lournal of ImmunologicalMelhods, 155(1992)257-265

257

© 1992ElsevierSciencePublishersB.V, All tightsicscfved0027.-1759/~/$05.00

JIM 06480

Flow cytometric analysis of transport activity in lymphocytes electroporated with a fluorescent organic anion dye Joseph E. Dinchuk ~, Keith A. Kelley b, and Gerald N. Callahan c ° Imtltut¢ for MolecularBiologicagsand the ~ In.tit.re for Arthritis ¢mdAutoimmuni~l, MilesResearch Center.400 Morgan Laae, West Haven, CT 06516, USA, and e Delxa'tment of Pathology, Colo'ado Slate University, Fort Collin~ CO 80523, USA

(Received23 March1992,accepted 18May 1992)

Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in |yraphocytes, Cloned tymphoma cells and normal and activated human T ceils were loaded with a membrane-imponneaILt, organic anion dye (Lucifer Yellow) by electroporatlon. D ~ efflux in lymphocytes was rapid, ener~-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, eleetroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, ~ e loss occurred in viable, functionally competent cells. These experiments demonstrate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages. Key words: Eleetroporation;Lucif©rYellow;Lymphooj1.es;Organicaniontransport;Probvnccid

Introduction Ion channels play a major role in initiating, regulating, and supporting many cellular immune functions (Chandy et at., I985). Recent evidence that macrophagos possess organic anion transporters led to the suggestion that these transporters may play a role in cellular immune rune-

Correspondence w: J.E. Dinchuk,DuPont MerckPhmmaceuticals, 500 SouthRidgewayAvenue,Olcnolden,PA 19836, USA. Tel.:(215)237-7755;Fax:(215)237.7865. Abbreviations: cOVA, chickenovalbumln;DMEMh, Dulbccco's modified Eagle's medium with high glucose; PBS, Dulhecco'scalcium-and magnesium-freephosphate-buffered saline;Th. T helpercell.

tion (Steinberg et al., 1987). Since lyraphoc~es generate many of the same organic anions (e.g., leukotrienes and arachidonate metabolites) as macrophages, we decided to investigate whether lymphooytes possess organic anion transporters and if so, if they exh~ited different rates of dye flux inactivated vs. normal I~aphoc~tes. Several reports haw described the use of organic anion dyes such as Lucifer Yellow to document and study organic anion transport in macrophages and polarized epithelia (Steinberg et al., 1987; Di Virgilio et ai., 1988; Gerard et al., 1990; Lipman et al., 1990). Lucifer Yellow is a low MW, non-toxic, organic anionic fluorescent dye that been used in a variety of tracer studies where membrane impormcance is a prerequisite (Stewart, t981; Honig and Hume, 1986; Bosel et

258 al, 1989; Griffiths et al., 1989) Previously, scrape-loading or ATP (5 raM) permeabilization has been used to load Lucifer Yellow into maerophages (EI-Fouly et al., 1987; Steinberg et al., 1987; Di Virgilio et al., 1988). In those studies, dye effiux was determined by isolating and lysing cells at defined time points and monitoring released fluorescence with a fluorescence spectrophotometer. As l~mphocytes are maintained in suspension, they axe not amenable to scrape-loading. In addition, concentrations of ATP as low as 20 /tM have been shown to be highly toxic to normal lymphoc,fies (Di Virgilio et al., 1989) and induce a large number of effects on treated cel|s (Di Virgilio et al., 1989; EI-Moatassim et al., 199D). Thus, we found existing methods for loading Lucifer Yellow into cells unsuitable. To model organic anion transport in lymphocytes, we used electroporation and flow cytometery to introduce Lucifer Yellow into cells and examine dye effiux (i.e., transport) activity under a variety of eonditiolls.

