Abstract
Leukocytes are recruited at the site of infection or injury as a part of the innate immune system, and play a very critical role in fighting the invading microorganisms and/or healing wounds. Neutrophils are the most abundant leukocytes in healthy humans and are the principal cell types that arrive at the target site in the initial phase of this process. Previous studies from our laboratory have shown that the amino acid glutamate is a novel chemotaxis-inducing factor for human neutrophils. In this report, we provide evidences that clearly demonstrate that the glutamate-induced neutrophil cell migration activity is mediated by the class I metabotropic glutamate receptors. Our results further show that a specific integrin β2 (ITG β2) receptor, namely LFA1 (αLβ2) is activated upon glutamate treatment and is required for further downstream signaling events leading to increased migration of human neutrophil cells. Following glutamate stimulation, LFA1 is phosphorylated by the Src Kinase Lck at the Y735 residue, which triggers a downstream signaling cascade leading to activation of PI3K, Syk, Vav and finally the Rho family GTPase, Rac2. Interestingly, glutamate was previously found to be present in elevated levels in wound fluid. Furthermore, glutamate level was also found to go up following inflammation. Taken together, our study suggests a novel mode of neutrophil recruitment to the target site following an infection or injury.
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Acknowledgments
The authors are grateful to Dr. Sandipan Ganguly, NICED (National Institute of Cholera And Enteric Diseases), Kolkata, India for the confocal microscopy as well as Mr. Kanchan Karmakar for his help during the study. The authors are highly obliged to Prof. Gopal Kundu, NCCS (National Centre for Cell Science), Pune, India for providing Lck and Syk inhibitors and to Prof Gary Bokoch, Scripps Research Institute, for providing the GST-PBD construct and Rac2 antibody. We are extremely thankful to Dr. S. Nath and Dr. G. P. Srivasatava, Albert David, Ltd., Kolkata, India for the outstanding cooperation received during the project, and Mr. Sanjay Dudhoria and Mr. Santanu Paul for help with the HPLC system. We also most gratefully acknowledge Dr. Amitabha Majumdar, (Weill Medical College of Cornell University, New York, USA) for the Alexa Phalloidin and Dr. Geetanjali Sundaram, University of Calcutta, India, for help with the flow cytometry. Finally, the authors greatly acknowledge the kind cooperation rendered during the work from the members of the Chattopadhyay laboratory as well as various volunteers who donated their blood to sustain the study.
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R. Gupta and S. Palchaudhuri contributed equally to this article.
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726_2012_1400_MOESM1_ESM.tif
Supplementary material 1. Figure S1 HPLC analysis profile of FITC-glutamate conjugate (a) and only FITC (b). The conjugate was purified by running the reaction mixture in HPLC and collecting only the active fraction (TIFF 915 kb)
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Gupta, R., Palchaudhuri, S. & Chattopadhyay, D. Glutamate induces neutrophil cell migration by activating class I metabotropic glutamate receptors. Amino Acids 44, 757–767 (2013). https://doi.org/10.1007/s00726-012-1400-1
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DOI: https://doi.org/10.1007/s00726-012-1400-1