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In vitro effects of mycophenolic acid on cell cycle and activation of human lymphocytes
Clinica Chimica Acta 300 (2000) 23–28 www.elsevier.com / locate / clinchim
In vitro effects of mycophenolic acid on cell cycle and activation of human lymphocytes Angelika Heinschink*, Markus Raab, Heide Daxecker, ¨ Andrea Griesmacher, Mathias M. Muller Institute of Laboratory Diagnostics and Ludwig Boltzmann Institute for Cardiothoracic Research, Kaiser Franz Josef Hospital, Kundratstraße 3, A-1100 Vienna, Austria Received 13 April 2000; accepted 19 April 2000
Abstract The immunosuppressant mycophenolic acid (MPA) selectively inhibits proliferation of T- and B-lymphocytes by blocking inosine 59-monophosphate-dehydrogenase (IMPDH), the key enzyme for de-novo-synthesis of guanine nucleotides. In an in vitro study the effects of MPA on human peripheral blood lymphocyte activation markers and on cell cycle characteristics were investigated. Mononuclear cells from healthy volunteers were incubated with phytohaemagglutinin (PHA) and increasing doses of MPA. After 72 h incubation an aliquot of the cells was stained with propidium iodide and measured by FACS analyses to assess the DNA shape. In addition, the expression of the activation markers HLA-DR and CD25 on T- and B-lymphocytes was determined by flow cytometry analysis. PHA stimulation led to a significant increase of the S-phase of cell cycle. PHA stimulation clearly increased mean fluorescence intensity (MFI) of HLA-DR expression on B-lymphocytes. PHA stimulation also elevated the number of CD25 positive B-lymphocytes. Expression of HLA-DR on T-lymphocytes was not influenced by PHA, whereas CD25 expression and MFI significantly increased. All the observed PHA induced effects were reduced by co-incubation with increasing doses of MPA. The data presented show that in vitro the immunosuppressive effect of MPA can be demonstrated using FACS technology on a cellular level. MPA leads to an inhibition of cell cycle proliferation in peripheral blood lymphocytes. 2000 Elsevier Science B.V. All rights reserved. Keywords: Mycophenolic acid; Activation markers; Cell cycle
Abbreviations: FACS, fluorescence activated cell scanner; IL-2, interleukin-2; IMPDH, inosine 59-monophosphate-dehydrogenase; MFI, mean fluorescence intensity; MPA, mycophenolic acid; PHA, phytohaemagglutinin *Corresponding author. Fax: 1 43-160-191-3309. E-mail address: [email protected] (A. Heinschink). 0009-8981 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00297-7
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A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
1. Introduction MPA is an immunosuppressive agent that mediates its effect by inhibition of inosine monophosphate dehydrogenase (IMPDH), the key enzyme for de novo synthesis of guanine nucleotides in lymphocytes [1–3]. MPA disturbs the homeostasis of nucleotide concentration and induces a reduction in intracellular guanine nucleotide pools. At the same time adenine nucleotide pools are increasing, thereby blocking de novo DNA synthesis and proliferation of lymphocytes, subsequently causing a reduced immunological response after stimulation. The aim of the present in vitro study was to investigate the influence of MPA on the cell cycle in PHA stimulated blood lymphocytes and whether the inhibition of the lymphocytic function by MPA can be demonstrated by a decreased expression of activation markers.
2. Materials and methods
2.1. Reagents PHA and MPA were purchased from SIGMA–ALDERICH Handels GmbH (Austria); RPMI-1640 with glutamax, fetal calf serum (FCS), penicillin, streptomycin and phosphate buffered saline (PBS) were obtained from Gibco BRL Life Technologies (Scotland); DNA kit and antibodies to HLA-DR, CD25, CD3, CD19 were from Becton Dickinson Immunocytometry Systems (California); LeucoSep was purchased from Greiner GmbH (Germany); Ficoll-Paque from Pharmacia Biotech AB (Sweden).
2.2. Experimental studies Blood samples from human volunteers containing EDTA were centrifuged over a Ficoll-Paque gradient. The harvested mononuclear cells from samples with identical blood group were washed in PBS and re-suspended in full medium – RPMI-1640 supplemented with 10% FCS, penicillin and streptomycin – at a final concentration of 1.0 3 10 6 cells per ml PHA (2.5 mg / 10 6 cells) for stimulation. Increasing concentrations of MPA from 0 to 1 mmol / l (0–320 mg / l) were added for investigating the immunosuppressive effect. Cells were incubated at 378C in a humidified CO 2 incubator for 72 h. Control cells were incubated for 72 h in the absence of PHA.
