Anti-alpha4beta1 integrin antibody induces receptor internalization and does not impair the function of circulating neutrophilic leukocytes.
OBJECTIVE
A compelling strategy for treatment of spinal cord injury is the blockade of integrin-mediated leukocyte extravasation using a monoclonal antibody (mAb) against the alpha4 subunit of the alpha4beta1-integrin. However, little is known with respect to neutrophil function following anti-alpha4 mAb treatment. This study assessed the effects of anti-alpha4 mAb binding on neutrophil activation [reactive oxygen species (ROS) production], function (phagocytic activity) and anti-alpha4-mAb/alpha4beta1-integrin-complex internalization.


METHODS
Resting, primed or stimulated rat neutrophils were incubated ex vivo with anti-alpha4 mAb or isotype-control antibody. ROS production, phagocytic activity, and anti-alpha4-mAb/alpha4beta1-integrin-complex internalization were determined by flow cytometry using dihydrorhodamine (DHR1,2,3), fluorescent microspheres, and indirect immunolabeling, respectively.


RESULTS
Brief (0.5 h) incubation of resting, primed or activated neutrophils with anti-alpha4 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-alpha4-mAb/alpha4beta1-integrin-complex was internalized after 1 h of anti-alpha4 mAb treatment and remained internalized up to 6 h.


CONCLUSIONS
Neutrophil ROS production and phagocytic function remain unaltered after brief anti-alpha4 mAb exposure, demonstrating that use of this mAb as a treatment should not adversely affect important beneficial roles of these cells.
Fleming
Jennifer C
JC
Schulich School of Medicine and Dentistry, Robarts Research Institute, The University of Western Ontario, London, ON, Canada. jflemi8@uwo.ca
Bao
Feng
F
Cepinskas
Gediminas
G
Weaver
Lynne C
LC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000627:therapeutic use; D002634:Chemotaxis, Leukocyte / Q000276:immunology; D005260:Female; D039041:Integrin alpha4beta1 / Q000276:immunology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000276:immunology*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013119:Spinal Cord Injuries / Q000276:immunology / Q000628:therapy; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D000911:Antibodies, Monoclonal; D039041:Integrin alpha4beta1; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013755:Tetradecanoylphorbol Acetate