Anti-α4β1 integrin antibody induces receptor internalization and does not impair the function of circulating neutrophilic leukocytes

A compelling strategy for treatment of spinal cord injury is the blockade of integrin-mediated leukocyte extravasation using a monoclonal antibody (mAb) against the alpha 4 subunit of the alpha 4 beta 1-integrin. However, little is known with respect to neutrophil function following anti-alpha 4 mAb treatment. This study assessed the effects of anti-alpha 4 mAb binding on neutrophil activation [reactive oxygen species (ROS) production], function (phagocytic activity) and anti-alpha 4-mAb/alpha 4 beta 1-integrin-complex internalization. Resting, primed or stimulated rat neutrophils were incubated ex vivo with anti-alpha 4 mAb or isotype-control antibody. ROS production, phagocytic activity, and anti-alpha 4-mAb/alpha 4 beta 1-integrin-complex internalization were determined by flow cytometry using dihydrorhodamine (DHR1,2,3), fluorescent microspheres, and indirect immunolabeling, respectively. Brief (0.5 h) incubation of resting, primed or activated neutrophils with anti-alpha 4 mAb had no effect on ROS production and did not change neutrophil phagocytic activity. However, prolonged incubation (2 h), assessed only in resting neutrophils, increased ROS production. The anti-alpha 4-mAb/alpha 4 beta 1-integrin-complex was internalized after 1 h of anti-alpha 4 mAb treatment and remained internalized up to 6 h. Neutrophil ROS production and phagocytic function remain unaltered after brief anti-alpha 4 mAb exposure, demonstrating that use of this mAb as a treatment should not adversely affect important beneficial roles of these cells.