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

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Abstract

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 α4 subunit of the α4β1-integrin. However, little is known with respect to neutrophil function following anti-α4 mAb treatment. This study assessed the effects of anti-α4 mAb binding on neutrophil activation [reactive oxygen species (ROS) production], function (phagocytic activity) and anti-α4-mAb/α4β1-integrin-complex internalization.

Methods

Resting, primed or stimulated rat neutrophils were incubated ex vivo with anti-α4 mAb or isotype-control antibody. ROS production, phagocytic activity, and anti-α4-mAb/α4β1-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-α4 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-α4-mAb/α4β1-integrin-complex was internalized after 1 h of anti-α4 mAb treatment and remained internalized up to 6 h.

Conclusion

Neutrophil ROS production and phagocytic function remain unaltered after brief anti-α4 mAb exposure, demonstrating that use of this mAb as a treatment should not adversely affect important beneficial roles of these cells.

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Acknowledgments

This research was supported by grants from the Canadian Institutes of Health Research, the Ontario Neurotrauma Foundation and the Lawson Health Research Institute. JC Fleming was supported by studentships from the Ontario Neurotrauma Foundation and the Canadian Institutes of Health Research. The authors are indebted to Drs. Greg Dekaban and Kristin Chadwick for their superb advice about flow cytometry. The authors also thank Drs. Arthur Brown, Feng Bao, and Canio Polosa for their constructive suggestions regarding this research.

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Authors and Affiliations

  1. Schulich School of Medicine and Dentistry, Robarts Research Institute, The University of Western Ontario, 100 Perth Drive, PO Box 5015, London, ON, N6A 5K8, Canada

    Jennifer C. Fleming, Feng Bao & Lynne C. Weaver

  2. Center for Critical Illness Research, Lawson Health Research Institute, Victoria Research Laboratories, 800 Commissioners Road E, London, ON, N6A 4G5, Canada

    Jennifer C. Fleming & Gediminas Cepinskas

Authors
  1. Jennifer C. Fleming
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  2. Feng Bao
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  3. Gediminas Cepinskas
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  4. Lynne C. Weaver
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Correspondence to Jennifer C. Fleming.

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Responsible Editor: L. Li.

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Fleming, J.C., Bao, F., Cepinskas, G. et al. Anti-α4β1 integrin antibody induces receptor internalization and does not impair the function of circulating neutrophilic leukocytes. Inflamm. Res. 59, 647–657 (2010). https://doi.org/10.1007/s00011-010-0177-5

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  • DOI: https://doi.org/10.1007/s00011-010-0177-5

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