Our results indicate ~hat electroporated, Lucifer Yellow-loaded lymphocytes are both viable and functionally competent. We provide evidence that elimination of dye occurs via energy-dependent, probenecid-inhibitable organic anion transporters that are constitutively active in lymphocytes. The presence of organic anion transporters in I~m~phoo/tesmay be important in the export of organic anions such as lactate, lenkotfienes, and prostagiandins. These transporters may hold special significance in the removal of pharmacologically relevant organic anions (e.g., AZT) from the cytoplasm of iymphocytes.

Materials .qnd methods

Cell lines Jurkat cells (a transformed human T lymphocyte line) were obtained from the ATCC and maintained in continuous culture in RPMI 1640 supplemented with 10% FCS and 2 mM Lglutamine (sRPMI). Resting and activated T ceils were prepared from freshly drawn human peripheral blood by the e~throcyte-rosette method (Blue et at., 1986). Resting T cells were cultured overnight in sRPMI prior to use in these experi-

merits. Activated T cells were prepared by culturing resting T celts for 3 days in sRMPI containing 1 : 100 PHA (Gibco 4670-0576AC) and 20 U/ml recombinant IL-2 (Boehringer-Mannheim #1011456). These cells were split 1:2 every 3rd day with sRPMI containing 1L-2 but no PHA, and used for dye efflux studies on days 12-15 of culture. The cloned, BALB/c-derived, cOVA-specific, l-Ad-restricted Th hybridoma, 3D0-54.8 (Shimonkcvitz et al., 1983) was kindly supplied by Dr. P. Mm'rack of the Howard Hughes Medical Institute at the National Jewish Center for Immunology and Respiratory Medicine, Denver, CO. This cell llne was derived from the fusion of I]ALB/c anti-cOVA T cell blasts with the BW 5147 (AKR-derived) thymoma. The 3D0.54.8 cell line was maintained in lymphocyte culture medium ((DMEMh containing 10% newborn-calf serum (Irvine Scientific, Santa Ann, CA)), 0.1 mM nonessential amino acids (Gibeo, Grand Island, NY), 1 mM sodium p~n~vate, 2 mM glutamine, 50 mM gentamicin, and 5 x 10 -~ M 2-ME. A20-2J cells are la +, H-2 d, B cell I.~nnphoma cells derived from a spontaneously arising tumor in BALB/c mice (Kim et al., 1979; Kappler et al., 1982) (gift of Dr. J. Kappler, National Jewish Hospital, Denver, CO). These cells were maintained in lymphoc~e culture medium. The CTLL cell line (gift of J. Allison, UC Berkeley) is an IL-2-dependent, tumor-specific, cytotoxic T cell line derived from C57BL/6 mice (Oillis and Smith, 1977). These cells were maintained in lymphocyte culture medium supplemented with 25 U/ml recombinant human IL-2 (Amgen Biologicals, Thousand Oaks, CA).

Electroporation conditions Cells were isolated from suspension cultures and washed 2-3 times in cold (4°C) PBS w/o Ca 2÷ or Mg2÷ and suspended at a concentration of approximately 5 × 106 cells/ml in cold D-PBS (w/o Ca ~+ or Mg 2+) containing 0.5% Lucifer Yellow CH (Sigma Chemical Co. St. Louis, Me). Exactly 1.0 ml of this suspension was placed into a Bin-Rod Gene Pulser cuvette and incubated on ice for approximately 5 rain prior to e|ectroporalion. The samples wore subjected to a single pulse of 600 V (25 p.F) in a Bin-Rod Gene-Pulser

259 (Bio-Rad Laboratories, Richmond, CA) with the exception of resting human peripheral T cells which received a single pulse of 700 V (25/tF). Electwporated celts were then incubated on ice for approximately 5 rain. The cells were then pipetted into a 15 ml centrifuge tube containing 10 ml of cold (4°C) PBS and centrifuged at 750 x 8 for 5 rain. The cell pellet was resuspended in cold PBS and washed once more prior to the tr.~nsfer of electroporated cells to culture media. Flow cytom~r~cmeazurements

