Agents and Actions vol. 9, 5/6 (1979) Birkh/iuser Verlag, Basel
549
Inhibition of Purine Nucleoside Phosphorylase Activity and of T-Cell Function with Allopurinol-Riboside by Y. NISHIDA, N. KAMATANI, K. TANIMOTO and I. AKAOKA Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan 113
Abstraet Allopurinol-riboside competitively inh~its the action of purlne nueleoslde phosphorylase on inosine in vitro with a Ki of 277 /Jmol. After simple incubation of allopurinolriboside with PNP, allopurinol was not formed. Lymphocyte blastogenesis induced by PHA and Con A was significantly suppressed by aUopurinol-riboslde in a concentration-dependent manner. When LPS was used as a mitogen, the inhibition of allopurinol-riboslde on lymphocyte proliferation was less marked. Humoral immunity was not suppressed by allopurinol-riboside. In contrast, cellular immunity was significantly suppressed by aUopurinolrlboside in vivo. These results suggested that allopurinolriboside is a drug which produces a model of PNP deficiency, and that it may be a useful inhibitor of cellular immunity.
Introduction Recently, some children with severe combined immunodeficiency have been reported to have adenosine deaminase (ADA) [1-4] or purine nucleoside phosphorylase (PNP) deficiency [5]. In ADA-deficient children, both Tcell and B-cell mediated immunities were impaired. The children with PNP deficiency have defective T-cell immunity but intact B-cell immunity. Coformycin [6] and erythro-9-(2-hydroxy-3nonyl) adenine hydrochloride [7] inhibit ADA activity, and have been used in in vitro studies to make an experimental model of ADA deficiency. In the present report the effects of allopurinol-riboside were studied on PNP activity in vitro and on lymphocyte proliferation induced by mitogens. The effects of allopurinol-riboside on humoral and cellular immunity to sheep red blood cells (SRBC) were also studied in mice. The results showed that allopurinol-riboside was useful as an inhibitor of PNP and as a T-cell inhibitor.
Materials and methods Enzyme assay PNP activity was determined according to the spectrophotometric method of KALCKAR [8]. The sample cuvette (final volume 3 ml) contained from ~0 to ] m M inosine as a substrate in 0.05 M phosphate buffer, pH 7.5, with or without 1.5 mM allopurinol-riboside. After addition of 20/tl of xanthine oxidase (8 p/ml: P-L Biochemicals Inc.) and 0.025 /~ of PNP (25 ~t/ml: Boehringer Mannheim) to the reaction mixture, the increase in optical density at 293 nm was recorded. Xanthine oxidase activity was not inhibited by allopurinol-riboside. Allopurinol-riboside was donated by Kyowa Hakko Corp. Ltd. Conversion of allopurlnolMboside 10 mg of aUopurinol-riboside dissolved in 1.0 ml, 0.05 M, phosphate buffer was incubated with 50/A of PNP (25 p/ml Boehringer Mannheim) for 30 min at 37~ The reaction mixture was spotted on Eastman (Kodak) thin-layer chromatography plates and developed with butanol:methanol :water :25% NH4OH (60:20:20:1 v/v). The purines were located on the chromatogram under ultraviolet light. The reaction mixture was also applied to a high-pressure liquid chromatogram (Hitachi 633) using Hitachi castam gel 2618 and eluted with 0.4 N-ammonium acetate, pH 4.0, at a pressure of 100 kg/cm 2.
