Diven, WF

Sanghvi, A

Starzl, T

Todo, S

Warty, VS

English

PubMed

Starzl, TE

Name not available

Increased cyclosporine uptake by cells pretreated with FK506 and evidence for binding of both drugs to a common intracellular protein

D-Scholarship@Pitt

Increased Cyclosporine Uptake by Cells Pretreated with FK506 and Evidence for Binding of Both Drugs to a Common Intracellular Protein A. Sanghvi. V.S. Warty. W.F. Diven. S. Todo. and T. Starzl FK506 is a newly described potent immunosuppressive agent. In vitro immunologic studies have indicated a syner-gism between FKS06 and cyclosporine. Our studies have indicated that the cellular uptake of cyclosporine is enhanced by pretreatment of peripheral blood Iymphcoytes for 2 to 22 hours with FKS06. Maximum enhancement of cyclosporin uptake was observed in the presence of cquimolar concentra-tions of both drugs. Equivalent enhancement was observed with peripheral blood lymphocytes that were freshly isolated or stimulated with phytohemagglutinin. This observation may offer an explanation for the observed immunologic synergism when both drugs arc coadministered. Also. uptake and binding characteristics of FKS06 were evaluated in human peripheral blood lymphocytes. The saturating con-centration of FK506 in these experiments was found to be approxImately 0.5 /lM. Scatchard analysis of binding data indicated two binding sites with ~ - 3.9 ± 1.8 x IQ-'M (high affinity) and K.. - 5.2 ± 0.8 x IQ-'M (low affinity). After incubations of cells with cyclosporine and FKS06. cells were disrupted by sonication and cell contents were analyzed on a Bio-Gel P-60 molecular sieve column. The elution profile indicated that both drugs migrated with an intracellu-lar protein with an approximate molecular weight of 18.000 to 19.000 dalton. These data imply that the intracellular transport of these drugs may involve similar. if not identical. proteins. Several studies have indicated that FKS06. a novel com-pound. is a potent immunosuppressive agent. Recent in vitro studies have suggested that FK506 possesses immunosup-pressIve properties similar to those of cyclosporine A. imply-ing that the mechanisms of action of these two drugs may be similar as well. The immunosuppressive effect of FK506 is several hundredfold greater than that of cyclosporine A. I •1 As previously demonstrated for cyclosporine A. the inhibitory effect of FKS06 is seen in mixed leukocyte culture (MLC) and on secondary proliferation of allorcactive T cells har-vested from MLC or propagated from organ transplant biopsies.J The published data further show that immunosup-pression by FKS06 may be mediated through an inhibition of interleukin-2 release.1- J again emphasizing the similarities between cyclosporm A and FK506. AIMS We attempted to (1) study the kinetics of binding and uptake of FK506 bv the human penpheral blood lymphocytes. (2) invcstlgate ;nd characterize the intracellular binding of both drugs. and (3) evaluate CsA uptake by human perIpheral blood lymphocytes pretreated with FK506. MATERIALS AND METHODS Tritiated CsA (Sandoz Pharmaceuticals. NY) and FKS06 (Fujisawa. Japan) were used as ethanolic solutions. Culture media and antibiotics were obtained from Glaco (Grand Island. NY). Bio-Gel P·6 and P-60 were obtained from Bic-Rad (Richmond. CAl. All other reagents and supplies came from Fisher Scientific (Pittsburgh, PAl. Peripheral blood mononuclear cells were isolated as described previously.