Measurements of Lucifer Yellow fluorescence and propldium iodide exclusion were made on a FACScan flow cytometer operating Consort 30 software (Becton Dickinson lmmunocytomctry Systems, San Jose, CA). Excitation of Lucifer Yellow and propidium iodide was provided by an

argon ion laser emitting at 488 run. Fluorescence signals were separated through the use o f dichroic fiIters; a 30 nm bandwidth centered at 530 nm for

Lucifer Yellow and a 42 nm bandwidth centered at 585 for propidium iodide. Logarithmic mean channel values for Lucifer Yellow fluorescence were calcnlated for viable cells (cells which exhibited fluorescence above the unstained control in the propidium iodide channel were excluded from the analyses) and converted to linear mean values according to the procedure described by Schnfid et al. (t988) Briefly, each of the channels of a 256 'channel histogram displaying the signals derived from a logarithmic amplifier is related to an intensity level by taking the channel number as the exponent of a base. The base is derived from the number of decades of dynamic resolutio, provided by the logarithmic amplifier. By raising

Yellow in PBS as de~fiheci in the text, The ~lls were washed free 04r e~ss dye and either fixed immed~rely (A) or after 2 h in culture (.B) in a 2.0% .solutionof fo~naldchyclc in PBS, Fornlalc~hyde FP,ation 'locks' this orlPm~ dye in ~ wit[mat no~ice~ effeetJnl~ it~ fluot~scegge characteristics. A punclate fluorescence that rc~mb]es cytop'.asn~ oqEanc]lesis p~sene Jn cell,, thaE

appear to havelostcylop~asmicfluorescence(B). Bar - 20/~m,

260 ~te base to the channel numbers from 0 to 255, a series of linear values is created that spans the intensity range from 1 to 10,000 for a four-decade amplifier and thus aLlows a direct comparison of intensity differences.

Microscopic studies of electroporated cel[~ Luc.er Yellow-loaded cells were washed two timesrin chilled (4°C) PBS and ¢itber flied immediately (in a solution of 2.0% formaldehyde in

PBS, pH 7.2) or cultured in lympho,=Tte culture medium at 37°C in a humkl environment of 95% CO2/5% 02. Fixed material was prepared ~Or fluorescence microscopy by washing in PBS, pc]. leting, and resuspend~ng cells in PBS, A drop of concentrated cell ~suspension was placed on a clean microscope slideand a coverslip was added. Cells w e r e studied and photographed with a Zeiss epi-fluoresconce, microscope using an excitation bandpass filter at 450-490 nm, a beam splitter at

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o[ electroporatk), on cell ~abfli~. Celts ele¢Imporated in the p r e ~ n ~ of 0.5% Lu~fer Yellow we;'e processed as iadicaled in the text and suspend©d in culture medium at 37°C in a humid environment of 95% CO~/5% 02. A]i~uots of ~urkatt o¢iI, dcrbrcd from the same el~ttopotation euvette were zemo~ed,after O, 2, 3, 4 and 8 h, stained with propidi:zm i~tide and anal'z'zedby two-color flow cytometw for Lucifer Yellow retention and ~ability. The FACScan images (a, C, and L:) correspond to time points 0, 2 and 4 on the graph ( A ) RI;. 2. Effect

261

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vant figures. Cultures were monitored for viability and Lucifer Yellow fluorescence.