Cell cultures 30 ml of venous blood was collected from a healthy subject in a heparinized syringe. The lymphocytes were separated by discontinuous centrifugation in Ficoll-hypaque solution as previously described [9] and washed three times in large volumes of TC199 solution (Chiba-ken, Serum Laboratory). The lymphocytes were rewashed with medium RPMI-1640 (Grand Island Biological Company) containing 10% foetal calf serum, 200 p/ml penicillin G and 100 pg/ml streptomycin, and resuspended at 1 x 106 cells/ml in the medium. Cultures were carried out in sterile microplates with round-bottom wells and were done in triplicate. I x 10~ of responding cells in 100 ~1 of culture medium which contained the various concentrations of sterilized allopurinol-riboside were put into each well. 100 ~1 of mitogens dissolved in culture medium of equal volume of medium as a
5 50
Inhibition of Purine Nucleoside Phosphorylase Activity and of T-Cell Function with Allopurinol-Riboside
control were added to the culture tubes on the first day of the culture. Their final concentrations were as follows: 4 pg/ml of phytohemagglutinin (PHA: Difco Laboratories), 50 pg/ml of concanavalin A (Con A: Pharmacia Fine Chemicals), 1 : 100 diluted pokeweed mitogen (PWM: Grand Island Biological Company), and 1000 pg/ml lipopolysaccharide B (LPS: Difco Laboratories). They were incubated for 5 days at 37~ in a humidified atmosphere with 5% CO 2 and 95% air. 1/tCi of H3-thymidine (42 Ci/mmoh Radiochemieal Centre) was added to each well for a further 24 h incubation. Cells were harvested on glass-fiber filter paper using a Millipore semi-automated harvester. Radioactivities of thymidine incorporated into lymphocyte D N A were measured in a liquid-scintillation counter and expressed as counts per 10 min.
I 3~ x[ ~ tgex
OOO000
= ~ 961
9
~ 481
O0
9
O0000
9
241
O0
tz ! 61
Humoral and cellular immunity Mice were used at 4 to 6 weeks of age. SRBC were obtained commercially from Nippon Bio-test Laboratories and were stored at 4~ The SRBC were washed three times with sterile physiological saline before use, and finally suspended at the desired concentration in saline. Humoral immunity was measured as the hemagglutinin titers. 1 x l0 s washed SRBC in a volume of 500 ~tl was injected intraperitoneally into 20 mice. 10 mice were injected intramuscularly with allopurinol-riboside in a dose of 1 mg/g body wt./day for 7 successive days. Seven days after injection, blood was collected by heart puncture and serum was separated. Hemagglutinin titers were measured in microtitration trays using 0.1 ml of serum diluted serially in getatin-veronal buffer to which an equal volume of 1% SRBC was added. The trays stood overnight at 4~ Cellular immunity was studied according to the method of LAGRANGE et al. [10]. 5 • 105 SRBC was injected intraperitoneally into 20 mice. Allopurinol-riboside (1 mg/g body wt./day) was given to the 10 mice for 5 successive days. Five days after injection, 1 • 108 SRBC in a volume of 50/21 was injected into the left foot pad of the mice. Twentyfour hours after this treatment, the increase of left foot pad weight was measured.
Results Allopurinol-riboside competitively suppressed PNP action on inosine. 50% inhibition was achieved at 277 ~mol of allopurinol-riboside.
Control
AIIoourinol-
riboside
Figure 1 Hemagglutinin titers to SRBC in mice treated with allopurinol-riboside and controls. m9 z0.0
.~ 15.0
I~
~_ ~ t0.0
5.0 Qtl
control
ABopurinolriboside
Figure 2 Delayed-type hypersensitivity to SRBC in mice treated with allopurinol-riboside and controls.
Table 1 Effect of allopurinol-riboside on the incorporation of 3H:thymidine into mitogen-stimulated lymphocytes. Allopurinolriboside
Mitogen
(mM)
Unstimulated
PHA
Con A
0 2.5 5 10
9 4,305 4,393 6,815
315,162_+ 13,355 141,679 -+ 20,122 67,533 _+ 6,901 15,849 + 1,503
32,213 15,410 10,010 7,499
_+ 386 -+ 948 _+ 894 + 720
PWM _+ 1,061 -+ 963 _+ 2,101 _+ 422
Results expressed in net counts per 10 min and per 1 x 105 cells. (Mean + SD.)
