Proc. Nati. Acad. Sci. USAVol. 88, pp. 9297-9301, October 1991Medical Sciences

Human immunodeficiency virus can infect the apical andbasolateral surfaces of human colonic epithelial cells

(AIDS/CD4/porous bottom dishes/colonic epithelium)

JACQUES FANTINI, NOUARA YAHI, AND JEAN-CLAUDE CHERMANNInstitut National de la Sante et de la Recherche M&dicale, Unite 322, Unit of Retrovirus and Associated Diseases, B.P. 33, 13273 Marseille Cedex 9, France

Communicated by Robert C. Gallo, July 25, 1991 (received for review May 1, 1991)

ABSTRACT The gastrointestinal tract is considered to bea major route of infection for the human immunodeficiencyvirus (HV). To understand the interaction of HIV withepithelial cells of the intestinal mucosa, we have studied theinfection of a human colon cancer cel clone HT-29-D4. Theenterocyte-like differentiation of this clone can be modulated invitro according to the concentration of glucose. We show that:(i) undifferentiated HT-29-D4 cells can be infected by HIVtypes 1 and 2 (HIV-1 and HIV-2) strains with no subsequenteffect on cell growth; (ii) undifferentiated HT-29-D4 cellsexpress a CD4-related antigen bearing epitopes of the immu-noglobulin-like variable (V) region domains V1 and V2 ofCD4but lacking the epitope known to be involved in HIV enveloperecognition; (iN) differentiated HT-29-D4 cells can be infectedby HIV after an interaction with either the apical brush bordermembrane (luminal side) or the basolateral side (serosal side);(iv) the CD4-lke molecule is restricted to the basolateraldomain of differentiated cells; and (v) the infection is notinhibited by anti-CD4 monoclonal antibodies (mAbs) OKT4,OKT4A, Leu-3a, B14, 13-B-8-2, S-T4 or S-T40. We concludethat epithelial intestinal cells may represent a major site ofentry for HIV. Infection of these epithelial cells may occur viathe basolateral membrane by HIV-bearing lymphocytes ormacrophages of the lamina propria and via the apical mem-brane by HIV present in the bowel lumen. This infection mayremain silent for up to 9 months, and the virus can be rescuedby cocultivation with lymphoid cells. These data may give anexplanation for the long latent seronegative state that mayoccur in a HIV-infected individual.

The gastrointestinal tract is a putative site of entry for thehuman immunodeficiency virus (HIV), the etiologic agent ofAIDS and associated diseases. The ability of HIV to infectrectal or colonic epithelial cells has been shown in vivo by insitu hybridization (1, 2) and in vitro by using normal (3) ortumoral (4) epithelial cells of colonic origin that are permis-sive for HIV infection.These polarized epithelial cells display two clearly distinguish-

able plasma membrane domains delimited by tight junctions: anapical domain facing the intestinal lumen and a basolateraldomain facing the internal milieu (5). These two domains havestriking differences in protein and lipid composition (6). Thisarchitectural organization is essential for the expression of thevectorial functions of epithelial tissues. Since the entry ofvirusesinto epithelial cells is frequently restricted to a specific membranedomain (7-11), it is important to study the polarity of HIVinfection in intestinal epithelial cells.Cloned HT-29-D4 was derived from a human adenocarci-

noma cell line (12-15) and constitutes a unique model for thestudy of cellular events correlated with epithelial differenti-ation. These cells differentiate quickly after a minor change

ofthe culture medium (namely, the replacement ofglucose bygalactose) (16). In this report we show that both undifferen-tiated and differentiated HT-29-D4 cells were sensitive toHIV infection. The infection of differentiated cells was notrestricted to a specific membrane domain and suggests thatepithelial cells of the bowel can be infected by the viruspresent in the lumen or in the lamina propria. Furthermore,since CD4-like molecules are preferentially expressed on thebasolateral membrane of HT-29-D4 cells, our results areconsistent with the existence of an apically restricted alter-native receptor for HIV envelope.

