HIV-1 Spreads from Lymphocytes to Normal Human Keratinocytes Suitable for Autologous and Allogenic Transplantation

Dunia Ramarli, Adriana Giri:* Salvatore Reina,t Ornella Poffe, Ranieri Cancedda,* Oliviero Varnier,*

Giuseppe Tridente, and Michele De Luca:j: Institute of Immunology and Infectious Diseases , University of Verona, Verona; *Laboratory of Molecular Virology, Advanced

Biotechnology Center, Genova; "!" Institute of Microbiology Istituto di Microbiologia, Un iversity of Genova; and :j: Unit of Epithelial

Biology and Biotechnology, Advanced Biotechno logy Center, Genova, Italy

Normal human keratinocytes can reconstitute i11 vitl'O

cohesive sheets of epithelium suitable for grafting onto patients. Despite the widespread use of au­tografts and allografts, no data are yet available on productive infection by human immunodeficiency virus (HIV-1) ofhuman keratinocytes. To address this point, we challenged keratinocytes at the second passage of culture with HTL V -IIIn virus by cell-free and cell-tnediated inoculum. Viral entry was not achieved by cell-free inoculum, thus demonstrating that cultured keratinocytes do not provide the mem­brane requirements for viral binding and/or internal­ization. By contrast, the cell-mediated inoculum overcame specific receptor constraints, leading to viral integration and productive infection. The p24g"g viral protein was transiently released in the culture

N anna l human keratinocytes isolated from a skin

biopsy and cultivated unde r proper conditions

form colonies, each colo ny b eing the progeny of a

single basal keratinocyte [1 ]. Keratinocytes located

at the periphery of the colony migrate outwards

and g row und er control of specific growth f.1ctors [2). whereas

those loca ted toward the center of the colony and committed to

terminal differentiation lose their adhesive m achin ery [3 ). inc rease

in size, migrate upwards, and ex press diffe rentiatio n-specific mark­

ers [4). Keratinocytc colonies wi ll eventua ll y fuse, givin g rise to a

stratifi ed squamou s epithe lium closely resemblin g normal hum an

epide rmis and maintaining the d ifferentiation patte rn of its i11 l!illo

counterpart; these have been used as autologous, permanent grafts

on patients [5,6). T ransp lanted auto logous cul tured keratinocytes

(autografts) have been u sed widel y for the perman ent coverage of

third-degree burns and large skin defects (rev iewed in [7]). Deep

partial skin thickness burns and limite d skin defects, su ch as chronic

leg ulce rs, arc also success fully treated with allogenic c ultured

epithelium (a ll ografts) [8). Allografts, which can be v iably frozen

and sto red, d o n ot "take" pe rman ently, but stimulate re- epitheli-

Manusc ript received November 1, 1994; fina l revi sion rece ived july 14,

'1995; acce ptecl for publ ication August 3. 1995. It<.!pri nt requests to: Dr. Dunia rl .. amarli . Istituto di Immunologia e

Malattie In fettive. Poli cl inico llorgoroma, Via dell e Mcnegone, 37134

Verona , Ita ly.

supernatant, although at low level. The viral progeny produced by infected keratinocytes was rescued and amplified by co-culture experiments performed with the HIV-1 high sensitive CEM-SS human T-cellline. Viral integration, p24g"g production, and secondary transmission to lymphoid cells was further confirmed with keratinocytes infected at the fourth passage of culture. Taken together, our results dentonstrate that cultured keratinocytes can be infected by HTL V -Illn virus, which can be maintained in sem.i-latent form for several passages after inoculum and rescued to full replication by a proper target. The i11 vitl'O dem­onstration of lympho-epithelial HIV-1 spreadings warns against the use of inappropriately screened biopsies for the preparation of skin grafts. Key word:

it~Jectioll .. ] l11vest Dermatol 105:644-647, 1995

alization by resident keratinocytes thro ugh the secretion of several

polypeptides in the wound Auids [9) . Despite the widespread u se of

autografts and allografts, no data are yet avai lable o n human

immunodeficiency virus (HIV) productive infection of human

keratinocytes. We assayed keratinocyte permiss iv ity for HlV-1

infection by ce ll -free and cell-mediated inocula. H e re w e show that

H IV -l ente rs human keratinocytes through heterologous cell-cell

contacts, integrates in the keratinocyte genome , and rep licates a

vira l progeny able to infec t lymphoid cell s.