A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
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2.3. Flow cytometric analysis After 72 h of incubation an aliquot of the cells was stained with propidium iodide (PI) and measured by Fluorescence Activated Cell Scanner (FACS)analysis (Facs Scan, Becton Dickinson) to assess DNA state. For evaluation of cell cycle standard software ‘‘Mode-fit’’ (Becton Dickinson) was used. Gates were set to differentiate between G0–G1, S-phase and G2-M, with apoptotic cells appearing to the left of the G0–G1 peak. Differential staining with anti-CD3 and anti-CD19 antibodies as well as the measurement of lymphocyte activation markers HLA-DR and CD25 (interleukin-2-receptor) were carried out by FACS analysis. The extent of the expression of HLA-DR and CD25 are shown as MFI.
2.4. Statistical analysis All experiments were performed in duplicate on at least five separate occasions. Differences between the groups were tested for statistical significance by using analysis of variance. All calculations were carried out with the statistical software package SAS / STAT [4]. P-values of , 0.001 and , 0.05 were considered statistically significant. Values are expressed as mean6S.D.
3. Results The influence of MPA on PHA-stimulated cell cycle and the expression of HLA-DR and CD25 on peripheral human lymphocytes is described in detail in Table 1.
3.1. Cell cycle PHA stimulation of lymphocytes led to a significant (P , 0.001) averaged increase of the 5-phase from 0.6 up to 24%. Co-incubation with MPA showed a concentration dependent inhibition of DNA synthesis: in samples treated with 1 mmol / l MPA we found values analogous to the controls. PHA stimulation of lymphocytes led to a decrease of G0–G1-phase, treatment with 1 mmol / l MPA reversed the PHA induced effect. G2-M-phase exhibited a characteristic PHA increase, MPA again suppressed G2-M.
A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
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Table 1 Effect of mycophenolic acid (MPA) on PHA-stimulated cell cycle and expression of HLA-DR and CD2S ( 5 Il-2 receptor) on peripheral human lymphocytes a Controls
PHA
PHA 1 MPA 0.1 mmol / l
PHA 1 MPA 0.5 mmol / l
PHA 1 MPA 1 mmol / l
Cell count (million / ml)
1.060.17
1.360.188
1.160.16 1
0.7860.13*
0.7860.1*
Cell cycle DNA G0–G1 (%) DNA S (%) DNA G2-M (%)
94.667.9 0.660.7 2.461.2
76.269.1 24.568.08 3.762.6
76.868.5 22.565.6 3.062.0
81.364.9 15.563.1 3.061.7
95.563.2 1 1.960.7* 1.360.6
B-lymphocytes activation markers HLA-DR 1 (%) 93.963.3 HLA-DR 1 (MFI) 7686375 CD25 1 (%) 38.8614.9 CD25 1 (MFI) 37.2612.0
94.366.8 250269478 89.7610.38 73064058
93.266.0 26536990 87.069.9 7326288
90.266.3 15896388 1 59.1611.7 1 157676.7 1
89.067.6 14816420 1 63.1612.9 1 123646.6*
T-lymphocytes activation markers HLA-DR 1 (%) 6.262.9 HLA-DR 1 (MFI) 57.2619.4 CD25 1 (%) 7.361.7 CD25 1 (MFI) 164694.5
35.1615.28 72.9626.3 80.4610.58 147566378
32.5613.7 73.1628.7 75.568.9 8916448 1
29.5615.9 68.6622.8 47.5613.5* 459612*
27.0610.9 77.1623.4 46.569.2* 284655.1*
a
Significance in comparison to the controls (8P , 0.001) and to PHA (*P , 0.001, 1 P , 0.05).