~spo,~e tO u,,t~e,,p,~,~tatio;~ by ele~rat~d Th cells,. Tbe~ abifity of Lucifer Yellow-cl¢ctroporated Th cells to generate IL-2 in response.to antigen presentation by the l-Ad-positive. B cell lyrephoton, A20-2J, was determined essentially as described previously (Kappler et al.,-1981). Briefly, A20-2.1 presenting cells were added to the wells of a 24-well culture plato 0 0 s ~:olL~/woI!in ~ 0 /tl lymphocyte vulture medium), cOVA, i mS/well in 300/~1 lympliocyte culture medium, was added to experimental wells. Lymphocyte culture medium alone was added to control wells. I-Ad restricted, eOVA-spe¢ific Th h.vbridoma cells (313054.8) were washed 3 × in PBS, and suspended in PBS containing 0.5% Lucifer Yellow. Approximately 5 × l0 s cells from each group were electroporated, washed 3× in PBS, and resuspended in lymphocyte culR~re medium. Approximately 105 electropor~ted and non-electroporated 3D054.8 cells were added• separately tO experimental and control wells and incubated for 24 h at 37~C. The supernatant from each well was collected and 100 /zl added to the wells of a 96.well microtlter plate containing 4 x 10 3 CTLL ¢¢1h suspended in 100/~! of lymphocyte ~ulture medium. After a further 24 h at 3"PC, the effects of supernatants on DNA replication by CTLL cells were determined by the addition of 1/~Ci of [~H]thymidine/well and measuring incorporation of [3H]thymidino into the :cells 24 h later.

Results

Effect~ of electroporation on cdlular, dye retention, viability, and function Light microsco,p~canalysis of 3D054.8 and A20 / 21 cells Trials reveal/~d fi00 V, 25 /zF to be a good compromise between cell viability and dye loading (data not shown). When electroporated under these conditions, approximately 85% of cells exhibited Lucifer Yellow fluorescence after electropotation and washing (Figs. 1A and 2B). This number:dropped to apprcaimately 50% at 30 min and 15% at 3 h after electroporation (Fig. 2B). As determined by trypan blue exclusion, viability of murine cell lines immediatdy after electroporation at 600 V 25 pF, was 90% and dropped to approximately 80% by 3 h post.electmporation (data not shown). Flow ¢ytometric analysis of human cell lines indicates that the number of viable (PI negative) cells remained constant at about 85%. For all cell lines studied, the level of

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Effect of temperature, probenecia~ sulfinpyrazone, and dinitropheaoi on dye retention Cells were loaded with Lucifer Yellow by elcetro~ration (600 V, 25 aF) and washed as usual. Cells (A20/2,1 and 313054.8) were then placed into lymphocyte culture media or into.sRPMl (all other cells) or into the respective media containing either one of the organic anion transporter [nhibitors (pr0benecid or sulfml~razone, Sigma Chemical Co.) or the antimetabolite 2,d-DNP (Sigma Chemical Co.). in some. cnltures, electroporated :cells were split and incubated ai both 37°C and 4°C. The concentrations of inhibRors and times of incubation are indicated in the role-

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~TMI~ Fig~3. Response to anlisen presentation in cleclrol~ratcd cells. Untreated (NON-EP) and electroporated (EP), Lucifer YelIow-10aded 3D0'14.8 c¢11swere co-cultured fox 24 b with A20.7,$ cells and either culture mcdium t - A G ) or culture medium plus chicken ovalbumin (+AG) and evaluatedfor Ihe . abiliPi of 3D054.8 cells to produce inter]euldn-2.in rcspomc to Spe,ciflc anligen presentation by the A20/2J cells, 11,-2 levels were determined by incubating CTLL cells with the 24 hour supematants of the above co-~Jltures,m'Kl.measuring DNA

synthesis in CTLL cells by uptake ef Iritialed thymidiae. Viability of 3D054.8LCCIIS ill ![heelectropotatndgroup seas approa:~mately80% at 3 h afterelectroporation.

