duffy blood group antigen [ 10, I I].

Moreover, the beta-chemokines macrophage in- flammatory protein lor (MIP-la), MIP-I p and RANTES can inhibit infection of CD4’ T cells by primary non-syncytium-inducing (NSI) HIV- 1 strains at the virus entry stage, and also block envelop-mediated cell to cell membrane fusion [ 12, 13 I. CD4’ T cells from some HIV- 1 -exposed but not infected individuals cannot fuse with NSI HIV-I strains while producing high levels of beta-

Binding of HIV-l to RBCs involves the IDARC 437

chemokines [ 141. Expression of the beta-chemo- kine receptor CCR-5 in CD4’, non-permissive hu- man and non-human cells renders them suscepti- ble to infection by HIV-l NSI strains [ 151, and allows HIV- 1 -env mediated membrane fusion. CCR-5 is a second receptor for NSI primary viru- ses [ 12, 13, 161. A cofactor required for infection with a virus adapted to grow in transformed T cell lines was recently identified and named fusin.

The purpose of the present work was to check whether if RBCs can bind an NSI strain of HIV-l and whether binding is mediated by DARC pro- tein which is known to be a receptor for RAN- TES. We further investigated the inhibition by C- C chemokines MIP-la, MIP-lb and RANTES of HIV-I binding to RBC.

supplemented with 10% human serum. Cells were sub- rsequently washed twice. peletted and adjusted to one million per sample. Activated PBMCs were incubated for 90 min uith I mL of viral supernatant (1: IO), with either normal RBCs. or RBCs incubated with HIV. or RBCs incubated with HIV-I followed by a wash or the supernatant from RBCs incubated with HIV-l and also washed. Cells were then washed and cultured for va- rious times in culture medium supplemented with 100 IU/mL cf IL-2. Peripheral blood leucocytes (PBLs) were counted in acetic acid and by trypan blue exclu- <ion every 3 days.

Viral monitoring

MATERIALS AND METHODS

Viral produci.ion was monitored by p24 enzyme-linked immunosorbent assay (ELISA) using the kit from Du- pont de Netrours (Les Ullis, France).

Cells RESULTS

Peripheral blood mononuclear cells (PBMCs) were puri- fied on Ficoll hypaque gradient from blood samples of healthy donors. Red cells were purified from peripheral blood by filtration on a Pall “BPF4” bea efficiency leu- cocyte removal blood processing filter system. Red cells were taken from either Duffy-positive (Fya+/Fyb+) Cau- casian or Duffy negative (Fya-/Fyb-) black African do- nors.

RBCs can bind HIV-l virions

Virus

HIV-l was a supernatant from H9 cell line infected by a NSI strain of HIV-I with p24 antigenemia of 70 ng/mL.

Chemokines

As monitored by p24 ELISA, viral production by PBMCs infected in vitro with HIV-l was found to be effective. P24 antigenemia become positive after 3 day culturing of infected cells. HIV-I p24 values after 20 days - culturing of PBMCs infec- ted by an NSI strain of HIV-l shown in.figur-e 1. By contrast in PBMCs infected in presence of blood purified RElC originating from Caucasian donors and carrying the Duffy antigen (Fya’/Fyb’) on their cell surface p24 antigenemia values remai- ned very low. This can be accounted for by the

C-C chemokines MIP- la, MIP-1P and RANTES were purchased from R&D (Cambridge, UK)

Red cell incubation with HIV-l

One million RBCs were incubated with I : IO dilution of HIV-l in 1 mL of culture medium. After I h, cells were or were not washed 3x and incubated for 1 h in I mL of culture medium. Cells were sedimented and the supernatant collected. For the experiments with chemo- kines, RBCs were incubated for I h with I ug/mL of a mixture of chemokines (MIP-la, MIP-ID and/or RANTES) and washed twice prior to their incubation with HIV- I.

Cell infection

Fresh PBMCs were activated for 48 h with phytohe- magglutinine (PHA)-p (3 ug/mL) in culture medium

Fig 1. Infection of activated PBMCs by an NSI strain of HIV- I. Infection \*as performed in presence or absence of Duffy- positive (Fya’/Fyh’) RBCs, and p24 antigenemia was measu- red 20 days l.rter.

A Lachgar et al

q Day7 n Day 14

Fig 2. Measure of p24 production by in vitro HIV-l infected PBMCs. Activated PBMCs were infected with either HIV-1 supernatant (Control). or incubated with RBCs that were pre- viously incubated with HIV-l and washed several times before incubation with the activated PBMCs, or incubated also with a 1 h supernatant from the RBCs described above (see materiel & methods). RBCs were from Duffy-positive Fy(+) or Duf- fy-negative Fy(-) donors. HIV-1 production was monitored at 7 and 14 days of infec!.ion by measuring of p24 production.

fact that Duffy antigen-carrying RBCs may bind HIV virions thus r’educing infection of PBMCs.