MATERIALS AND METHODSCell Culture. The human colic adenocarcinoma cell lines T

84 (provided by B. Verrier, Marseille), HT-29 (ATCC HTB38) and the clonal subpopulation HT-29-D4 (12) were rou-tinely grown in Dulbecco's modified Eagle's medium/Ham'sF-12 medium, 1:1 (vol/vol), supplemented with 10% heat-inactivated fetal calf serum and 15 mM Hepes, pH 7.4. Cellswere harvested from the culture flasks by a 10-min treatmentwith trypsin-EDTA (modified Puck's saline solution contain-ing 0.5 g of trypsin and 0.2 g of EDTA per liter). The cellsuspension was then added to an equal volume of culturemedium, centrifuged, and resuspended in fresh medium. Toinduce differentiation, HT-29-D4 cells were cultured in glu-cose-free medium containing 5 mM galactose as described(12). When indicated, cells were cultured on Nuclepore filter(pore size, 3 Am) in Transwell cell culture chambers (Costar)as described (13). Human lymphoblastoid T cells CEM andSup T1 were grown in RPMI 1640 supplemented with 10%(vol/vol) heat-inactivated calf serum.

Virus Infection. The viruses used were the prototype HIV type1 strain HIV-1-BRU (17, 18), the highly cytopathic strain HIV-1-NDK (19), and HIV type 2 strain HIV-2-ROD (20) maintainedin aCEM continuous cell line. Exponentially growing HT-29-D4cells were exposed for 16 hr to HIV-1 orHIV-2 [103 tissue culture50o infective dose (TCIICW)] in serum-supplemented culturemedium with Polybrene at 2 Ag/ml. After extensive washing,cells were harvested with trypsin-EDTA and subcultured at adensity of 5 x 104 cells per cm2.

Reverse Trnscriptase (RIT) Assay. Levels of RT activity inculture supernatants were determined by the method ofRey etal.(21) and Willey et al. (22), slightly modified. Culture supernatants(1 ml) were centrifuged in a Beckman TL100 centrifuge (rotorTLA.100.2) for 5 min at 95,000 rpm, and the pelleted virus waslysed by 0.5% Triton X-100. Aliquots (10 A.l) oflysate were addedto 40 Al of a mixture containing poly(rA) and oligo(dT) (Phar-macia; each at 0.25 A unit/ml), 20 mM MgCl2, 2 mM dithio-threitol, and 2.5 ACi of (methyl-3H)dTTP (Amersham; specificactivity, 30 Ci/mmol; 1 Ci = 37 GBq). After 1 hr of incubation

Abbreviations: HIV, human immunodeficiency virus; HIV-1 andHIV-2, HIV types 1 and 2; mAb, monoclonal antibody; RT, reversetranscriptase; TNF, tumor necrosis factor; V, variable region;TCID5o, tissue culture 50% infective dose.

9297

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at 37C, the reaction mixture was deposited onto DE 81 filterpaper (Whatman) and allowed to dry at room temperature. Thefilters were washed in 5% Na2HPO4 followed by 70%6 ethanol,dried, and placed in a scintillation counter (Packard 1600 CA) formeasurement of radioactivity.Polymerase Chain Reaction (PCR). HIV-infected cells (5 X

106 cells per ml) were lysed overnight in a buffer containingNonidet P-40, Tween 20, and proteinase K. The reaction wasstopped by incubation at 950C for 10 min. Samples (25 1.l)were submitted to HIV-specific DNA amplification withnucleotide primers from the pol gene (nucleotides 3208-3235and 3739-3766) (CIS BioInternational, Grenoble, France)and 2 units of Taq polymerase (Cetus) per ml.The following sequence of incubations was used: 5 min at

940C for initial denaturation and then 30 cycles (1 min at 94TC,45 s at 550C, and 2 min at 720C), followed by 5 min at 720Cbefore cooling at 40C. After separation by agarose gel elec-trophoresis, the amplified products were analyzed by South-ern blot hybridization with a radiolabeled DNA probe spe-cific for a pol sequence internal to the primer pair.

i n n Llng. Immunofluorescence was per-formed on living HT-29-D4 cells as described (16) by usinganti-CD4 monoclonal antibodies (mAbs) OKT4A from OrthoDiagnostics, Leu 3a from Becton Dickinson, and B14 and 13-B-8-2 from Immunotech (Luminy, France). mAbs S-T4 and S-T40were from Sanofi (Paris), and OKT4 was from Ortho Diagnos-tics.