3.2. Activation markers of B- and T-lymphocytes 3.2.1. B-lymphocytes The number of HLA-DR positive B-lymphocytes was constant, independent of the conditions of incubation. However, PHA stimulation led to an increase of MFI (P , 0.001) from 768 up to 2502. Increasing concentrations of MPA showed a continuous decrease in the MFI; 1 mmol / l MPA decreased HLA-DR expression by 41% compared to PHA stimulated lymphocytes (P , 0.05) PHA stimulation led to a significant increase of CD25 positive B-lymphocytes from 38% up to 90% (P , 0.001). Co-incubation with MPA showed again a dose-dependent decrease. For the MFI we found the corresponding increases and decreases with stimulation and suppression. 3.2.2. T-lymphocytes PHA activation led to an increase of HLA-DR positive T-lymphocytes from 6% up to 35% (P , 0.001) Simultaneous inhibition with MPA indicated a small decrease in HLA-DR positive T-lymphocytes. MFl of HLA-DR positive T-cells remained almost unchanged.
A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
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The expression of CD25 on T-lymphocytes increased under PHA stimulation from 7% up to 80% (P , 0.001) and was reduced to 46% (P , 0.001) upon co-incubation with MPA. This increase under PHA stimulation and the dosedependent inhibition under MPA was also observed for MFI of CD25 positive T-cells.
4. Discussion These results demonstrate that in vitro PHA activation of peripheral blood lymphocytes could be studied elegantly and easily by the determination of cell cycle stage and detection of expressed surface activation markers using flow cytometry. In addition we could prove the immunosuppressive effect of MPA on a cellular level: MPA leads to an inhibition of DNA synthesis and by that to proliferation of lymphocytes. The investigation of the cell cycle characteristics and inhibition of S-phase by MPA is concomitant with the inhibition of thymidine incorporation demonstrated recently [5,6]. As a possible mechanism the MPA induced GTP depletion in lymphocytes inhibits the induction of cyclin D3, a major component of cyclin-dependent kinase (CDK), and decreases the degradation of p27 kip1 , a CDK inhibitor, resulting in the detention of the cell cycle in the early to mid-G1 phase [7]. After PHA stimulation and following immunoinhibition with MPA T- and B-lymphocytes react with changes of HLA-DR and CD25 expression in the same direction. Under PHA stimulation the expression of the interleukin-2-receptor (CD25) better characterizes the functional condition of lymphocytes than does HLA-DR. These in vitro results show the possibility that the biological effects of immunosuppressive drugs on cellular level can also be measured in the blood of patients. Cellular and humoral mechanisms are involved in graft rejection leading to release of cytokines, mediators, acute-phase proteins, activators of the complement system as well as to activation of immunocompetent cells. The graft is finally destroyed by combined specific actions of cytotoxic T-cells and proinflammatory cytokines, a variety of antibodies and unspecific proteolysis due to the activation of the complement system as well as of granulocytes. The in vitro model described mimics the activation of lymphocytes by PHA stimulation as seen in transplant recipients during acute infections and rejections. It was shown that MPA selectively exhibits anti-proliferative effects in T- and B-cells by decrease of activation markers and S-phase. The MPA doses used in vitro are found in patients treated with MPA. Applying FACS analysis in transplant recipients under immunosuppressive therapy can be used for the detection of activation status of peripheral blood lymphocytes in order to determine infection and / or rejection episodes and the overall effect of the therapeutic regimen.
A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
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Acknowledgement This study was supported by the EU project BMH4-CT98-379.
References [1] Allison AC, Eugui EM. The design and development of an immunosuppressive drug, mycophenolic acid. Springer Semin Immunopathol 1993;14:353–80. [2] Eugui EM, Almquist SJ, Muller CD, Allison AC. Lymphocyte-selective cytostatic and immunosuppressive effects of mycophenolic acid in vitro: role of deoxyguanosine nucleotide depletion. Scand J Immunol 1991;33:161–75. ¨ [3] Griesmacher A, Weigel G, Seebacher G, Muller MM. IMP-dehydrogenase inhibition in human lymphocytes and lymphoblasts by mycophenolic acid and mycophenolic acid glucuronide. Clin Chem 1997;43(12):2312–7. [4] SAS Institute. SAS / STAT user’s guide, version 6, 4th ed. Cary-NC: SAS Institute Inc. 1989. [5] Nowak I, Shaw LM. Mycophenolic acid binding to human serum albumin: characterization and relationship to pharmacodynamics. Clin Chem 1995;41:1011–7. ¨ [6] Allison AC, Kowalski WJ, Muller CD, Eugui EM. Mechanism of action of mycophenolic acid. NY Acad Sci 1993;696:63–87. [7] Laliberte´ J, Yee A, Xiong Y, Mitchell BS. Effects of guanine nucleotide depletion on cell cycle progression in human T-lymphocytes. Blood 1998;91(8):2896.
