Diven, W

Duquesnoy, R

Makowka, L

Sanghvi, A

Starzl, TE

Warty, V

Zeevi, A

PubMed

Name not available

FK-506 enhances cyclosporine uptake by peripheral blood lymphocytes

D-Scholarship@Pitt

( ( ( t 110r FK-S06 Enhances Cyclosporine Uptake by Peripheral Blood Lymphocytes A. Sanghvi, V. Warty, A. Zeevi, W. Diven, R. Duquesnoy, L. Makowka, and T.E. Starzl T HE PROCESS of cellular uptake and metabolism of cyclosporine (CsA) is poorly understood. Previously, we have shown that the transport of CsA in blood occurs in association with plasma Iipoproteins,l with low-density lipoprotein (LOL), and with high-density lipoprotein, each transporting about 32% of the total amount of CsA in blood. This observation raises the possibility that further cellular metabolism of CsA, at least in part, may take place in conjunction with LOL metabolism through its specific, high-affinity cell surface receptor. In this report, we present results of studies that indicate that (1) [lH]_ labeled CsA is taken up, in an apparently specific manner, by peripheral blood lympho-cytes (PBL) when incubated with the [lH] CsA-LOL complex; and (2) cellular uptake of [lH] CsA is enhanced when PBL, whether freshly isolated or stimulated with phyto-hemagglutinin (PHA), are incubated in a medium containing both [lH] CsA and FK-506 (FK) in a 1:1 (wt/wt) proportion. MATERiAlS AND METHODS Tritiated dihydrocyclosporine A (60 mCi/mmoI) and CsA standards were obtained from Sandoz, Ltd (Basel, Switzerland) as ethanolic solutions. FK was a gift from Fujisawa Pharmaceutical Co, Ltd (Osaka, Japan). Methanolic solution of FK was used in the experiments. Culture media and antibiotics were obtained from GlBCO (Grand Island, NY). All other reagents and supplies were from Fisher Scientific Co, Pittsburgh. Lipoproteins Human LOL (d, 1.019 to 1.063) was prepared from blood collected in 0.1 % EOT A from healthy subjects who had fasted overnight. Lipoproteins were fractionated by sequential ftoatation in a Beckman preparative ultracen-trifuge (Beckman Instruments, Inc, Fullerton, CAl according to Havel et aJ.2 Protein determinations were performed by the Bradford method.' Cells Peripheral blood mononuclear cells were isolated as previously described· from approllimately 100 mL of blood obtained from healthy volunteers for each experi-ment. The monocyte content of these cells was depleted by the method of Jerrells et al. S Cell viability was assessed by using the dye ellclusion method.' PHA-Induced Activation of PBL Lymphocyte cultures were set up in 24-well cluster plates (5 x 10' cells/well) in 1 mL of tissue culture medium supplemented with 10% human serum, 20% interleukin-2, and PHA (0.0125 I'g/mL). After 96 hours, the cells were harvested and washed twice and the cell concentration determined. Alloreactive T cell clones were generated from mixed lymphocyte cultures. The uptake of CsA by these cells in various experiments was determined as described in the following sections. Lipoprotein-CsA Complex LOL (100 I'g in I mL RPMI 1640 medium) was added dropwise with stirring to an ethanolic ['H) CsA solution (SO I'L). The mixture was incubated with constant shak-ing for one hour at room temperature. The specific activity of the [3H] CsA-LOL oomplex thus obtained in three separate preparations was 20 ± 0.8 x ut dpm/I'g LOL. The purity of LOL was assessed by sodium dodecyl sulfate gel electrophoresis. CsA Uptake by PBL Lymphocytes were incubated in RPM I 1640 medium containing 1 % bovine serum albumin (BSA) in the pres-ence or absence of C,A and/or FK for 24 hours. After these preincubations, the cells were washed twice with ice-cold 0.85% NaCI solution and then resuspended in 1 % From th~ D~pa"m~nts of Pathology and Surg~ry, Univ~rlity of Pitl$burgh School of M~diciM. Support~d in part by Grantl HL-36416 and AI-13467 from th~ Nationallnstitut~1 of H~alth. B~th~sda. MD. Adduls r~print r~qu~sts to A. Sanghvi, PhD, ~pa"­m~nt of Pathology. Univerlity of Pitl$burgh School of M~diciM. 776 Scaif~ Hall. Pittsburgh, PA 15161. C 1987 by GruM & Stratton. Inc. 0041-1345/87/1905-6009$03.00/0 r,.".",."r.tion ~, V04 XIX. No 5, Suppl6 (October'. 1987: pp 46-49 46 t 1 ~ i-t t 46 BSA·RPMI m~-dium. The cells were placed in culture wells (approximately 10' cells/well) and exposed to increasing molar concentrations of ['HJ CsA (specific activity. 