· Monocytes were removed by means of an adhering technique. Lymphocytes were harvested. washed twice with ice-cold saline. and then resuspended in culture medium. Both the CsA and FKS06 then were added to the cell suspension. and cells were incubated for I hour at 37°C in a himidified mixture of 95% O2:5% CO2, Transport fluxes were stopped by adding 10 vol cold saline. Cells were washed twice with same cold saline to remove the excess of both drugs. The cell pellet was resuspended in ice-cold Tris buffer (20 mM. pH 7.2) containing 2-mercaptoetbanol and sodium azide. The cells were disrupted by sonic oscillations. and cell debris was removed by means of centrifugation (40.000 g for IS min). A l-mL supernatant sample was centrifuged on a mini Bio-Gel P-6 column to separate protein-ligand complex from the free ligand that is completely retained in the column. The intracellular protein-CsA and FKS06 complex then was chromatographed on a Bio-Gel P-60 column (pre· calibrated with the appropriate molecular weight protein markers). and the fractions were used to determine CsA and FKS06 for protein contents. Lymphocytes were incubated with different concentra-tions of FKS06 for I hour at noc. Cells were washed three times with cold saline and incubated in 0.2 mL in KOH for I hour at 70°C. The digested solution was used for the measurement of FK506 by means of enzyme immunoassay and protein concentration. In another set of experiments. cells were incubated in 1 % BSA-RPMI 1640 medium in the presence of I /lg/mL FKS06 under culture conditions. Cells were washed and exposed to different concentrations of CsA and FKS06 for 2 From the Department. of Pathology (Sanghvi. Warty. and Divenl and Surgery (Todo and Slarzll. UniverSity of Plttsburgn School of Medicine. Pittsburgn. Pennsylvania. Addre •• repnnt reque ... to A. Sanghvi. PhD. Director Clinical Cheml.lry·CLSI CHP Main Tower. Aoom 6846. 3705 Fifth Avenue al DeSoto Street. Pittaburoh. PA 15213·2583. USA. ~ 1989 by Appleton & Lanoe. Inc. 0041·1346/89/$3.001 +0 1050 rra"."Mnt.tiOll Proce.alfttla, Vol 21. No 1 (Februaryl. 1988: pp 106()o1042 INCREASED CYCLOSPORINE UPTAKE AND FK506 hours. Uptake of CsA by the cells under these conditions was evaluated by a measure of radioactivity. RESULTS The concentration dependence of intracellular accumulation and binding of FKS06 by human peripheral blood lympho-cytes were studied. Cells were incubated in different concen-trations of FK506 for 1 hour at 37°C. The uptake of FK506 by PBl was seen as a saturable process. with saturation occurring at approximately 0.5 ~M concentration of the drug (Fig I). Scatchard analysis of the binding data was consis-tent with two distinct classes of binding sites. one with I<.! -3.9 ± 1.8 x 1O-'M for the high-affinity sites and the second with I<.! - 5.2 ± 0.8 x 1O-6M for the low-affinity sites. This analysis further revealed that there arc 5.6 :t 1.0 x 10· and 2.5 ± 0.9 x 106 high- and low-affinity binding sites. respec-tively. per cell. In another set of experiments. peripheral blood lympho-cytes (100 x 106 ) were incubated with 2 ~g FK506 and 1.5 Ilg CsA in culture medium for 1 hour. Intracellular contents were analyzed for binding of both drugs to macromolecules. Intracellular accumulation of both drugs was rapid. and both appeared specifically associated with intracellular macro-molecule(s). Figure 2 illustrates the elution profile of both cyclosporin and FK506 on a calibrated Bio Gel P-60 column. The calibration curve of the molecular-weight markers-ovalbumin. carbonic anhydrase. cytochrome C. and vitamin en ..J ..J W u <D 0 .... --<D , 0 .... x en w ..J 0 :E c z ::::I 0 m <D 0 L.O :::.:: u. 0.5 04 0.3 0.2 0.1 _o-0 / 0 j 0.12 0.24 Q36 0.48 0.6 Q9 FK S06,MOLES X 1O-6/L MEDIUM FIG 1. 1051 100 M 0 0 50 -0 25 e 200 :: ... Il. ::: 0 " 20 W 150 ci ~ 10 .., " ;0: ~ « '" ..J 5 1t ;;, 0 15 u 100 .