Inhibition of Infection by Anti-CD4 mAbs. Cells werepreincubated for 1 hr at 40C and infected in the presence of

Proc. Natl. Acad. Sci. USA 88 (1991)

mAbs (10 ,ug/ml) as described by Rey et al. (23). Ten daysafter infection, HIV infection was assessed by cocultivationwith lymphoblastoid T cells.

RESULTS AND DISCUSSION

Immunofluorescence Localization of CD4 in HT-29-D4Cells. CD4 is a 55-kDa glycoprotein found predominantly onthe surface ofT lymphocytes. The extracellular region of themolecule consists of four domains sharing sequence homol-ogy with the variable (V) region of immunoglobulin lightchains (24). The binding site for HIV envelope glycoproteingpl20 is located within the V1 domain (25).We have studied the expression of CD4 on HT-29-D4 cells

by indirect immunofluorescence, using a panel of mAbs:OKT4A and Leu 3a, which recognize the binding site forgpl20 in the V1 domain; 13-B-8-2, which binds the V1 domainwithout interfering with gpl20 binding; and B14, which bindsto the V2 domain (26).

Undifferentiated HT-29-D4 cells displayed a uniform la-beling with B14 (Fig. 1 A and B) and 13-B-8-2 (not shown) butwere not labeled by OKT4A (Fig. 1 C and D) or Leu 3a (notshown). When cultured in glucose-free galactose-containingmedium, HT-29-D4 cells formed a regular monolayer ofdifferentiated cells exhibiting the features of normal colonicepithelial cells (12, 13). These cells can be cultured onpermeable filters in culture chambers, a technique allowingan independent access of the apical and the basolateraldomain (Fig. 2). This model was used successfully to dem-

'-^X ya, . *4

-~~~~~~~L''

w4104 v F

FIG. 1. CD4 expression on HT-29-D4 cells. When grown in glucose-containing medium, HT-29-D4 cells were not polarized, and a uniformimmunofluorescent staining was obtained with B14 (A) but not with OKT4A (C) anti-CD4 mAbs. In differentiated HT-29-D4 cells grown inglucose-free medium on permeable substratum, labeling with B14 was restricted to the basolateral membrane (E). Phase-contrast micrographscorresponding to A, C, and E are given in B, D, and F, respectively. (Bar = 10 Am.)

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Proc. NatL. Acad. Sci. USA 88 (1991) 9299

1-

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-~~~~-'axons~~~~~~~~)it. ....................&

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LAMINA PROPRIA

FIG. 2. Schematic representation of the colonic epithelium andthe in vitro model of differentiated HT-29-D4 cells. (Upper) Cellculture chamber (in vitro model). The apical side ofthe cells faces theupper compartment. Identifications: 1, apical compartment; 2, HT-29-D4 monolayer; 3, polycarbonate porous filter (3-,um pore size); 4,basal compartment. (Lower) Colonic surface epithelium (in vivosituation). Identifications: 5, epithelial absorbing colonocytes; 6,goblet cells secreting mucus; 7, basal membrane; 8, lamina propria(including lymphocytes, macrophages, and a few eosinophils); 9,intraepithelial lymphocyte.

onstrate the segregation of functional vasoactive intestinalpeptide receptors at the basolateral domain ofHT-29-D4 cells(27). Fig. 1 E and F show the characteristic staining patternof B14 antibodies on the basolateral surface of an intactHT-29-D4 monolayer. A similar pattern of fluorescence wasobtained with 13-B-8-2, but OKT4A and Leu 3A did not labelthe basolateral membrane. We failed to detect any significantapical labeling with the four anti-CD4 mAbs used in thisstudy. These results further characterize the CD4-like anti-gen expressed at the basolateral side of HT-29-D4 cells. ThisCD4-related molecule is a 60-kDa protein (16) that possessesat least the V2 and V1 domains of CD4 but lacks the epitoperecognized by OKT4A and Leu 3a.

