Assessment of mechanisms of action of immunosuppressive drugs using novel whole blood assays

Assessment of Mechanisms of Action of Immunosuppressive Drugs Using Novel Whole Blood Assays M.J. Barten, J.F. Gummert, T. van Gelder, R. Shorthouse, and R.E. Morris

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HOLE BLOOD ASSAYS are preferred because they better approximate the complex in vivo relationships among drug concentration, the binding of drug to plasma constituents and cellular targets, and the interaction among immune cells and their products. Additionally, the whole blood technique is more rapid and requires smaller sample volumes than methods that rely on purified lymphocytes.1 We assessed lymphocyte proliferation by measuring [3H]-TdR incorporation and also assessed the expression of the T-lymphocyte surface antigens CD11a, CD25, CD54, and CD71 with flow cytometry analysis. We stimulated whole blood with concanavalin A (Con A) and phorbol 13-myristate 12-acetate (PMA) plus anti-CD28 (CD28) and determined the relative potencies of different immunosuppressive drugs on inhibition of lymphocyte function.

MATERIALS AND METHODS Blood was obtained from Lewis rats (Charles River Laboratories, Wilmington, Mass, USA). Different concentrations of cyclosporine (CsA, Novartis, East Hanover, NJ, USA), tacrolimus (TRL, Fujisawa Pharmaceuticals Inc Ltd, Osaka, Japan), sirolimus (SRL, Weyth-Ayerst Research, Princeton, NJ, USA), and mycophenolic acid (MPA, Sigma Chemical Co, St Louis, Mo, USA) were added to whole blood. Diluted blood was stimulated with either Con A (15 ␮g/mL, Vector Laboratories, Inc, Burlingame, Calif, USA) or PMA (50 ng/mL, Sigma Chemical Co, St Louis, Mo, USA) plus anti-CD28 (10 ␮g/mL, Clone: JJ319; Pharmingen, San Diego, Calif, USA). Lymphocyte proliferation was measured by [3H]-TdR (1 ␮Ci, 6.7 Ci/mol, New England Nuclear, Boston, Mass, USA) incorporation. Lymphocyte surface antigen expression was measured by flow cytometric analysis using directly labeled FITC and PE or biotin-labeled mAbs (Pharmingen San Diego, Calif, USA) against Pan-T cell (Ox52), CD11a, CD25, CD54, and CD71. Streptavidin PECy5 (Dako Corporation, Carpinteria, Calif, USA) was added to bind to biotin-labeled mAb. Five thousand events of each sample were analyzed with an Epics XL-MCL flow cytometer (Coulter Corporation, Miami, Fla, USA). All data are expressed as mean ⫾ standard error of mean (SEM). The effects of all drugs were calculated as follows: % inhibition ⫽ [1 ⫺ (with drug/without drug)] ⫻ 100. Drug concentrations that produce 50% inhibition (IC50) were calculated after fitting the concentration-effect curves in an Imax sigmoidal pharmacodynamic model (WinNonlin 1.1, Scientific Consulting, Inc, Cary, NC, USA).

RESULTS

SRL was the drug that most potently inhibited lymphocyte proliferation and activation in our whole blood assays followed by TRL, CsA, and MPA. SRL more potently inhibited lymphocyte function after PMA ⫹ CD28 stimulation ([3H]TdR: 0.6 ⫾ 0.2 nmol/L and CD25: 0.8 ⫾ 0.1 nmol/L) than after Con A stimulation ([3H]TdR: 8.1 ⫾ 1.6 nmol/L and CD25: 7.8 ⫾ 1.2 nmol/L). We observed higher IC50 values for TRL after Con A stimulation ([3H]TdR: 26 ⫾ 0.6 nmol/L and CD25: 23 ⫾ 0.6 nmol/L) compared to IC50 values after PMA ⫹ CD28 stimulation ([3H]TdR: 12 ⫾ 0.7 nmol/L and CD25: 19 ⫾ 2 nmol/L). The IC50 values for CsA after Con A stimulation were [3H]TdR: 550 ⫾ 24 nmol/L and CD25: 578 ⫾ 31 nmol/L, and after PMA ⫹ CD28 stimulation were [3H]TdR: 385 ⫾ 17 nmol/L and CD25: 721 ⫾ 54 nmol/L. The potency of MPA for inhibition of immune function was greater after Con A stimulation (431 ⫾ 4 nmol/L) compared to the potency after PMA ⫹ CD28 stimulation (985 ⫾ 20 nmol/L). In addition to its antiproliferative effect, MPA showed an effective dosedependent suppression of the expression of various lymphocyte surface antigens (eg, CD25: Con A: 896 ⫾ 45 nmol/L and PMA ⫹ CD28: 1032 ⫾ 128 nmol/L). There were high correlations between all drug concentrations and the magnitudes of inhibition of all lymphocyte functions for both mitogen assays (r2 ⫽ 0.83– 0.99). DISCUSSION

We define mechanisms of action of immunosuppressive drugs on mitogen-induced lymphocyte proliferation and lymphocyte activation in novel whole blood assays. The relative potencies of all drugs were identical to the relative differences in their effective immunosuppressive doses in vivo. Although other assays have shown that signaling From Transplantation Immunology, Department of Cardiothoracic Surgery, Stanford University Medical School, Stanford, California, USA. This work was supported by the Hedco Foundation and the Ralph and Marian Falk Trust. Address reprint requests to R.E. Morris, MD, Transplantation Immunology, Department of Cardiothoracic Surgery, Stanford University, Stanford, California 94305-5407.

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Transplantation Proceedings, 33, 2119–2120 (2001)

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through PMA ⫹ CD28 is resistant to CsA and TRL, we demonstrated that CsA and TRL do inhibit lymphocyte functions after PMA ⫹ CD28 stimulation. We described a simple and reproducible technique for measuring lymphocyte functions in whole blood. Our results demonstrated the advantages of investigating mechanism of actions of immunosuppressive drugs in whole blood. Therefore, whole

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blood assays of lymphocyte functions could be useful in drug development programs.

REFERENCE 1. Gummert JF, Barten MJ, Sherwood SW, et al: J Pharmacol Exp Ther 291:1100, 1999