900 to 1.100 dpm/pmol) and equivalent concen· trations of cold CsA and FK. as indicated in the legends to the figures. for one hour at 37°C in the humidified atmosphere of 95% O 2:5% COl' Transport flux was stopped by adding ice-cold saline and cooling the mixture further for ten minutes over ice. The cells were washed three times with cold saline and incubated in 0.2 mL i N KOH for one hour at 70°C. The digested solution was used for the measurement of radioactivity and protein determinations. The cell content of FK was measured by an enzyme immunoassay method obtained from Fujisawa. In experiments when the [lH] CsA-LOL complex was used, the results are expressed as picomoies of CsA accumulated per milligram cell protein as a function of micrograms of LOL protein and the corresponding amount of ['H] CsA contained in the ['HJ CsA-LOL complex. Nonspecific (receptor-independent) binding of LOL to the cells in these experiments was determined by incubating the cells in the presence of IOO-fold excess of cold LOL as well as cold CsA. RESULTS CsA uptake by lymphocytes from two healthy donors was studied by incubating cells with the [3H] CsA-LDL complex. The kinet-ics of the uptake of CsA in these experiments are shown in Fig 1, panels A and B. In both individuals studied, the CsA uptake appears to reach saturation at an LDL concentration of about 50 J.LgjmL medium. The correspond-ing CsA concentration is about 900 ng/mL. In a previous study4 on the cell surface binding and subsequent metabolism of LDL by T and B lymphocytes, we have observed that the 200 i I r :100 I ; u A B 100 so SANGHVI ET AI.. LDL receptor is saturated at 50 J.Lg LDLjmL medium. Moreover, eHJ CsA uptake by lym-phocytes was suppressed in the presence of cold CsA and LDL. Combined, these observa-tions suggest that internalization of lipopro-tein-associated CsA may occur via the high-affinity specific receptor for LDL on the cell surface. The experimental conditions used in these studies imply that the CsA measure-ments shown reflect both the processes of cell surface binding and internalization. Cell u la r CsA uptake in the a bsence of LD L was studied in lymphocytes from two healthy volunteers (other than for Figs 1 and 2). The results of the experiments shown in Fig 2, panels A and B, indicate that; as in the presence of LDL, [lH] CsA uptake in this instance may also involve a saturable process. Because a specific cell surface receptor for CsA has not been identified to date whereas characteristics of a. specific cytosolic pro-tein~ycJophilin7-that binds CsA have been described, the data in Figs 3 and 4 may be most compatible with the binding of internal-ized CsA to cycJophilin or similar protein(s).8 When a 100-fold excess of cold CsA was included in these incubations. the cellular uptake of labeled CsA declined significantly, which suggested a competitive process. The same was true when a lOO-fold excess of FK (with respect to the [3H] CsA concentration) was present in the incubations_ These data would imply that both FK and CsA may be competing for binding to the same cytosolic S 10 IS so 1S 100 S 10 2S so 7S 100 Fig 1_ Cellular uptake of CsA in PSl incubated with the ['HI CsA-lDl complex for one hour. The open end closed symbols represent the tou .. .00 nonspeeific IlDl receptor-Independent) upuke of ['HI CsA. respectively. P,nels A.oo 8 .... resutts in two subJect8. ~ LDL I'l101'£11,...,.. 3.1'2 1.44 5.' 1.2 10.' 14.4 I Llk. PIlOTE ...... ' .... ~.12 144 U 1.2 to.' t4.4 C'ICl.OPOMC ~ .. SANGHVI ET AL ated at 50llg LDL/mL H] CsA uptake by Iym-sed in the presence of 1mbined, these observa-.:rnalization of Iipopro-lay occur via the high-or for LDL on the cell ntal conditions used in lat the CsA measure-th the processes of cell .ernalization. in the absence of LDL ;ytes from two healthy for Figs I and 2). The ents shown in Fig 2, icate that, as in the I CsA uptake in this Ie a saturable process. surface receptor for ,ified to date whereas 1ecific cytosolic pro-t binds CsA have been :;"igs 3 and 4 may be e binding of internal-)r similar protein(s).8 ,s of cold CsA was :>a tions, the cellular .ecHned significantly, )etitive process. The aO-fold excess of FK CsA concentration) bat ions. These data 'K and CsA may be ) the same cytosolic 1 . Cellular uptake of Pal incubated with the \-lDl complex for one rhe open and doaed repreaent the tOlllI specific (LDl receptor-dent) uptake of rtf) pective/y. Pan ... A end lutta in two aubjecta . . -...... '-~ ... .....