Duffy antigen as a receptor of HIV-l on RBCs

To check whether the Duffy receptor may func- tion as a receptor for the NSI HIV-l, we incuba- ted either Duffy-positive (Fya+/Fyb+) RBCs from Caucasian donors or Duffy-negative (Fya’/Fyb’) RBCs from black African donors, with HIV-l su- pernatants. RBCs were of either species sub- sequently washed 3x with RPM1 and incubated for 1 h with 1 mL of culture medium. spun down and supernatants collected. Following this activa- ted PBMCs were incubated with either 1 mL of HIV-l supernatant, or RBCs which were incuba- ted with HIV-I and washed or with the superna- tants collected above. Viral load was monitored after 7 and 14 days of cell culturing. In ,figure 2, we see that PBMCs infected with HIV-l super- natant have produced p24 antigen pointing to their effective infection. By contrast, PBMCs in- cubated with Duffy--negative RBCs or with the su- pernatant of the latter did not produce any p24 antigen. As expected, PBMCs incubated with Duf- fy-positive RBCs were infected and produced high amounts of ~24 antigen. This is consistent with the assumption that Duffy-positive RBCs bind HIV-l virions to release them into the cu- ture and infect PBMCs. Importantly, purified PBMCs were not infected when they were incu- bated with a supernatant collected from Duffy-

Fig 3. C-C chemokines inhibition of the binding of HIV-l to the RBCs. RBCs from a Duffy-positive donor were incubated without (RBC-HIV) or either with a mixture of C-C chemo- kines MIPlc( MIPlP and RANTES (RBC-MMR-HIV), or with MlPl c1 (RBC-MIPI a-HIV) or RANTES (RBC-RANTES-HIV) alone. RBCs were then incubated with a NSI HIV-I supernatant and washed several times before being added to -activated PBMCs. Direct infection of PBMCs bv HIV-I supernatant was used as control. HIV-I production wa; moni:ored at 7 and 14 days of infection by measuring of p24 production.

positive RBCs incubated with HIV-l and washed several times, following incubation, which shows that HIV-l virions are tightly bound to RBCs and not free in the culture medium.

Evidence for the role of DARC in binding HIV- 1 to RBCs

To clarify how HIV-l virions bind to RBCs, we incubated for 1 h Duffy-positive RBCs with a mixture of C-C chemokines consisting of MIP- 101, MIP-1 p and RANTES (MMR lpg/mL) after which they were washed 3x prior to there incu- bation with HIV-l as described above. In figure 3 is reported p24 antigen production by either PBMCs infected by HIV-l as a control and PBR4Cs incubated with Duffy-positive RBCs in- cubated with HIV-l before being washed or RBCs incubated with C-C chemokines before being washed and incubated with HIV-l. From this fig- ure it results that RBCs incubated with HIV-l bind the virus to transfer it to PBMCs in culture. Binding was inhibited by pre-incubating RBCs with C-C chemokines, as shown b.y the lack of p24 antigenemia up to 2 weeks. Thjs is in agree- ment with the assumption that HIV should bind to RBCs via the DARC protein which is a recep- tor for C-C chemokines. In addition, it is well known that C-C chemokine receptors on mono- cytes/macrophages or T cells are co-receptors for HIV-I.

Binding of HIV-l to RBCs involves thr: DARC 439

DISCUSSION

In this study we have investigated the possible binding of HIV-1 viral particles to RBCs and the mechanism by which HIV-I can bind to these cells. We have shown that RBCs originating from healthy Caucasian donors and carrying the Duffy antigen protein at their surface bind NSI HIV-l virions to subsequently release them in the culture medium, which can account for the infection of PBMCs. Besides, RBCs from healthy black do- nors that do not carry the Duffy antigen at their surface did not bind HIV-1 virions. This is con- sistent with the assumption that HIV-l particles bind to RBCs via DARC, which was described as a common receptor for C-C chemokines and the malaria parasite Plasrnodium vivax.

More importantly, several authors have reported chemokine class differences in binding to the Duffy antigen [ 171. For instance, RANTES exhi- bits a high affinity for the Duffy antigen [3]? as opposed to MIP-1 cx and /3, which do not bind to this receptor [3, 181. In the present report, we have shown that pre-incubation of Duffy-positive RBCs with MIP-lo does not inhibit binding of HIV-l particles to these cells as RANTES does. These data are in line with the affinity of NSI HIV-l particles for the DARC antigen.

Furthermore, recent data have shown that the Duffy antigen receptor for chemokines is expres- sed on endothelial cell membranes [B, 93 as well as on few subsets of neurons in the central nerv- ous system [B]. This finding together with our present results can help understand how HIV-l can enter some CD4- cells as is the case of neu- rons. It can be postulated that HIV-l might reach the central nervous system via the RBCs which act as vectors, and HIV-I particles by red cells or by microglial cells [ 191, could penetrate neurons. These data suggest a role for RBCs as a possible reservoir of HIV-l virions.

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