MATERIALS AND METHODS

Cells and Cell Lines Normal human epidermal keratinocytl!S were

obtained &om skin biopsies of HIV-1 se ronegative healthy volunteers and culti vated on a fl!eder laye r o f letha ll y irradiated 3T3-J 2 ce lls (a gift of H. Green, Harvard Medical School, Boston, MA) as previously described [1 ]. In brief, skin biopsies werl! minced and trypsinized (0.05'1,, trypsin/ 0.01 'Yo ethylenediaminctetraacetic acid) at 3 7°C for 3 h. Cells were collected

every 30 min , plated (2.5 X 1 0'1/cm2) on 3T3-j2 cells (2.4 X 1 O'/cm2

) and cultured in humidified atmosphere at s•y,, C 0 2 in kera tinocytc growth medium: Dulbecco 's modified Engle's medium and Ham's F12 media (3:1 mixture) (ICN, Mi lano, Italy) containing 'I 0% fetal bovine serum (PAA, Technogcnetics , Mi lano, Italy), insulin (5 J.l.g / ml) (Sigma, Milano, Italy), transferrin (5 J.l.g/ml) (Sigma), adenine (0 .1 8 mM) (Sigma), hydrocortisone (0.4 J.l.g/ ml) (Sigma) , cholera toxin (0.1 nM) (Sigma). triiodothyronine (2 nM) (Sigma) , epidermal growth f.1ctor (1 0 ng/ ml) (Austral Biological, San Ramon CA), glutamine (4 mM), and penicillin-streptomycin (50 IU/ ml). Sub-conflue nt prin1ary c ultures were passaged in secondary cultures by trypsin-ethylenediaminetetraacetic acid tre:ltmcnt. H9 human T-cell lin~

0022-202X/95/SU9.50 • SS DI0022-202X(95}00361-4 • Copyright © 1995 by The Society for Investigative Dermatology. Inc.

644

VOL 105. NO. NOVEM UER. 1995

ce ll - tree

day from 4 7 9 11 4 7 9 11 ino c ulum

culture 3'd 4th 5th 6th p assage

p 24gag/ 0 33 :!: 0 0 0 0 0 0 106 ce ll s 29

':" I u <!

"" u 1-0 0

A

B

c

HIV- 1 INFECTION OF KER.ATINOCYTES 645

N I N

I I

u :I: <! u

<! u

cell mediated 0 u

D

E

4 7 9 11

0 23 <7.8 0 F

Figure 1. HTLV -III0 viral strain enters c ultured keratinocytcs by cell-mediated but not cell-free inoculum. C ultured kera tinocytes were chall e n ged with HTLV-111 11 virus by cell-free and ce ll-mediated inoculum . C dls and cell-free supernatants were collected at inte rval s from viral exposure to eval ua te viral integration and p24·'"·' prod uc ti on, re spective ly assessed by DNA amplificati on o fHIV-1 seq uences and enzyme-linked immunosorb ent assay.

• A and B , gag sequence amplifi cation in celll ysatcs co rresponding to 5 X 1 o·• keratinocytes chall enged at the second passage of culture. Samples were co llected at day 4, 7, 9, and 11, corresponding to keratinocytes at the th ird, fourth, fifth , and sixth passage from cell-free and cell-medi ated inoculum . Positive and nega tive control s arc respectively represented by AC H-2 T cells co rresponding to I 0 provira l copies and unin fected cell lysa te (CTR). C. p24·' '" production of second-passage kcratin ocytcs after ccll-tn cdiatcd inoc u lmn . D 11nd E, gn,~ seque nce antplifi cation in cell lysatcs co rrespo nding to 5 X I o·' kcratinocytcs challenged at the fourth passage of cul ture . Sam ples were coll ected at da y 4, 7, 9 . and 11 , corres ponding to keratinocytes at the fifth , sixth, seventh. and eighth passage fro n1 ccll-ntc diated inoculum . Positi ve and negative contro ls arc represented by ACI-I-2 corres ponding to 10 and 1 provira l copies and unin fcctcd ce ll lysate (C TR). F, p24-'"·' production of fourth-passage keratinocytes afte r cell-mediated inoculum . Ge l ex posure was 3 h in A and D an d ove rni ght in B and E . Primer pairs were 145-431 in A and Band 38-39 in D and E.