--..:~:,., ... ".- '......-, I I ROLE OF FK IN CYClOSPORINE UPTAKE BY PBL 47 A B Fig 2. Cellular uptake of CsA in PBl incubated with ['HI CsA (0--<)' and in the pres-ence of a tOO-fold excesa of FK (t;-t;) and unlabeled CsA ( ... - ... ,. Panels A and Bare results in two subjects. 0,5 1,0 ZO ~L OSPOMCE ",10-' 1111 0.5 1'0 Z,(, '1.0 CYCLOSPORINE (,.,0 .... ' components, though not necessarily the same '!Sites. In Fig 3 are shown the effects of including equivalent amounts of either FK or cold CsA (1:1, wt/wt) together with [3Hl CsA at each point in the incubation medium on the cellular uptake of [3H] CsA (panel A). For panels B and C, the experimental conditions were iden-tical to those in panel A except that cells were preincubated for 24 hours with either 10 Ilg CsA/mL (panel B) or with 1 Ilg FK/mL (panel C). Subsequently. the cells were washed and incubated with [lH] CsA as described in Materials and Methods. Under all three conditions, the cellular uptake of [3H] CsA was higher at each concentration of [3H] CsA in the incubation in the presence of equivalent amounts of FK than in its absence. When the cells are preincubated with 10 Ilg CsA, the absolute uptake of [3H] CsA is reduced in all cases relative to panels A and C, thereby suggesting that some of the cellular A B '00 CsA binding sites are taken up by unlabeled CsA. However, even under these conditions, inclusion of FK in the medium is able to induce a higher [lH] CsA uptake. Preincuba-tion of cells in the presence of 1 Ilg FK does not seem to significantly stimulate further the increased CsA uptake (panel C). PHA-stimulated lymphocytes exhibit a generally increased [3H] CsA uptake (Fig 4, panels A, B, and C), compared with unstimu-lated cells. Conditions for these experiments were identical to those for experiments in Fig 3 except that preincubations with 10 Ilg CsA and 1 I'g FK/mL of medium were for 72 hours. Again, the uptake of PH] CsA is enhanced when the incubations contain amounts of FK equivalent to the amounts of [3H] CsA at each point. . DISCUSSION Several results presented here support the following observations: (I) the drug FK, when c 1.0 1.0 4.0 ~t"I~1 Fig 3. lymphocytel were preincubated in ", BSA in RPMI (panel A) and 1% BSA in RPMI containing 101'9 CIA/ml (panel B, or 1 lig FK/ml (panel C) for 24 hours. The cellula. uptake of rtf! CsA was then measured in the absence (0--<)) and presence of equivalent amounts of FK (t;-t;) and cold CIA ( ... - ... , • .-. indicates displacement of rtf) CaA in the preaence of a 100-fold exce .. of cold CaA. 48 SANGHVI ET Al A B c ". 10 10 to 1.0 4.0 1.0 10 4.0 ~1I·1O"U1 C"«l..DSJIIQIIIM:lr."""'l ,1'Q.QSI'I'OMC (IUO".' Fig 4. Lymphocytes were preincubeted in RPMI medium containing PHA (panel AI. and 10 I'g CsA I mL (panel BI and 1 I'g FKI ml (panel CI for 72 hours. The cellular uptake of rHI CsA was then measured in the absence (~I and presence of equivalent amounts of FK (fl.-fl.1 and cold CsA (.t.-.). present in incubations of lymphocytes with [JH] CsA in equivalent concentrations, enhances the cellular uptake of CsA; and (2) the cellular uptake of CsA appears to involve both specific as well as nonspecific processes, the former taking place when the drug is associated with lipoprotein. In this instance, the CsA-LDL complex may be interiorized via the high-affinity, specific cell surface receptor for LDL. Support for this concept derives partly from the calculations of disso-ciation constants for various experiments reported here. The double reciprocal plots of data9 from these experiments yield two quite distinct apparent dissociation constants (KD). For the cellular uptake of CsA complexed with LDL, KD - 2.3 and 0.6 x 10-9 mol/L (Fig 1, panels A and B, respectively). For the data in all of the rest of the experiments, KD -2 X 10-7 mol/L (Table 1). The Ko of 10-9 mol/L for CsA when in association with LDL certainly suggests high-affinity binding. The Table 1. Binding of C.A to lymphocytes Experiment A. ['H) CsA-LOL + PBl (Fig 1) B. rH) CsA + PBl (Figs 2 and 3) + FK (Fig 3) c. eH) CsA + PHA-1Itimulated PBL + FK (Fig 4) K., (moIIL) 2.3 x 10-1 0.6 x 10-" 2.0 :!: 0.5 X 10-7 1.9:!: 0.3 x 10-7 2.0 :!: 0.3 X 10-7 2.1 ± 0.1 x 10-7 For experiments B and C, the values expressed are means ± SO; n - 5 and 3, respectively. rate-limiting step in the LDL pathway is the binding of LDL to a specific high-affinity receptor on the cell surface. 