In vitro effects of mycophenolic acid on cell cycle and activation of human lymphocytes Angelika Heinschink*, Markus Raab, Heide Daxecker, ¨ Andrea Griesmacher, Mathias M. Muller Institute of Laboratory Diagnostics and Ludwig Boltzmann Institute for Cardiothoracic Research, Kaiser Franz Josef Hospital, Kundratstraße 3, A-1100 Vienna, Austria Received 13 April 2000; accepted 19 April 2000
Abstract The immunosuppressant mycophenolic acid (MPA) selectively inhibits proliferation of T- and B-lymphocytes by blocking inosine 59-monophosphate-dehydrogenase (IMPDH), the key enzyme for de-novo-synthesis of guanine nucleotides. In an in vitro study the effects of MPA on human peripheral blood lymphocyte activation markers and on cell cycle characteristics were investigated. Mononuclear cells from healthy volunteers were incubated with phytohaemagglutinin (PHA) and increasing doses of MPA. After 72 h incubation an aliquot of the cells was stained with propidium iodide and measured by FACS analyses to assess the DNA shape. In addition, the expression of the activation markers HLA-DR and CD25 on T- and B-lymphocytes was determined by flow cytometry analysis. PHA stimulation led to a significant increase of the S-phase of cell cycle. PHA stimulation clearly increased mean fluorescence intensity (MFI) of HLA-DR expression on B-lymphocytes. PHA stimulation also elevated the number of CD25 positive B-lymphocytes. Expression of HLA-DR on T-lymphocytes was not influenced by PHA, whereas CD25 expression and MFI significantly increased. All the observed PHA induced effects were reduced by co-incubation with increasing doses of MPA. The data presented show that in vitro the immunosuppressive effect of MPA can be demonstrated using FACS technology on a cellular level. MPA leads to an inhibition of cell cycle proliferation in peripheral blood lymphocytes. 2000 Elsevier Science B.V. All rights reserved. Keywords: Mycophenolic acid; Activation markers; Cell cycle
Abbreviations: FACS, fluorescence activated cell scanner; IL-2, interleukin-2; IMPDH, inosine 59-monophosphate-dehydrogenase; MFI, mean fluorescence intensity; MPA, mycophenolic acid; PHA, phytohaemagglutinin *Corresponding author. Fax: 1 43-160-191-3309. E-mail address: [email protected] (A. Heinschink). 0009-8981 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00297-7
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A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
1. Introduction MPA is an immunosuppressive agent that mediates its effect by inhibition of inosine monophosphate dehydrogenase (IMPDH), the key enzyme for de novo synthesis of guanine nucleotides in lymphocytes [1–3]. MPA disturbs the homeostasis of nucleotide concentration and induces a reduction in intracellular guanine nucleotide pools. At the same time adenine nucleotide pools are increasing, thereby blocking de novo DNA synthesis and proliferation of lymphocytes, subsequently causing a reduced immunological response after stimulation. The aim of the present in vitro study was to investigate the influence of MPA on the cell cycle in PHA stimulated blood lymphocytes and whether the inhibition of the lymphocytic function by MPA can be demonstrated by a decreased expression of activation markers.
2. Materials and methods
2.1. Reagents PHA and MPA were purchased from SIGMA–ALDERICH Handels GmbH (Austria); RPMI-1640 with glutamax, fetal calf serum (FCS), penicillin, streptomycin and phosphate buffered saline (PBS) were obtained from Gibco BRL Life Technologies (Scotland); DNA kit and antibodies to HLA-DR, CD25, CD3, CD19 were from Becton Dickinson Immunocytometry Systems (California); LeucoSep was purchased from Greiner GmbH (Germany); Ficoll-Paque from Pharmacia Biotech AB (Sweden).
2.2. Experimental studies Blood samples from human volunteers containing EDTA were centrifuged over a Ficoll-Paque gradient. The harvested mononuclear cells from samples with identical blood group were washed in PBS and re-suspended in full medium – RPMI-1640 supplemented with 10% FCS, penicillin and streptomycin – at a final concentration of 1.0 3 10 6 cells per ml PHA (2.5 mg / 10 6 cells) for stimulation. Increasing concentrations of MPA from 0 to 1 mmol / l (0–320 mg / l) were added for investigating the immunosuppressive effect. Cells were incubated at 378C in a humidified CO 2 incubator for 72 h. Control cells were incubated for 72 h in the absence of PHA.