36,204 42,743 33,208 11,869
LPS + 958 _+ 2,515 + 7,103 _+ 3,470
25,181 23,873 19,104 16,283
+ 8,460 + 3,598 • 3,623 _+ 1,170
Inhibition of Purine Nucleoside Phosphorylase Activity and of T-Cell Function with Allopurinol-Riboside
After simple incubation of allopurinolriboside with PNP in vitro, allopurinol could not be detected using high-pressure liquid chromatography and/or Eastman Kodak thin-layer chromatography. The results of (AH) thymidine incorporation in lymphocytes stimulated with various mitogens are shown in Table 1. The mitogen-induced proliferation of human lymphocytes was in general suppressed by allopurinol-riboside. The inhibitory effect was dose-dependent. Blastogenesis induced by PHA and Con A was markedly inhibited by allopurinol-riboside, while its inhibition of that induced by PWM and LPS was less marked. Hemagglutinin titers are shown in Figure 1. There was no significant difference in the hemagglutinin titer between the control mice group and the group treated with aUopurinolriboside. In contrast, the increase of foot pad weight in the mice treated with allopurinol-riboside was significantly lower, 4.3 _+ 1.4 mg, than the average increase of 17,9 + 3.3 mg in the controls (Fig. 2). Discussion Children with PNP deficiency have been shown to have defective T-cell immunity but intact B-cell immunity, but the causal relationship between depressed immunity and these enzyme deficiencies has not yet completely been elucidated. The role of purine metabolizing enzymes such as ADA and PNP in the immune response is of particular interest. In children with ADA deficiency, both T-cell and B-cell functions are impaired. The deficiency of ADA may lead to the intracellular accumulation of adenosine and/or D-adenosine. The increased concentration of adenosine and/or Dadenosine might be related to the pathogenesis of the immune dysfunction on this disease as excess adenosine in lymphocyte culture is toxic [2, 11]. Coformycin, a competitive inhibitor of ADA, has been demonstrated to suppress lymphocyte blastogenesis [6], and this agent has been used in vitro to produce a condition similar to ADA deficiency. PNP converts inosine and guanosine to their respective free purine bases. Deficiency of PNP may lead to the accumulation of intracellular inosine, guanosine and D-guanosine. High concentration of guanosine is also known to suppress
55 1
lymphocyte proliferation [13]. Indeed, elevated levels of inosine have been confirmed in the plasma of children with PNP deficiency [14]. The reason for the normal B-cell function in these patients remains unclear. Allopurinol-riboside significantly suppressed PNP activity in vitro. This suppressing effect may be due to binding to the active site of PNP by allopurinol-riboside in a manner which inhibits its activity and results in competitive suppression. These results indicate the usefulness of alMpurinol-riboside as an inhibitor of PNP. PHA and Con A are known to stimulate T-cells preferentially. Allopurinol-riboside produced dramatic suppression in lymphocyte blastogenesis stimulated by PHA and Con A. On the contrary, lymphocyte proliferation caused by a selective Bcell stimulant, LPS, was not inhibited by allopurinol-riboside to the same degree as the PHA and Con A responses. There results suggested that allopurinol-riboside mainly suppresses the Tcell function. Humoral immunity was not suppressed by allopurinol-riboside but cellular immunity was significantly suppressed by allopurinol-riboside in an in vivo experiment. Allopurinol-riboside in an adequate dose may make an experimental model of PNP deficiency and might be a useful and powerful inhibitor for cellular immunity. Received 9 May 1979.
References [1] E.R. GIBLETT, J.E. ANDERSON, F. COHEN, B. POLLARA and H.J. MEUWISSEN, Adenosine-Deaminase Deficiency in Two Patients with Severely Impaired Cellularlmmunity, Lancet 2, 1067-1069 (1972). [2] C.R. SCOTT, S-H. CHEN and E.R. GmLETr, Detection of the Carrier State in Combined lmmunodefieiency Disease Associated with Adenosine Deaminase Deficiency, J. Clin. Invest. 53, 1194-1196 (1974). [3] J. YOUNT, P. NICHOLS, J.D. OCHS, S.P. HAMMER, C.R. SCOTT, S-H. CHEN, E.R. GmLETr and R.J. WEDGEWOOD, Absence of Erythrocyte Adenosine Deaminase Associated with Severe Combined Immunodefieiency, J. Pediatr. 84, 173-177 (1974). [4] R. PARKMAN, E.W. GELFAND, F.S. ROSEN, A. SANDERSON and R. HIRSCHHORN,Severe Combined Immunodeficiency and Adenosine Deaminase Deficiency, N. Engl. J. Ned. 292, 714-719 (1975). [5] E.R. GIBLETF, A.V. AMMANN, D.W. WARA, R. SANDMAN and LK. DIAMOND, Nucleoside-Phosphorylase Deficiency in a Child with Severely Defective T-Cell Immunity and Normal B-Celt Immunity, Lancet 1, 1010-1013 (1975). [6] F.F. SNYDER,J. MENDELSOHN and J.E. SEEGMILLER, Adenosine Metabolism in Phytohemagglutinin-Stimulated Human Lymphocytes, J. Clin. Invest. 58, 654666 (1976).
