Novel assays of multiple lymphocyte functions in whole blood measure: New mechanisms of action of mycophenolate mofetil in vivo

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Abstract

The antiproliferative effects of MMF are believed to be the mechanism of its immunosuppressive action. We further investigated the mechanisms of action by assessing the pharmacodynamics (PD) of MMF in treated animals using whole blood assays not only of lymphocyte proliferation but also of activation. In vitro, different MPA concentrations were added to rat whole blood. In vivo, Lewis rats were treated with single doses of 5, 10 or 20 mg/kg MMF (n=6 rats/dose group). Blood was obtained before and at different times after drug administration. For both in vitro and in vivo studies, different mitogens with calcium-dependent (TCR) or -independent (co-stimulatory) pathways of lymphocyte activation were added to the blood for stimulation. Proliferation was measured by [3H]TdR incorporation and by flow cytometric detection of DNA content. Activation was measured by changes in T cell surface expression of CD25, CD134, CD71, CD11a and CD54. In vitro and in vivo studies showed a dose-dependent inhibition by MPA and MMF, respectively, of lymphocyte proliferation and surface antigen expression. We observed high correlations between MMF PD effects over time with both MMF dose and MPA plasma concentrations in vivo. We show that MMF, apart from its antiproliferative effect, induced a dose-dependent suppression of calcium-dependent and -independent stimulated expression of important lymphocyte cell surface antigens. These data suggest that the ex vivo assessment of immune function in whole blood can uncover new mechanisms of MMF action. Our results demonstrated that the measurement of the PD is a means to assess the functional effects of MMF after its administration in vivo.

Introduction

After absorption from the gut, MMF is rapidly de-esterified to its active metabolite MPA [1]. MPA inhibits the key enzyme inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway of guanine nucleotide biosynthesis, which is a requisite complement to the salvage pathway for T and B cells to undergo mitogenic transformation [2], [3]. The antiproliferative effect of MPA is explained by the reduction of intracellular guanosine levels observed in many in vitro studies with purified lymphocytes in different species [4], [5], [6]. Recently, it was shown that MPA also decreases ATP pools in mitogen-stimulated T lymphocytes [7]. Earlier studies have shown antiproliferative effects of MMF or MPA in vivo [8], [9].

To learn more about the immunosuppressive mechanisms of action of MMF administered in vivo, we used whole blood cultures instead of purified peripheral blood lymphocytes. The use of whole blood better reflects the environment in which the drug is acting in vivo than using purified peripheral lymphocytes. The use of whole blood overcomes the loss of drug-target binding, the alteration of drug distribution among plasma proteins and red blood cells, and the selective loss of cells, which is caused by isolation of immune cells. Additionally, the whole blood technique is more rapid and requires smaller sample volumes than methods that rely on purified lymphocytes [10], [11].

In earlier studies, the PD effect of immunosuppressive drugs in Con A-stimulated whole blood was measured by [3H]TdR incorporation [12], [13], [14]. But since the immune function is also determined by direct cell–cell interaction through surface antigens with adhesive, co-stimulatory or inhibitory functions [15], we exploited the specificity, sensitivity of flow cytometry to determine PD of MPA over time in MPA treated rats to assess proliferation and the expression of T cell surface antigens, CD25 and CD134. We observed in this study a high correlation between PD and PK [16]. Next, we used these whole blood assays to assess PD of MPA treated heart transplanted rats and found a high correlation between PD and histologic severity of rejection [17].

The whole blood assays used in these studies were limited: (1) blood was stimulated by one mitogen (Con A); (2) only single- or two-color flow cytometry was used; and (3) only two cell surface antigens were detected. We optimized our assays to reduce these limitations, using mitogens which signal through calcium-dependent (Con A, PMA+IONO) or calcium-independent (PMA+anti-CD28) pathways, using a three-color flow cytometry technique and measured the expression of many different cell surface receptors (CD11a, CD25, CD54, CD71 and CD134) with important functions in the immune response [18].

Although our previous work investigated the PD effects of MPA the present study used MMF since this drug is approved for use in clinical transplantation. We designed this study to uncover the mechanism of action of MMF and to determine the correlation between MMF PD and PK. To achieve these goals, we used our optimized whole blood assays to measure lymphocyte proliferation by [3H]TdR incorporation and flow cytometric detection of PCNA expression and DNA content. The cyclin PCNA is an auxiliary protein necessary for DNA polymerase and is maximally expressed in the mid-S-phase of the cell cycle [19].