A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
25
2.3. Flow cytometric analysis After 72 h of incubation an aliquot of the cells was stained with propidium iodide (PI) and measured by Fluorescence Activated Cell Scanner (FACS)analysis (Facs Scan, Becton Dickinson) to assess DNA state. For evaluation of cell cycle standard software ‘‘Mode-fit’’ (Becton Dickinson) was used. Gates were set to differentiate between G0–G1, S-phase and G2-M, with apoptotic cells appearing to the left of the G0–G1 peak. Differential staining with anti-CD3 and anti-CD19 antibodies as well as the measurement of lymphocyte activation markers HLA-DR and CD25 (interleukin-2-receptor) were carried out by FACS analysis. The extent of the expression of HLA-DR and CD25 are shown as MFI.
2.4. Statistical analysis All experiments were performed in duplicate on at least five separate occasions. Differences between the groups were tested for statistical significance by using analysis of variance. All calculations were carried out with the statistical software package SAS / STAT [4]. P-values of , 0.001 and , 0.05 were considered statistically significant. Values are expressed as mean6S.D.
3. Results The influence of MPA on PHA-stimulated cell cycle and the expression of HLA-DR and CD25 on peripheral human lymphocytes is described in detail in Table 1.
3.1. Cell cycle PHA stimulation of lymphocytes led to a significant (P , 0.001) averaged increase of the 5-phase from 0.6 up to 24%. Co-incubation with MPA showed a concentration dependent inhibition of DNA synthesis: in samples treated with 1 mmol / l MPA we found values analogous to the controls. PHA stimulation of lymphocytes led to a decrease of G0–G1-phase, treatment with 1 mmol / l MPA reversed the PHA induced effect. G2-M-phase exhibited a characteristic PHA increase, MPA again suppressed G2-M.
A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
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Table 1 Effect of mycophenolic acid (MPA) on PHA-stimulated cell cycle and expression of HLA-DR and CD2S ( 5 Il-2 receptor) on peripheral human lymphocytes a Controls
PHA
PHA 1 MPA 0.1 mmol / l
PHA 1 MPA 0.5 mmol / l
PHA 1 MPA 1 mmol / l
Cell count (million / ml)
1.060.17
1.360.188
1.160.16 1
0.7860.13*
0.7860.1*
Cell cycle DNA G0–G1 (%) DNA S (%) DNA G2-M (%)
94.667.9 0.660.7 2.461.2
76.269.1 24.568.08 3.762.6
76.868.5 22.565.6 3.062.0
81.364.9 15.563.1 3.061.7
95.563.2 1 1.960.7* 1.360.6
B-lymphocytes activation markers HLA-DR 1 (%) 93.963.3 HLA-DR 1 (MFI) 7686375 CD25 1 (%) 38.8614.9 CD25 1 (MFI) 37.2612.0
94.366.8 250269478 89.7610.38 73064058
93.266.0 26536990 87.069.9 7326288
90.266.3 15896388 1 59.1611.7 1 157676.7 1
89.067.6 14816420 1 63.1612.9 1 123646.6*
T-lymphocytes activation markers HLA-DR 1 (%) 6.262.9 HLA-DR 1 (MFI) 57.2619.4 CD25 1 (%) 7.361.7 CD25 1 (MFI) 164694.5
35.1615.28 72.9626.3 80.4610.58 147566378
32.5613.7 73.1628.7 75.568.9 8916448 1
29.5615.9 68.6622.8 47.5613.5* 459612*
27.0610.9 77.1623.4 46.569.2* 284655.1*
a
Significance in comparison to the controls (8P , 0.001) and to PHA (*P , 0.001, 1 P , 0.05).
3.2. Activation markers of B- and T-lymphocytes 3.2.1. B-lymphocytes The number of HLA-DR positive B-lymphocytes was constant, independent of the conditions of incubation. However, PHA stimulation led to an increase of MFI (P , 0.001) from 768 up to 2502. Increasing concentrations of MPA showed a continuous decrease in the MFI; 1 mmol / l MPA decreased HLA-DR expression by 41% compared to PHA stimulated lymphocytes (P , 0.05) PHA stimulation led to a significant increase of CD25 positive B-lymphocytes from 38% up to 90% (P , 0.001). Co-incubation with MPA showed again a dose-dependent decrease. For the MFI we found the corresponding increases and decreases with stimulation and suppression. 3.2.2. T-lymphocytes PHA activation led to an increase of HLA-DR positive T-lymphocytes from 6% up to 35% (P , 0.001) Simultaneous inhibition with MPA indicated a small decrease in HLA-DR positive T-lymphocytes. MFl of HLA-DR positive T-cells remained almost unchanged.