Infection of HT-29-D4 Cells by HIV. In a first set ofexperiments, we have studied the sensitivity of undifferen-tiated HT-29-D4 cells for HIV infection. HIV-1-NDK wasable to replicate in HT-29-D4 cells as demonstrated by: (i) thefact that cell-free supernatants were infectious for HT-29,HT-29-D4, and peripheral blood lymphocytes; (ii) the detec-tion of a significant RT activity in cell-free supernatants; and(iii) the budding ofmature viral particles as shown by electronmicroscope studies. In contrast, HIV-1-BRU and HIV-2-ROD did not replicate in HT-29-D4 cells. No viral antigenwas produced as shown by radioimmunoprecipitation assay,and viral production was rescued after cocultivation withlymphoid cells (Fig. 3) but not after treatment with 2'-deoxy-5-iodouridine, tumor necrosis factor a, or dexamethasone.Confirmation of silent infection of HT-29-D4 cells by HIV-1-BRU (Fig. 4) and HIV-2-ROD (not shown) was obtained bygene amplification analysis with pol primers and chronicallyinfected HT-29 cells and lymphoblastoid CEM cells as pos-itive controls. The presence of a proviral DNA genome inthese nonproducer cells suggests a block at the transcription

* NDK* BRUA ROD

HT29-D4

Time, days

FIG. 3. RT activity in medium from cells cocultured with HIV-infected HT-29-D4 cells. HT-29-D4 cells were infected with HIV-1-NDK (e), HIV-1-BRU (W), or HIV-2-ROD (A) as described. At thethird passage (day 10 after infection), HT-29-D4 cells were coculti-vated with a clonal lymphoid cell line (CEM, clone 5) at a density of5 x 105 cells per ml. After 24 hr, CEM cells were harvested, culturedin separate flasks, and tested for RT activity at selected intervals.

level. Interestingly, cell growth was not altered in HT-29-D4infected cells. One can rule out the possibility that thetransmission of infection to indicator T cells may be due tocarry-over virus from the inoculum because: (i) the super-natant from latently infected HT-29-D4 cells was not infec-tious, (ii) the CD4- human colonic adenocarcinoma cell lineT84, known to be not susceptible to HIV infection (28), wasnegative by PCR analysis; and (iii) virus could be rescued bycocultivation 9 months after infection, demonstrating thepersistence of HIV in HT-29-D4 cells.The role ofthe CD4 molecule as a HIV receptor for colonic

cells was investigated by examining the susceptibility ofHIV-1-BRU, HIV-1-NDK, and HIV-2-ROD to anti-CD4mAbs. Saturating concentration (10 Fg/ml) of OKT4 (anti-V3/V4 domains); B14 (anti-V2); S-T40 (anti-V1/V2); S-T4,13.B.8.2., Leu 3a, and OKT4A (anti-Vi); and a low virus titer(10 TCID50) were used in these experiments. Under theseconditions, attempts to block infection of HT-29-D4 cells byHIV-1 and HIV-2 strains were unsuccessful.

BRU NDK NDK

D4 HT29 Do HT29 T84

w@0

BRUCEM

O POL0 HIVI

559 bp

FIG. 4. Detection of HIV DNA sequences by PCR and Southernblot hybridization. Thirty days after exposure to HIV, cells werelysed and the extract was subjected to HIV-specific DNA amplifi-cation with nucleotide primers from the pol gene as described.Chronically infected lymphoblastoid CEM and noncloned HT-29cells were used as positive controls. The 559-base-pair amplifiedfragment was not detected in T84 cells exposed to HIV-1-NDK or inmock-infected cells (not shown).

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Table 1. RT activity recovered after 24 hr of incubation of HIVintroduced on the apical or basolateral side of differentiatedHT-29-D4 monolayers

Compartment

Condition of incubation Apical Basolateral

HIV added apicallyFilter 1 262,874 536Filter 2 218,946 582Control* 378,200 235,200

HIV added basolaterallyFilter 1 246 306,034Filter 2 310 198,332Control* 36,600 385,800

Results are expressed in cpm for two distinct representativeexperiments (filters 1 and 2).*The control was the filter alone (i.e., without the cell monolayer).The initial virus input was 800,000 cpm (5 x 105 TCID50 per ml).