We also measured the expression of different T lymphocyte surface antigens with three-color flow cytometric detection. CD25, α-chain of the IL-2 receptor, is expressed in the early phase after T-cell activation. The clonal proliferation of activated T cells depends upon the expression of this receptor and resting lymphocytes do not express CD25 [20]. Expression of CD134 (OX40), a member of the nerve growth factor/tumor necrosis superfamily (NGF/TNF), is restricted to expression on activated CD4 lymphoblasts [21] and is involved in adhesion of activated T cells to vascular endothelial cells [22]. Expression of CD71, the transferrin receptor, must be increased to internalize the iron required for DNA synthesis and proliferation [23]. CD11a (LFA-1 α-chain), is the counter-receptor for CD54 (ICAM-1) and both are bidirectionally expressed on the cell surface (ICAM-1 on APC bind LFA-1 on T cells and LFA-1 on APC bind ICAM-1 on T cells) [24]. LFA-1 is the ligand for ICAM-1 and provides a co-stimulatory signal for T-cell receptor-mediated activation of resting T cells [25].

The mechanisms of action and effects of immunosuppressive drugs on immune cells have been primarily defined from in vitro studies. The effects of MPA in vitro on the expression of CD25 are controversial. MPA has been reported either to have no effect [26], [27] or to inhibit the expression of CD25 in antigen- or mitogen-activated purified lymphocytes [5]. In MLR cultures, MPA inhibits CD25 and CD71 on allostimulated T-cell subsets [28]. Earlier studies showed that MPA inhibits expression of adhesion molecules expressed on endothelial cells or monocytes [8], [29], but recent studies do not show MPA decreases glycosylation in Con A-stimulated PBL [30].

The inhibition of lymphocyte proliferation is presumed to be the most important immunosuppressive effect of MMF. However, our data showed that MMF inhibited the expression of many T cell surface activation antigens after stimulation of whole blood with different mitogens signaling through calcium-dependent and -independent pathways of T-cell activation. These new mechanisms of action of MMF may contribute significantly to its overall efficacy. In addition, we also showed a significant correlation between PD and PK of MMF.

Section snippets

Objective

Our primary objectives were to exploit improved whole blood assays of T cell functions to investigate the mechanisms of immunosuppressive action of MPA in vitro and the PD effects of MMF administered in vivo. More specifically, we used three-color flow cytometry to quantitate the suppression by MMF of T-cell activation caused by three distinct mitogenic stimuli. T-Cell activation was determined by quantitating proliferation and suppression of expression of cell surface growth factor receptors

Animals

Adult male Lewis (LEW, RT1I/CrlBR, viral antibody-free, Charles River Laboratories, Wilmington, MA) rats weighing 335–370 g were housed in polycarbonate microisolation cages. Standard diet and tap water were provided ad libitum and animals were acclimated under a 12-h light/dark cycle for 2 weeks before the study began. The study was approved by the institutional animal care and use committee. The animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’,

MPA potency and inhibition of lymphocyte functions in vitro

MPA was added to whole blood to produce six different blood concentrations: 0.25; 0.5; 1; 5; 10; and 100 μM (0.08, 0.16, 0.32, 1.6, 3.2 and 32 mg/l) and allowed to equilibrate for 30 min at 37°C. Blood was then diluted and stimulated with Con A, PMA+CD28 or PMA+IONO. We did not measure CD11a or CD54 expression after PMA+IONO-stimulation, because of the lack of adhesion molecule expression following stimulation with this mitogen [35].

There were clear concentration-dependent inhibitions of [3

Discussion

In this study we showed that MMF suppressed the expression of important surface antigens on T lymphocytes after calcium-dependent or -independent stimulation of whole blood. We also found high correlations between PD and PK of MMF.

The inability to observe effects of immunosuppressive drugs on immune cells in vivo has limited drug development, their optimum clinical use and the design and interpretation of clinical trials in transplantation. But now new technologies using small amounts of whole

Nomenclature

[3H]TdRTritium labeled thymidine
PBMCPeripheral blood mononuclear cells
PCNAProliferating cell nuclear antigen
IL-2 RInterleukin-2 receptor
NGF-/TNF-RNerve growth factor-/tumor necrosis factor receptor
LFA-1Leucocyte function antigen-1
ICAM-1Intercellular adhesion molecule-1
APCAntigen presenting cell
MLRMixed lymphocyte reaction
Con AConcanavalin A
PMAPhorbol 12-myristate 13-acetate
IONOIonomycin
MMFMycophenolate mofetil
MPAMycophenolic acid
PDPharmacodynamics
PKPharmacokinetics
HPLCHigh performance liquid

Acknowledgements

This work supported by Roche pharmaceutical company, NJ, USA and also by Hedco Foundation and the Ralph and Marian Falk Trust. Markus J. Barten was supported by the Novartis study grant of the European Society of Transplantation. Teun van Gelder was supported by the Foundation ‘Vereniging Trustfonds Erasmus Universiteit Rotterdam’ in the Netherlands and by the Dutch Kidney Foundation. Jan F. Gummert was supported by Deutsche Forschungsgemeinschaft grant Gu 472/1-1.

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