5 52
Inhibition of Purine Nucleoslde Phosphorylase Activity and of T-Cell Function with Allopurinol-Riboside
CARSON and J.E. SEEGMILLER, Effect of Adenosine Deaminase Inhibition Upon Human Lymphocyte Blastogenesis, J. Clin. Invest. 57, 274-282
[71 D.A.
(1976). [8] H.M. KALC~A~ Differential Spectrophotometry of
Purine Compounds by Means of Specific Enzymes, J. Biol. Chem. 167, 429-443 (1947). [91 A. BovuM, Separation of Leukocytes from Blood and Bone Marrow, Scand. J. Clin. Lab. Invest. 21 (suppl 97), 77-89 (1968). [10] P.H. LAGRANGE, G.B. MACKANESS and T.E. MILLER,
Influence of Dose and Route of A ntigen Injection of the Immunological Induction of T Cells, J. Exp. Med. 139, 528-542 (1974). [11] L.J. GUDAS, B. ULLMAN, A. COHEN and D.W. MARTIN Jr., Deoxyguanosine Toxicity in a Mouse T
Lymphoma: Relationship to Purine Nucleoside Phos-
phorylase-Associated Immune Dysfunction, Cell 14, 531-538 (1978). [121 R.P. AGARWAL, S.M. SAGAR and R.E. PARKS Jr.,
Adenosine Deaminase from Human Erythroeytes: Purification and Effects of Adenosine Analogs, Bio chem. Pharmacol. 24, 693-701 (1975). [13] K. ITO and H. UCHINO, Control of Pyrimidine Biosynthesis in Human Lymphoeytes: Inhibitory Effect of Guanine and Guanosine on Induction of Enzyme for Pyrimidine Biosynthesis de novo in Phytohemagglutinin-Stimulated Lymphocytes, J. Biol. Chem. 251, 1427-1430 (1976). [14] A. COHEN, G.E.J. STAAL, A.J. AMMANN and D.W. MARTIN Jr., OrotieAciduria in Two UnrelatedPatients
with Inherited Deficiencies of Purine Nucleoside Phosphorylase, J. Clin. Invest. 60, 491-494 (1977).
549
Inhibition of Purine Nucleoside Phosphorylase Activity and of T-Cell Function with Allopurinol-Riboside by Y. NISHIDA, N. KAMATANI, K. TANIMOTO and I. AKAOKA Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan 113
Abstraet Allopurinol-riboside competitively inh~its the action of purlne nueleoslde phosphorylase on inosine in vitro with a Ki of 277 /Jmol. After simple incubation of allopurinolriboside with PNP, allopurinol was not formed. Lymphocyte blastogenesis induced by PHA and Con A was significantly suppressed by aUopurinol-riboslde in a concentration-dependent manner. When LPS was used as a mitogen, the inhibition of allopurinol-riboslde on lymphocyte proliferation was less marked. Humoral immunity was not suppressed by allopurinol-riboside. In contrast, cellular immunity was significantly suppressed by aUopurinolrlboside in vivo. These results suggested that allopurinolriboside is a drug which produces a model of PNP deficiency, and that it may be a useful inhibitor of cellular immunity.
Introduction Recently, some children with severe combined immunodeficiency have been reported to have adenosine deaminase (ADA) [1-4] or purine nucleoside phosphorylase (PNP) deficiency [5]. In ADA-deficient children, both Tcell and B-cell mediated immunities were impaired. The children with PNP deficiency have defective T-cell immunity but intact B-cell immunity. Coformycin [6] and erythro-9-(2-hydroxy-3nonyl) adenine hydrochloride [7] inhibit ADA activity, and have been used in in vitro studies to make an experimental model of ADA deficiency. In the present report the effects of allopurinol-riboside were studied on PNP activity in vitro and on lymphocyte proliferation induced by mitogens. The effects of allopurinol-riboside on humoral and cellular immunity to sheep red blood cells (SRBC) were also studied in mice. The results showed that allopurinol-riboside was useful as an inhibitor of PNP and as a T-cell inhibitor.