(gift of R .C. Ga ll o, LTCB, N C I, NIH, Bethesda , MD) . CEM-SS hu man T-cell line (g.ift of Pete Nara, Frederick University, Rockville, IN), and HTLV-11 113 chron ica ll y infected 1-19 cell line we re grown in RI'MI 1640 m edium suppl emented w ith 1 0"/., FBS, glutam ine (4 mM), and antibio ti cs .

lm=unofluorescence T he fo llowing m onoclona l antibod ies (MoAbs) were u sed in ind irect irnrnun oAuorcsccncc: D 'l - ·12 (an ti-tnajor hi stocom­patibility complex (M H C) class II an tigens, gift of Dr. rt.S . Acco lla), Nal/34 (anti- COla, g ift of Dr. F. Ca labi). anti-CD3. and anti-CD45 (from Bccton-Dickinson , M ountain View, C A) . BrieAy, up to 1 O'' ce ll s were incubated on ice for 30 min with the va rio us MoAbs in the presence of 100 J.Lglrnl of tota l human lg (S igma), washed , stained in the same conditions with F(ab)2 Auo resceine iso thyocytra te-labeled goat anti-mo use (from Becton-Dickinson), washed, and sco red at the microscope.

Virus T he HTLV-111 13 v iral strain was host in the H 9 T-cel l line. Supernatan ts ob tained from cells grown up to 3 X 106 cells/ml were cell-clarified by centrifugation, fi ltered thro ugh 0 .45-J.Lm pore-size filters, and stored at - 80°C un ti l usc.

IDV Infection HTLV-111 13 infection expe ri me nts were performed by cell-free and ce ll - mediated inoculum of keratinocytes obtained fi·om the same flask at the second passage of cul ture and by cell-mediated inoculum of keratin ocytes at the fo urth passage of culture. For cell-free inocula, keratinocytes we re incubated ove rni ght in H9 cell supernatant (p24 viral prot e in, 250 ng/ml) at 37°C, washed, treated w ith trypsin- cthylencdi­aminetetraacetic ac id , and plated onto new Aasks . For cell-mediated inoc­ula, kerarinocytes were seeded at 3.5 X 10~ /cm 2 in six-well Costar plates and co- cul tured overni ght at 1:1 cell ratio w ith y-irrad ia tcd (4000 rad) 1-1 9

ce lls. At the end of the co-cul ture, H9 ce ll s were rem oved by multiple w ashes and adherent keratin ocytes were trypsini scd and seede d o n to nc\v plates. C ell aliquots (1 O'' cells) were sta ined before platin g with M oA bs rccoguizing the C D 3 aud CD45 lymphoid antigens. Keratiuocyte cultures we re se ri all y passaged after viral exposure. Ce ll s and cell-free supern atants were collected from each passage to fo llow viral integra tion. p24 viral protein production (p24·'''·') and viral progeny infecti vity. Ce ll death of y-irradiatcd H 9 cells was o bserved 4 d after treatment. as assessed in separate culture.

