Effect of the Anti-TNF Agents, Adalimumab, Etanercept, Infliximab, and Certolizumab PEGOL (CDP870) on the Induction of Apoptosis in Activated Peripheral Blood Lymphocytes and Monocytes

Purpose: There are contradictory data in the literature regarding the effect of anti-TNF agents on the apoptosis of leukocytes. This is the first study to compare the effect of the anti-TNF agents etanercept, infliximab, adalimumab, and certolizumab pegol on the induction of apoptosis directly in vitro in normal activated lymphocytes and monocytes. Methods: Leukocytes were stained in whole blood with the directly fluoresceinated anti-TNF reagents or relevant controls and analyzed by flow cytometry after isotonic lysis of red cells. Peripheral blood mononuclear cells were prepared using density gradient centrifugation. Lymphocytes were then isolated from this population by negative selection and monocytes by positive selection using MACS beads. Lymphocytes were cultured with anti-CD3 and anti-CD28 for 24 hours and the monocytes with IL-4 and GMCSF. The effect of the anti-TNF agents on apoptosis of these cells over this period was determined by Annexin V binding using flow cytometry. Results: All four anti-TNF agents bound to peripheral blood neutrophils, monocytes, and a subset of lymphocytes in comparison with negative control reagents. Infliximab, adalimumab, and etanercept were equally potent at causing apoptosis of the activated monocytes and lymphocytes in this system. Certolizumab pegol did not mediate apoptosis of any cells above the level of the negative control. Conclusions: This study confirms the data in the literature regarding the induction of apoptosis of activated monocytes and lymphocytes in vitro by infliximab and adalimumab. However in our system, etanercept was found to mediate mononuclear cell apoptosis in a similar manner to infliximab and adalimumab, which contradicts other published reports. Therefore, our study indicates that the exact conditions used for in vitro experiments may significantly affect the results obtained. These data also raise the possibility that the lack of efficacy seen with etanercept in Crohn's disease is not owing to an inability to cause apoptosis. Certolizumab pegol is the only anti-TNF agent that, under the conditions tested, does not kill leukocytes in vitro by apoptosis.