Materials and methods Enzyme assay PNP activity was determined according to the spectrophotometric method of KALCKAR [8]. The sample cuvette (final volume 3 ml) contained from ~0 to ] m M inosine as a substrate in 0.05 M phosphate buffer, pH 7.5, with or without 1.5 mM allopurinol-riboside. After addition of 20/tl of xanthine oxidase (8 p/ml: P-L Biochemicals Inc.) and 0.025 /~ of PNP (25 ~t/ml: Boehringer Mannheim) to the reaction mixture, the increase in optical density at 293 nm was recorded. Xanthine oxidase activity was not inhibited by allopurinol-riboside. Allopurinol-riboside was donated by Kyowa Hakko Corp. Ltd. Conversion of allopurlnolMboside 10 mg of aUopurinol-riboside dissolved in 1.0 ml, 0.05 M, phosphate buffer was incubated with 50/A of PNP (25 p/ml Boehringer Mannheim) for 30 min at 37~ The reaction mixture was spotted on Eastman (Kodak) thin-layer chromatography plates and developed with butanol:methanol :water :25% NH4OH (60:20:20:1 v/v). The purines were located on the chromatogram under ultraviolet light. The reaction mixture was also applied to a high-pressure liquid chromatogram (Hitachi 633) using Hitachi castam gel 2618 and eluted with 0.4 N-ammonium acetate, pH 4.0, at a pressure of 100 kg/cm 2.
Cell cultures 30 ml of venous blood was collected from a healthy subject in a heparinized syringe. The lymphocytes were separated by discontinuous centrifugation in Ficoll-hypaque solution as previously described [9] and washed three times in large volumes of TC199 solution (Chiba-ken, Serum Laboratory). The lymphocytes were rewashed with medium RPMI-1640 (Grand Island Biological Company) containing 10% foetal calf serum, 200 p/ml penicillin G and 100 pg/ml streptomycin, and resuspended at 1 x 106 cells/ml in the medium. Cultures were carried out in sterile microplates with round-bottom wells and were done in triplicate. I x 10~ of responding cells in 100 ~1 of culture medium which contained the various concentrations of sterilized allopurinol-riboside were put into each well. 100 ~1 of mitogens dissolved in culture medium of equal volume of medium as a
5 50
Inhibition of Purine Nucleoside Phosphorylase Activity and of T-Cell Function with Allopurinol-Riboside
control were added to the culture tubes on the first day of the culture. Their final concentrations were as follows: 4 pg/ml of phytohemagglutinin (PHA: Difco Laboratories), 50 pg/ml of concanavalin A (Con A: Pharmacia Fine Chemicals), 1 : 100 diluted pokeweed mitogen (PWM: Grand Island Biological Company), and 1000 pg/ml lipopolysaccharide B (LPS: Difco Laboratories). They were incubated for 5 days at 37~ in a humidified atmosphere with 5% CO 2 and 95% air. 1/tCi of H3-thymidine (42 Ci/mmoh Radiochemieal Centre) was added to each well for a further 24 h incubation. Cells were harvested on glass-fiber filter paper using a Millipore semi-automated harvester. Radioactivities of thymidine incorporated into lymphocyte D N A were measured in a liquid-scintillation counter and expressed as counts per 10 min.
I 3~ x[ ~ tgex
OOO000
= ~ 961
9
~ 481
O0
9
O0000
9
241
O0
tz ! 61
Humoral and cellular immunity Mice were used at 4 to 6 weeks of age. SRBC were obtained commercially from Nippon Bio-test Laboratories and were stored at 4~ The SRBC were washed three times with sterile physiological saline before use, and finally suspended at the desired concentration in saline. Humoral immunity was measured as the hemagglutinin titers. 1 x l0 s washed SRBC in a volume of 500 ~tl was injected intraperitoneally into 20 mice. 10 mice were injected intramuscularly with allopurinol-riboside in a dose of 1 mg/g body wt./day for 7 successive days. Seven days after injection, blood was collected by heart puncture and serum was separated. Hemagglutinin titers were measured in microtitration trays using 0.1 ml of serum diluted serially in getatin-veronal buffer to which an equal volume of 1% SRBC was added. The trays stood overnight at 4~ Cellular immunity was studied according to the method of LAGRANGE et al. [10]. 5 • 105 SRBC was injected intraperitoneally into 20 mice. Allopurinol-riboside (1 mg/g body wt./day) was given to the 10 mice for 5 successive days. Five days after injection, 1 • 108 SRBC in a volume of 50/21 was injected into the left foot pad of the mice. Twentyfour hours after this treatment, the increase of left foot pad weight was measured.