HIV- 1 DNA Sequence Amplification HIV- 1 gag-sequence amplifi ca­tion was done as previous ly described f I 0] . BricAy, ce ll lysa tcs (5 X 1 0~ /sa mple) suitability fo r polym erase amplifi cation were assessed by atnpli ficatio n of hutnan cytocro n1 c B sequences by using the prime r pair MVZ03 / 04 in 50 mM KC I; 10 mM Tris-H C I, pH 8.3; 3 .5 mM MgCI, ; 50 nM of each primer; 200 mM dNTPs: and 0.75 uni ts of Taq po limerasc (Perkin Elmer Corpo ratio n, N orwa lk , CT). Mixtures underwe nt 35 cycles of amplifi cation (9 4° 15", 42° 15", 72° 30") in a GeneAmp PCR System 9600 (Perkin Elmer). The cytocrome B 367-bp band was visualized on 2'Xo agarose gel. HIV-1 ,(!ag sequ ences were then atnp liti ed by usin g the pritn c r pairs SK145-4 31 o r 38-39 in the same wo rking bufre r w ith 2 .5 units of Taq polymerase (Perkin Elmer) . Mixtures underwent 35 cycles ampli fication (92° 1 0", 56° 15", 72° 1 0") in the same apparatus. Ampli fication products were hybridized in liquid phase to 32P-y :a:icnosinc t riph osph:He labeled SKI 02 o r SK 19 probe and ana lyzed on a 1 0'% po lyacrylamide gel. AC H-2 T -cell line, containing one prov iral co py per ce ll 111 ]. represented the pos itive contro l. Gels were exposed 3 h and o ve rnig ht at roon1 tcn1pcrature to Amersham H ype rfi lm films.

646 RAMARU ET A L THE JOU11..NAL OF INVESTIGATI VE DEI1..MATOL0GY

.. A ~ B =s -.: ... "'

... . >250 >250

Q s .... 11> lit .... ....

200 200

150 150

100 100

50 50

11 day from co-culture 4 7 9 11 day from co-culture

Figure 2. Infected keratinocytes transmit viral infection to lymphoid cells . The HIV-1 high sensitivity CEM-SS T-cellline was co-cultured with

keratinocytes at day 7 (e), 9 (•). and 11 (.'-) from cell-media ted inoculum . Lymphoid target ce ll s were then recovered, washed twice, and maintained in culture for 10 d. Cell-fi·ec supernatants were seri ally collected and assayed for p24·'"" production. A, p24·'"·' production (mean :':: SD) of CEM-SS co-cul tured

with keratinocytes infected at the second passage of culture . B, p24""·' production (mean :!:: SD) of CEM-SS co-cultured with keratin ocytcs infected at the

fourth passage of cul ture.

p24R".< Production Cell supernatants were assayed fo r p24·'"·' production by enzyme-linked immunosorbent assay according to manuf.1cturer's in­structions (Coul ter, Hialeah, FL). Negative samples were Polyethylen­glycol precipitated (15M polycthylen-glyco i/1 .5M NaCI) and re-examined in the same assay. Optica l density va lues were plotted on the stand,.rd curve and expressed as pg/1 O'' ce lls.

Viral Progeny Infectivity CEM-SS human T-cell li ne was co-cultured overnight at 1:1 ratio with adherent kcratinocytes at day 7, 9, and 11 from ini tia l inoculum . Lymphoid target cell s were then recovered, washed twice, and maintained in culture for 10 d. Cell-free supernatants were serially collected and assayed for p24·'"·' production.

RESULTS AND DISCUSSION

HTLV-IIIn Infection of Keratinocytes at the Second Pas­sage o f Culture T he permissivi ty to the HIV-1 infection of keratinocytes was first investigated on second-passage cul tures by cell-free and cell-mediated inocu la, perfo rm ed in parallel with keratinocytes obtai ned fi·om the sam e Aask. CD1 and MHC class II (Langerhans cells) antigens were undetectable by indirect immu­noAuorcscence on kera tinocytes obtained from different donors. Positive controls used were fi·es h unfraction ated thymocytes (CD1)

and PHA-lL- 2-activated T lymph ocytes (MHC cl ass ll) (not shown) . T he lack of detectable vi ral sequences afte r ce ll-free in oculum (Fig 1A,B) demonstrated that keratinocytes do n ot p rovide the m em bra ne microenvironment required for v iral bind­ing and/or in terna lization and confirmed the absence of HIV-1-susceptible Langerhans cells in the cultures. In agreement w ith previous reports [1 2, 13], however, direct contact between infected 1-:19 cells and ke ratin ocyte monolayers overca m e specific receptor constraints, leading to the amplification of at least 1 0 v iral copies per 5 X 104 cultured cell s in the samples collected at day 4, 7, 9, and