A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
27
The expression of CD25 on T-lymphocytes increased under PHA stimulation from 7% up to 80% (P , 0.001) and was reduced to 46% (P , 0.001) upon co-incubation with MPA. This increase under PHA stimulation and the dosedependent inhibition under MPA was also observed for MFI of CD25 positive T-cells.
4. Discussion These results demonstrate that in vitro PHA activation of peripheral blood lymphocytes could be studied elegantly and easily by the determination of cell cycle stage and detection of expressed surface activation markers using flow cytometry. In addition we could prove the immunosuppressive effect of MPA on a cellular level: MPA leads to an inhibition of DNA synthesis and by that to proliferation of lymphocytes. The investigation of the cell cycle characteristics and inhibition of S-phase by MPA is concomitant with the inhibition of thymidine incorporation demonstrated recently [5,6]. As a possible mechanism the MPA induced GTP depletion in lymphocytes inhibits the induction of cyclin D3, a major component of cyclin-dependent kinase (CDK), and decreases the degradation of p27 kip1 , a CDK inhibitor, resulting in the detention of the cell cycle in the early to mid-G1 phase [7]. After PHA stimulation and following immunoinhibition with MPA T- and B-lymphocytes react with changes of HLA-DR and CD25 expression in the same direction. Under PHA stimulation the expression of the interleukin-2-receptor (CD25) better characterizes the functional condition of lymphocytes than does HLA-DR. These in vitro results show the possibility that the biological effects of immunosuppressive drugs on cellular level can also be measured in the blood of patients. Cellular and humoral mechanisms are involved in graft rejection leading to release of cytokines, mediators, acute-phase proteins, activators of the complement system as well as to activation of immunocompetent cells. The graft is finally destroyed by combined specific actions of cytotoxic T-cells and proinflammatory cytokines, a variety of antibodies and unspecific proteolysis due to the activation of the complement system as well as of granulocytes. The in vitro model described mimics the activation of lymphocytes by PHA stimulation as seen in transplant recipients during acute infections and rejections. It was shown that MPA selectively exhibits anti-proliferative effects in T- and B-cells by decrease of activation markers and S-phase. The MPA doses used in vitro are found in patients treated with MPA. Applying FACS analysis in transplant recipients under immunosuppressive therapy can be used for the detection of activation status of peripheral blood lymphocytes in order to determine infection and / or rejection episodes and the overall effect of the therapeutic regimen.
A. Heinschink et al. / Clinica Chimica Acta 300 (2000) 23 – 28
28
Acknowledgement This study was supported by the EU project BMH4-CT98-379.
References [1] Allison AC, Eugui EM. The design and development of an immunosuppressive drug, mycophenolic acid. Springer Semin Immunopathol 1993;14:353–80. [2] Eugui EM, Almquist SJ, Muller CD, Allison AC. Lymphocyte-selective cytostatic and immunosuppressive effects of mycophenolic acid in vitro: role of deoxyguanosine nucleotide depletion. Scand J Immunol 1991;33:161–75. ¨ [3] Griesmacher A, Weigel G, Seebacher G, Muller MM. IMP-dehydrogenase inhibition in human lymphocytes and lymphoblasts by mycophenolic acid and mycophenolic acid glucuronide. Clin Chem 1997;43(12):2312–7. [4] SAS Institute. SAS / STAT user’s guide, version 6, 4th ed. Cary-NC: SAS Institute Inc. 1989. [5] Nowak I, Shaw LM. Mycophenolic acid binding to human serum albumin: characterization and relationship to pharmacodynamics. Clin Chem 1995;41:1011–7. ¨ [6] Allison AC, Kowalski WJ, Muller CD, Eugui EM. Mechanism of action of mycophenolic acid. NY Acad Sci 1993;696:63–87. [7] Laliberte´ J, Yee A, Xiong Y, Mitchell BS. Effects of guanine nucleotide depletion on cell cycle progression in human T-lymphocytes. Blood 1998;91(8):2896.