Results Allopurinol-riboside competitively suppressed PNP action on inosine. 50% inhibition was achieved at 277 ~mol of allopurinol-riboside.
Control
AIIoourinol-
riboside
Figure 1 Hemagglutinin titers to SRBC in mice treated with allopurinol-riboside and controls. m9 z0.0
.~ 15.0
I~
~_ ~ t0.0
5.0 Qtl
control
ABopurinolriboside
Figure 2 Delayed-type hypersensitivity to SRBC in mice treated with allopurinol-riboside and controls.
Table 1 Effect of allopurinol-riboside on the incorporation of 3H:thymidine into mitogen-stimulated lymphocytes. Allopurinolriboside
Mitogen
(mM)
Unstimulated
PHA
Con A
0 2.5 5 10
9 4,305 4,393 6,815
315,162_+ 13,355 141,679 -+ 20,122 67,533 _+ 6,901 15,849 + 1,503
32,213 15,410 10,010 7,499
_+ 386 -+ 948 _+ 894 + 720
PWM _+ 1,061 -+ 963 _+ 2,101 _+ 422
Results expressed in net counts per 10 min and per 1 x 105 cells. (Mean + SD.)
36,204 42,743 33,208 11,869
LPS + 958 _+ 2,515 + 7,103 _+ 3,470
25,181 23,873 19,104 16,283
+ 8,460 + 3,598 • 3,623 _+ 1,170
Inhibition of Purine Nucleoside Phosphorylase Activity and of T-Cell Function with Allopurinol-Riboside
After simple incubation of allopurinolriboside with PNP in vitro, allopurinol could not be detected using high-pressure liquid chromatography and/or Eastman Kodak thin-layer chromatography. The results of (AH) thymidine incorporation in lymphocytes stimulated with various mitogens are shown in Table 1. The mitogen-induced proliferation of human lymphocytes was in general suppressed by allopurinol-riboside. The inhibitory effect was dose-dependent. Blastogenesis induced by PHA and Con A was markedly inhibited by allopurinol-riboside, while its inhibition of that induced by PWM and LPS was less marked. Hemagglutinin titers are shown in Figure 1. There was no significant difference in the hemagglutinin titer between the control mice group and the group treated with aUopurinolriboside. In contrast, the increase of foot pad weight in the mice treated with allopurinol-riboside was significantly lower, 4.3 _+ 1.4 mg, than the average increase of 17,9 + 3.3 mg in the controls (Fig. 2). Discussion Children with PNP deficiency have been shown to have defective T-cell immunity but intact B-cell immunity, but the causal relationship between depressed immunity and these enzyme deficiencies has not yet completely been elucidated. The role of purine metabolizing enzymes such as ADA and PNP in the immune response is of particular interest. In children with ADA deficiency, both T-cell and B-cell functions are impaired. The deficiency of ADA may lead to the intracellular accumulation of adenosine and/or D-adenosine. The increased concentration of adenosine and/or Dadenosine might be related to the pathogenesis of the immune dysfunction on this disease as excess adenosine in lymphocyte culture is toxic [2, 11]. Coformycin, a competitive inhibitor of ADA, has been demonstrated to suppress lymphocyte blastogenesis [6], and this agent has been used in vitro to produce a condition similar to ADA deficiency. PNP converts inosine and guanosine to their respective free purine bases. Deficiency of PNP may lead to the accumulation of intracellular inosine, guanosine and D-guanosine. High concentration of guanosine is also known to suppress
55 1
lymphocyte proliferation [13]. Indeed, elevated levels of inosine have been confirmed in the plasma of children with PNP deficiency [14]. The reason for the normal B-cell function in these patients remains unclear. Allopurinol-riboside significantly suppressed PNP activity in vitro. This suppressing effect may be due to binding to the active site of PNP by allopurinol-riboside in a manner which inhibits its activity and results in competitive suppression. These results indicate the usefulness of alMpurinol-riboside as an inhibitor of PNP. PHA and Con A are known to stimulate T-cells preferentially. Allopurinol-riboside produced dramatic suppression in lymphocyte blastogenesis stimulated by PHA and Con A. On the contrary, lymphocyte proliferation caused by a selective Bcell stimulant, LPS, was not inhibited by allopurinol-riboside to the same degree as the PHA and Con A responses. There results suggested that allopurinol-riboside mainly suppresses the Tcell function. Humoral immunity was not suppressed by allopurinol-riboside but cellular immunity was significantly suppressed by allopurinol-riboside in an in vivo experiment. Allopurinol-riboside in an adequate dose may make an experimental model of PNP deficiency and might be a useful and powerful inhibitor for cellular immunity. Received 9 May 1979.