11 from cell-mediated inoculum corresponding to keratinocytes at the thi rd, fourth, fifth, and sixth passage (Fig 1 A,B), respective ly

(see Materials and Methods) . Residual 1-:1 9 ce ll contamin ation was ru led o ut given i) the tim e of J-1 9 death (4 d afte r irrad iation)

assessed in separate cu lture, ii) the absen ce of CD3 and CD45 lymphoid antigens in the ke ratinocytc culture assessed by indirect immun ofluore scence, iii ) the lack of p24·'"·" production at day 4

fi·om starting in ocu lum (sec be low), and iv) the increasing poly­m erase chain reaction sign als during the follow-up.

p24·""·" Production of Infected Keratinocytes Viral replica­tion occurred , albeit at low levels, in in fected keratinocytes . as demonstrated by the p24·""-" protein re leased in the culture super­natant. Indeed , p24·"''-" protein , undetectable at day 4 after inocu­

lum , became c lea rl y present at day 7 and return ed to undetectable leve ls at day 9 and 11 (F ig 1C). Identica l resul ts were o btain ed in

four different experim en ts perform ed w ith three difFe rent kerat.ino­cyte stt·a ins, and the peak of p24·"''·0 protein productio n was repro­ducibl y observed at the sa m e time point. Consistentl y w ith the low (or slow) viral rep lication rate, none of the infected cul tures showed viru s-induced cytopathic efFect during the culture observa­tion .

HTLV -IIIu Infection ofKeratinocytes at the Fourth Passage of Culture To exclude further the poss ib ili ty that rare and/or undetectabl e Langerhan s cells represented the 1-IIV-1 target in rhe ke ratinocyte secondary cultures, we performed cell-mediated in­fection s ofkeratin ocytes at the fourth passage of cul ture. CD1 + and MHC class 11 + cells were undetectable in the culture by indirecr immuno Au oresccnce. No difFerences were observed between kera­tinocytes at the second and at the fourth passage of culture in tcm1s of viral in tegration and p24·""·' production, thus demonstrating thar the permiss ivity to viral in tegration and/or early repli catio n events was reta ined du ring aging of the culture. Indeed, viral sequen ces were detected througho ut 11 days after in fec ti o n, fi·o m the fifth to the eigh th passage of culture (Fig 1D,E), ruling out the possibiLi ty

that Langerhans ce lls m ay account for ampli ficatio n signals. The peak of p24·""-" production (Fig 1F) was reproducibly observed at day 7 an d returned to undetectabl e levels at days 9 and 11 .

Secondary Infection of Lymphoid Cells with the Viral Progeny Produced by Infected Keratinocytcs To in vesti­gate w hethe r infectious virions were produced by second- and fourth-passa ge-infected ke ratinocytes and wheth e r the v iral prog­eny had m aintained the orig inal lymphotropism , we ca rried out secondary in fections of lymphoid ce ll s. T he l-IlY-sensitive CEM-S

VOL. 105 , NO. 5 NOVEM ll ER 1995

hum a n T - cell line was co-cultured overnight wi th keratinocytes I, obtain e d fi·om each passage perform ed afte r J-1 9 priming, recov­

ered , a nd gro wn in cul ture to fo ll ow th e p24·""·" pro du ctio n up to 11 d after contact. R esul ts o bta ined by using in fected ke ratin ocytes fro m the thit·d to the sixth passage o r fi·o m the fo urth to the e ighth passage are shown in F ig 2A a nd B , respecti ve ly. T he hum an T - cell line cou ld rescue and ampli fy to a full productive in fec tio n

, rhe viral parti cles produced by ke ra tinocytes in fec ted at both the seco nd and th e fo urth passage (A and B). Secondary tran smjss ion