Infection of Polarized Monolayers of HT-29-D4 Cells. In asecond set of experiments, glucose-deprived differentiatedcells were cultured on a permeable substratum where theyformed a leak-proof and electrically active polarized mono-layer (13) resembling the colonic epithelium (Fig. 2). It isimportant to note that the differentiation process of HT-29-D4 cells involved virtually the total population of cells inthe monolayer. A transepithelial electrical resistance of 250± 46 fl-cm2 indicated the uniform presence of tight junc-tions-a property of epithelial differentiation (13). The virus(strain HIV-1-NDK) was incubated in either the apical or thebasal compartment of the cell culture chamber. After 24 hr ofincubation, the residual RT activity in both compartmentswas measured (Table 1). Our results clearly establish that: (i)in the absence of the cellular monolayer, the virus can diffusefrom the apical to the basal compartment and, to a lesserextent, from the basal to the apical compartment; and (ii)HT-29-D4 cells form a tight monolayer that blocks thetransport of the virus from one side to the other.To assess whether the cells had been infected after an

apical on a basolateral contact (or both) with the virus,HT-29-D4 cell monolayers were harvested after three sub-cultures by trypsinization. Viral production was monitoreddirectly in the culture supernatant or indirectly after cocul-ture with lymphoid cells. A low but significant RT activity(10,000 cpm per ml) could be detected in the culture super-natants of HT-29-D4 cells exposed to apical or basolateralHIV. Viral production was greatly enhanced after cocultiva-tion in the presence oflymphoid cells for cells infected via theapical or the basolateral plasma membrane (Table 2).These data show that both undifferentiated and differen-

tiated HT-29-D4 cells were sensitive for HIV infection. In the

Table 2. RT activity (cpm/ml) in Sup T1 cells cocultivated for24 hr with HT-29-D4 cells infected via the apical or thebasolateral side

Day after Apical infection Basolateral infectioncoculture Filter 1 Filter 2 Filter 1 Filter 2

4 18,680 7,602 8,8% 9,2188 285,700 212,900 243,288 341,606

12 709,768 449,290 614,8% 610,532Filter-grown HT-29-D4 cell monolayers (transepithelial resistance

> 250 Q cm2) were incubated with HIV-1-NDK (7 x 103 TCID50 perml) added either in the apical or the basal compartment. After 24 hr,tight junctions were disrupted by a 1-hr treatment with calcium-freemedium, and the cells subsequently were harvested by trypsiniza-tion. After three successive passages (day 10 after infection), thecells were cocultivated with Sup T1 indicator cells (5 x 101 cells perml) for 24 hr. RT activity was measured in the cell-free supernatantof Sup T1 cells as described.

case of differentiated cells, the virus could enter nonprolif-erating cells via either the basolateral or apical membranedomain. Inhibition of infection by anti-CD4 monoclonalantibodies was not successful. Therefore, the mechanism ofentry of HIV through each membrane domain of HT-29-D4cells remains a challenging problem. It is possible that HIVbinds the CD4-like antigen characterized in the basolateralmembrane, but at a site distinct from OKT4A/Leu 3a epi-tope. Apical infection could occur following an interactionwith a brush border component immunologically related toCD4, as recently reported.*

In conclusion, we confirm that epithelial cells can beinfected by HIV (3, 4, 29). We show that resting colonicepithelial cells can be infected by HIV-1 and HIV-2 strains.The fact that this infection can remain silent could explainwhy some male homosexuals remain seronegative despiteanal intercourse with seropositive partners. The induction ofthe virus by lymphoid cells may result in the appearance ofanti-HIV antibodies a long time after contamination. Becausethe infection can occur apically in vitro, it is possible that thecolorectal epithelium represents a major site ofentry for HIVduring receptive anal intercourse. Moreover, infected cells inthe lamina propria (which contains T cells, macrophages, andeosinophils) can transmit HIV to epithelial intestinal cells viatheir basolateral membrane.

*Moyer, M. P., Huot, R., Wideman, D. & Martin, D., Fifth Inter-national Conference on AIDS, June 4-9, 1989, Montreal, p. 606(abstr. 90).

We thank Jean-Jacques Roccabianca for photographic works,Nadia Guettari for critical advice in PCR experiments, and Gene-vieve Plumion for typing the manuscript. Financial support fromInstitut National de la Sante et de la Recherche Medicale, AgenceNationale de Recherches sur le Sida, and the Association desArtistes contre le SIDA is fully acknowledged. J.F. is from theUniversity of Aix-Marseille I.

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