References [1] E.R. GIBLETT, J.E. ANDERSON, F. COHEN, B. POLLARA and H.J. MEUWISSEN, Adenosine-Deaminase Deficiency in Two Patients with Severely Impaired Cellularlmmunity, Lancet 2, 1067-1069 (1972). [2] C.R. SCOTT, S-H. CHEN and E.R. GmLETr, Detection of the Carrier State in Combined lmmunodefieiency Disease Associated with Adenosine Deaminase Deficiency, J. Clin. Invest. 53, 1194-1196 (1974). [3] J. YOUNT, P. NICHOLS, J.D. OCHS, S.P. HAMMER, C.R. SCOTT, S-H. CHEN, E.R. GmLETr and R.J. WEDGEWOOD, Absence of Erythrocyte Adenosine Deaminase Associated with Severe Combined Immunodefieiency, J. Pediatr. 84, 173-177 (1974). [4] R. PARKMAN, E.W. GELFAND, F.S. ROSEN, A. SANDERSON and R. HIRSCHHORN,Severe Combined Immunodeficiency and Adenosine Deaminase Deficiency, N. Engl. J. Ned. 292, 714-719 (1975). [5] E.R. GIBLETF, A.V. AMMANN, D.W. WARA, R. SANDMAN and LK. DIAMOND, Nucleoside-Phosphorylase Deficiency in a Child with Severely Defective T-Cell Immunity and Normal B-Celt Immunity, Lancet 1, 1010-1013 (1975). [6] F.F. SNYDER,J. MENDELSOHN and J.E. SEEGMILLER, Adenosine Metabolism in Phytohemagglutinin-Stimulated Human Lymphocytes, J. Clin. Invest. 58, 654666 (1976).
5 52
Inhibition of Purine Nucleoslde Phosphorylase Activity and of T-Cell Function with Allopurinol-Riboside
CARSON and J.E. SEEGMILLER, Effect of Adenosine Deaminase Inhibition Upon Human Lymphocyte Blastogenesis, J. Clin. Invest. 57, 274-282
[71 D.A.
(1976). [8] H.M. KALC~A~ Differential Spectrophotometry of
Purine Compounds by Means of Specific Enzymes, J. Biol. Chem. 167, 429-443 (1947). [91 A. BovuM, Separation of Leukocytes from Blood and Bone Marrow, Scand. J. Clin. Lab. Invest. 21 (suppl 97), 77-89 (1968). [10] P.H. LAGRANGE, G.B. MACKANESS and T.E. MILLER,
Influence of Dose and Route of A ntigen Injection of the Immunological Induction of T Cells, J. Exp. Med. 139, 528-542 (1974). [11] L.J. GUDAS, B. ULLMAN, A. COHEN and D.W. MARTIN Jr., Deoxyguanosine Toxicity in a Mouse T
Lymphoma: Relationship to Purine Nucleoside Phos-
phorylase-Associated Immune Dysfunction, Cell 14, 531-538 (1978). [121 R.P. AGARWAL, S.M. SAGAR and R.E. PARKS Jr.,
Adenosine Deaminase from Human Erythroeytes: Purification and Effects of Adenosine Analogs, Bio chem. Pharmacol. 24, 693-701 (1975). [13] K. ITO and H. UCHINO, Control of Pyrimidine Biosynthesis in Human Lymphoeytes: Inhibitory Effect of Guanine and Guanosine on Induction of Enzyme for Pyrimidine Biosynthesis de novo in Phytohemagglutinin-Stimulated Lymphocytes, J. Biol. Chem. 251, 1427-1430 (1976). [14] A. COHEN, G.E.J. STAAL, A.J. AMMANN and D.W. MARTIN Jr., OrotieAciduria in Two UnrelatedPatients
with Inherited Deficiencies of Purine Nucleoside Phosphorylase, J. Clin. Invest. 60, 491-494 (1977).