262 LY fluorescence decreased rapidly over the first 2 h and reached a plateau by 3 h (Figs. 2A and 2B).

leukin-2 in response to antigen presentation by the l-Ad-positive B cell lymphoma line A21F2J. Supernatants from interacting T helper cells and A20-2,1 cells (in the presence and absence of antigen) were tested for their ability to stimula*e DNA synthesis in the IL-2-dependent cell line CTLL (as measured by uptake of [3H]tbymidine). We found that electroporated, Lucifer Yellow-

Electropomted Th cells respo~,d normally to antigen presentation by I-A d restri,'ted cells Both electroporated and untreated 3D0-54.8 cells were tested for their abifity to produce inter-

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2 s 4 s ~Me (HRS) FiiJ. 4, Inhibition of dye effiux by orlanJc amen transport inhibitors. Prubenccid (2 raM) and snlf[npyrazonc {I mM) were inclu~d in culture media of Lucifer Yellow-loaded lymphocT,es n d dye efflux was monitored by FAC5 for Jurka'~t ceils (A) or resting and activated human peripherul T lymphocytus (B). Data are shown as the percent of maximum inhibition ¢,f :iye emux as a function of time. Maximum dye effi~ is defined as the difference in linear mean value between unstained cells and oelis immediately following ¢lectroperution with I..ucifor Yellow ( T - 0), Thus, alter 2 h in culture, ceils incubated in the presence o[ 2 mM probenecid ~lai~.d approximately 52% of their odsinal dye load (or 82~ for sulfinpytazone treated cells) vs. approximately 8% in non druB-treated controls.

263 loaded 3D0-54.8 cells retained the ability to produce IL-2 in response to antigen presentation by A20-2J cells (Fig. 3).

Inhibition of dye efflux from lymphocyte cell lines Probenecid (2 raM) and su[flnpyrazone (1 raM) inhibited the loss of Lucifer Yellow fluorescence from lymphocytes at doses consistent with the inhibition of active transport of organic anions previously demonstrated in macrophages and epithelia (2, 3, 5). Data are shown as the percent of maximum inhibition of dye effiux as a function of time (Figs. 4.4 and 4B). Similar inhibition by incubation at 4°C or in the presence of 1 mM 2,4.dinitrophenol (not shown) indicates that the efflux of Lucifer Yellow is an energy-dependent process. In addition, cells incubated in cell culture media containing 0.5% Lucifer Yellow fail to accumulate cytoplasmic fluorescence (data not shown).

Discussion Organic anion transport mechanisms exist in a variety of epithelia (Blomstedt and Aronson, 1980; Specter and Goetzl, 1985; Di Virgilio etal., 1990; Gerard e t a l . , 1990; Tsuji et aL, 1990) and macrophages (Steinberg etal., 1987; Di Virgilio etal., 1988; Lipman etal., 1990), and may play a role in the concentration or excretion of physiologically significant organic anions such as uric acid, lactate, prostaglandins, and lenkotrienes. Our results indicate that lymphocytes possess probenccid-inhibitable organic anion transporters that are analogous to those previously documented in polarized epithelia and macrophages.

Organic anion dyes such as carboxyfluorescein and Lucifer Yellow have been used as reliable indicators of organic anion transport in macrophages and epithelia (Steinberg et ai., 1987; Di Virgilio et al., 1988, 1990; Gerard etal., 1990; Tsuji et al., 1990). Electroporation proved to u¢ an effective means for loading lymphocytes with Lucifer Yellow because the great majority of treated cells remained viable and retained their functional capabilities after electroporation. Dye efflux from electroporated cells was monitoped over several hours in culture by flow ofiom-

e t ~ Our results indicate that lymphocytes eliminate cytoplasmic d~e in a manner similar to that observed for polarized epithelia (Gerard etal., 1990) and macrophages (Steinberg etal., 1987; Di Virgilio etal., 1988). Dye efflux was rapid (almost all fluorescence lost within 2 h) and was shown to be an active transport phenomenon by several criteria (dye efflux was inhibited at 4~C and by dinitrophenol, dye failed to accumulate in cells cultured in the presence of 0.5% Lucifer Yellow, and dye loss in ceils corresponded to an increase of dye in the media, data not shown). It is known that routine macrophages both sequester cytoplasmic Lucifer Yellow within endusomcs and secrete the dye into the extracellu lar medium and that prohenecid, the classical organic anion transport inhibitor, inhibits these