'o It may be expected, therefore, that this would be the initial step in the metabolism ofCsA bound to LDL. The significantly reduced cellular con-tent of [JH] CsA in the presence of cold LDL appears to strengthen this view. Whether such a binding of CsA-LDL to the LDL receptor followed by further metabolism along the LDL pathway occurs cannot be inferred from the experiments reported here. If true, how-ever. it could represent a significant pathway for CsA metabolism because some 32% of the drug in blood is bound to plasma LDL. ' The concentrations of CsA required to saturate the Table 2. Effect of Preincubation of PBl With FK on the Cellular Content of the Drug FK (ngt 1(" Cells) Subject Controj' Experimental· B. 0.05 0.07 b. 0.12 0.25 2 a. 0.19 0.38 b. 0.20 0.79 3 a. 0.49 0.69 b. 1.58 Not measured PSl were preincubated for 24 hours in 1 % BSA with and without (control) 1 I'g FK/ml medium. • After preincubation and washings, control and experi-mental cells were incubated with 1 and 4 x 10-7 mol/L, a and b respectively, concentrations of FK for one hour. The FK content in the cells was meas ... ed by enzym ...... inked immu-nosorbent assay. c SANGHVI ET AL 1.0 4.0 .. {IaK)"", and 10 IoIg CsA/mL (panel BI ured in the absence (Q--{)I le LOL pathway is the I specific high-affinity surface. lo It may be lat this would be the JOlism of esA bound to . reduced cellular con-presence of cold LOL lis view. Whether such , to the LOL receptor letabolism along the IOnot be inferred from :d here. If true, how-a significant pathway :ause some 32% of the to plasma LOL.l The :quired to saturate the ion of PBL With FK on the t of tha Drug ; lng/10' <:.Ilsl Experimental' 0.07 0.25 0.38 0.79 0.69 Not measured hours in 1 'J6 BSA with and Jium. hings. control and experi-I and 4 x 10-' mol/l. a of FK for one hot.. The FK j by anzyme-/inked immu-.• ,.--, ... -~-. -,." .... ,- .. ~, .,--,...'- ~·-.-..,..~v ROLE OF FK IN CYClOSPORINE UPTAKE BY PBL cellular mechanism(s) responsible for esA uptake are also different in the presence and absence of LDL, 900 ng/mL with LDL and about 300 ng/mL without LDL. This infor-mation further segregates the two processes. The Ko of 2 x 10-7 mol/L when eH] esA is incubated with lymphocytes in the absence of LDL is strikingly similar to the reported Ko's for esA binding to cyclophilin,7 calmo-dulin," and perhaps other cytosolic proteins. 8 This similarity implies that what is being measured in these experiments (ie, in the 49 absence of LDL) is the binding of [JH] esA to cytosolic proteins after internalization. Although the uptake of eH J esA is increased in the presence of FK, Ko remains unchanged (Table I). How FK promotes esA uptake by lymphocytes is not presently known. In preliminary experiments, we have observed that cells incubated with FK under various conditions accumulate the drug intra-cellularly (Table 2). The kinetics of the pro-cess of cellular uptake of FK are currently being investigated. REFERENCES I. Gurecki J, Warty V, Sanghvi A: Transplant Proc 17: 1997, 1985 2. Havel RJ, Eder HA, Bragdon JH: J Clin Invest 63:1345,1955 3. Bradford M: Anal Biochem 72:248, 1976 4. Sanghvi A, Warty V: Biochern J 227:397.1985 5. Jerrells TR, Dean JH, Richardson GL, et al: J Immunol Methods 32:11,1980 6. Kaplan ME, Clark C: J Imrnunol Methods 5:131, 1968 7. Handschumacher RE, Harding MW. Rice J, el al: Science 226:544, 1984 8. Fabre I, Fabre G, Lena N, et al: Biochem Pharma-col 35:4261, 1986 9. Gal 0, Simpson DR, Porter CS, et al: J Cell Bioi 92:597. 1982 10. Goldstein JL, Brown MS: J Bioi Chern 249:5153, 1974 11. Colombani PM, Robb A, Hess AD: Science 228:337, 1985 " " I • I I I 

FK-506 enhances cyclosporine uptake by peripheral blood lymphocytes.

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FK-506 enhances cyclosporine uptake by peripheral blood lymphocytes.

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