<31 0 w Il. ..J " 0 U1 :I <0 0 oJ U 50 >- 2 3 4 U ~ VelV o S 30 40 50 60 FRACTION FIG 2. Bl2_is shown as an inset. These data show that both cyclosporine A (lI.) and FK506 (e) elute association with protein or proteins with a molecular weight of about 18.000-19.000 dalton. Figure 3 shows the effects of including equivalent amounts of either FK or cold CsA (1: I. wt/wt) together with [lHJ CsA at each point in the incubation medium on the cellular uptake of [lHJ CsA (panel A). For panels Band C. the experimental conditions were identical to those in panel A except that cells were preincubated for 24 hours with 10 Ilg FK/ml (panel C). Subsequently. the cells were washed and incubated with [lH] CsA as described above. Under all three conditions. the cellular uptake of [JHJ CsA was higher at each concentration of eH] CsA in the incubation in the presence of equivalent amounts of FK than it was in its absence. When the cells were preincubated with 10 Ilg CsA. the absolute uptake of [JHJ CsA was reduced in all cases relative to panels A and C. suggesting that some of the cellular CsA binding sites are taken up by unlabeled CsA. However. even under these conditions. inclusion of FK in the medium was able to induce a higher [lHJ CsA uptake (Fig 3. panels A. B. and C) compared with unstimulated cells. Conditions for these experiments were identical to those for the experiments in Fig 3. except that preincubations with 10 Ilg CsA and I Ilg FK/ml of medium were for 72 hours. '0' I'; ,Q • 0 : 0 "') '0 CtCi~DDDDDK1 c,..-- ..... , ~ .... , l .......... -.. ................. _ ........................... _. ~ ....... ' ....... __ a ......... ......... __ flMtCM __ _. __ .... _ • __ ........... __ ca.......a. ~ ___ ...... CIA. __ ......... _ .. _c.A. FIG 3. -----------,052 Again. the uptake of IJH) CsA is enhanced when the Incuballons contain amounts of FK equivalent to the amounts of IlH) CsA at each point. DISCUSSION Cyclosporlne and FKS06 share a remarkable array of prop-ertIes despite the obvious differences in their molecular structures. Although the immunosuppressive effects of FKS06 are several hundredfold greater than those of cyclo-sporine A. the mechanism of immunosuppression of the two drugs is the same. i.e .• blockage of interleukin release. This report delineates further similarities between the two drugs. Both drugs exhibit saturation kinetics of binding to human peripheral blood lymphocytes. and both apparently bind to the same intracellular protein. I r it is indeed the same protein that both the drugs are bound to. the question of whether separate binding sites are involved in this process must be investigated. Our results also demonstrate that human peripheral blood lymphocytes exposed to FKS06 are able to take up more cyclosporine relative to cells that are not similarly exposed. Their sensitivity to cvclosporme in terms of MLC and primed lymphocyte tests is also increased. This evidence suggests that binding of one drug. FKS06. appears SANGHVI. WARTY. DIVEN ET AL to modify the cellular response to another drug. cyclosporine. resulting in a greater uptake of cyclosporine by the cell. CONCLUSIONS The binding and kinetics of uptake of FK506 by human peripheral blood lymphocyte exhibit a saturable process. with saturation occurring at a concentration of approxi. mately O.S I'M of the drug. Skatchard analysis of binding data (Fig I) indicates two distinct classes of binding sites for FKS06: (I) Kd - 3.9 := I.S x 1O-'M. high affinity. and (2) Kd - 5.2 :!: 0.8 x 10-6M. low affinity. Intracellularly. both drugs appear to bind to a common protein with an apparent molecular weight of IS.OOO to 19.000 dalton. REFERENCES I. Ochiai T. Nakajima K. Nagata M. Suzuki T. Asano T, Uematsu T. et al: Transplant Proc 19: 1284. 1987 2. Zccvi A. Duquesnoy R. Eiras G. Todo S. Makowka L. Starzl T: Surg Res Commun 1:315. 1987 3. Zeevi A. Duquesnoy R. Eiras G. Rabinowlch H. Todo S. Makowka L. Starz1 TE: Transplant Proc 19:40. 1987 4. Sanghvi A. Warty V: Biochem J 227:397. 1985 

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Increased cyclosporine uptake by cells pretreated with FK506 and evidence for binding of both drugs to a common intracellular protein

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