' from the second- passage-infected keratinocytes to lymphoid cells occu rred o nl y in the presen ce of low (but detectabl e) p24·""·" prod u c tio n (A), w hereas secondary tran smiss ion from the fo urth­passage-in fec ted keratinocytes to the same ta rget was observed no t on ly in the presence (B, day 7) , bu t a lso in th e absence of detectable levels of viral protein (B, days 9 and 11 ) . T he possibili ty that H 9 released viri ons acco unted fo r lymph oid cell infection can be exclude d o n the bas is of positive co-culture w ith the serially trypsirUzed keratinocytes at day 11 fi·o m ce ll-mediated in oculum (see M aterials and 1\ll.etlwds). Taken together , these resul ts dem on­strate t hat kera tinocyte m o nola yers m ay hos t in semi-l atent fo rm the HTLV-III 13 virus, w hich can sti ll be rescued to full replication by a p r oper ta rge t .

T h e m aj or site o f HlV-1 replication is the hum an lym pho id compartm ent [1 4) . It has been dem onstrated , how ever, th at HIV- 1 can infect a w ide range of human cell s of di ffe ren t embryonic o rig in [1 4) , including bra in ce ll s, cutaneous fibrobl as ts, chondrocy tes , and epi thelial ce ll s [1 4). Infec tion o f human epi thelia had been inves­tigated m ostl y in intestinal tum o r cell lin es by several studies showjng that lympho-epi the li al tra nsmiss ion m ay occur 11ia phago-

/ cytos is, direct fu sio n, and coated pit fo rmation ac ting as C D 4-ind e p e nden t ro utes fo r viral entry w hen the ta rget- virus carrying

1 cell contact is achieved [1 2, 13]. O ur results dem onstrate th at nonnal cultured human keratin ocytes can be pro ductive ly infected ill 11itro by an 1-llV-l m ono- and lympho tropic strain ;md may

• transmit , in turn, the viral infection to by-stander lymph o id cells, ( therefore suggestin g that cell adh esion co uld underlie viral spread­{ ing wjthin epi the lia l layers and lym pho-ep ithe lial in fect io n circui ts

as well . To date, n o strict regula tion is yet ava ilable concerning the screening of human keratinocyte dono rs. Even th o ugh the proba­bility of ill 11ivo-infected keratin ocytes in the asym ptom ati c popu­lation m ay be lo w , these o bservati ons warn aga ins t the use o f inappropriate ly screened skjn bio psies destined fo r the preparation of auto- and allografts.

I-I IV-1 INFECT ION OF KE itAT INOCYTES 647

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2. Darrandon Y. Green H: Ce ll mig:r:1tion is essential fOr suswincd g rowth of kcrari nocyte colo nies: the ro les o f transfo rming grow lh f: ~e.: wr-a· and e pide rma l growth facto r. Cd/ 50: 113 1- 11 3 7. 1987

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9. D i Ma rco E. Marho r M. Bo nd:m z:1 S. C urul i N. Marchisio PC . Cancedda R . De Luc;1 M: Nerve g rowth facro r (NG I::) binds ro norma l human kcratinocytes through hig h and low affmity receptors and stim ul:ucs the ir growth by :1 nove l amacrine loop. J IJicJ/ Clw11 2M;:22S38-22S -4 6. 1993

10. Giri AA. Lillo FF, Mc Dermo n JL, J:mn uzzi C. l"tisso S, Fo rn i G. Conccd i D R. Va micr OE: De tection of 1-ll V sequ ences in chil dren using radio m etric :111d colorirnetri c po lyn 1erase cl lai ll re:1c tio n. J J\ INJ VinJ/ -42 :41-4 -4 18. 1994

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12 . Phill ips DM. 13o urimbaiar AS: Mcch:m ism of I-I IV spread from lymphocytes ro epithe li a. VimltJ,g)' 186:26 1-273 . 1992

13. lJo urimh:1iar A ·. Phill ips DM: Tr:1 nsm iss ion of la lln an im num odel·i cie ncy vi rus fro 111 lll unoc)' tcs to epithel ia. ) A IDS 4:56-63, 199 1

1-4. Levy JA: Pathogenesis of human imm unodc ticicncy virus infectio n . J\lirrobinl Rn' 57:1 83-289. 1993