processes (Steinberg et al., 1987; Di Virgilio et al., 1988). In our study, Lucifer Yellow efflux was inhibited by the organic anion transporter inhibitms probenecid and sulfinpyrazone and dye appeared to be retained or accumulate within cytoplasmic vacuoles of electroporated lymphocytes. Thus, our results appear to agree with previous reports documenting the presence of organic anion transporters in cell types other than po!arized epithelia. Frobe~eeid has been used in animals and humans to inhibit the hepatic and renal clearance of organic anion antibiotics (Borga e t a l . , t986; Overbosch etaL, 1988; Allen et al., I990;, Hedaya e t a l , 1990; Tsuji et al., 1990). To have a similar effect on lymphocyte transport mechanisms would necessitate the modification of neutral cornpounds which pass the membrane barrier of lymphocytes and are subsequently processed to their anionic forms in the cytoplasm, as is the case for compounds like Zidovudine (Furman etaI., 1986; Morse etal., 1990; Sawchuck et al., 1990). In summary, electroporation is an effective way to load lymphocytes with organic anion dyes as the vast majority of Lucifer Yellow-loaded ceils were both viable and functionally compe. tent. Lucifer Yellow appears to be removed from lymphocytes via energy-dependent organic anion transporters. Lucifer Yellow efflux from lymphocytes was inhibited after incubation at ¢~C, in the presence of dimitrophenol or in media containing the organic anion pump inhibitors su[fiopyrazone

Morse, G.D., Lecbuer, J.L., Santora, J.A. and Rozek, S.L. (1990) Zidovudine Update; t990 DICP, Ann. Pharmacother. 24, 754. Overbou;h. D , Gulpen, C,V., Herman& J, and Mattie, H. (1988) The effect of probeneeid on the renal tubular excretion of benzylpenicillin. 81". J, Clin, PharmacoL 25. 51. Pitls, J.D. and Kam, E, (t985) Communication compaflments in mixed cell cultures. Exp. Cell Res. 156, 439. Sawchuk, R.J. and Hedaya, M.A. (19o,0) Mm:tuling the uptake of Zidovudine (AZT) into cerebroxpinal fluid. I. Effect of Ptobencc~. Pharn~e. Res. 7, 332. Schmir~, [., Schmid, P. and Giorgi. J.V. (1988) Conversion of loaarithmic channel numher~ into relative linear fluorescence intensity. Cylometry 9, 533. Shimonkevitz, R., Kappler, $., Marraek, P. and Grey, H. (1983) Antigen recognition by H-2 restricted T cel[s. I. Cell-free antigen processing. J. Exp. Mud. 158, 303. Spcetor, R, and Goctzl, E.J. (1985) Lcukotrienc C4 transport by the chomid plexus in vitro. Science 228, 325,

Steinberg, T.H., Newman, A.S. Swanson, £A. and Silversteln, S.C. (1957a) Maerophages posse~ probenecid.tnh~table organic ion transpocler that remove f[ttmcsccnt dyes from the cytoplasmic matrix, J. Cell Biol. 105, 2695, Steinberg, T.H., Newman, A_C;.,Swat~m, J.A. and Sitversl¢in, S.C. (198"/b) ATF'4-permeabili~es.the plama menlbfaae of mou~ maerophages to fluoc¢~nl d3~es.~. Biol, Chem, 262, 8884. Stewart. W,W. (1978) Funclional conneclioas between ~ l h as reveakd by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 14, T41. Stewart, W.W. 41981) Lacifer dyes - highly fluorescent d~es for biological tracing. Nalure 292, 17. Tsuji, A., Tetasaki, T., Tamai, 1. and Takcda, K. (19gO) [n vivo evidence for carrier-mediated uptake of heta-la~am antibiotics through organic anion trampocl systems in rat kidw'y and liver. J. Pharmacol. Exp. Ther. 253, 315,