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Hydrogen Sulfide Reduces Neutrophil Recruitment in Hind-Limb Ischemia-Reperfusion Injury in an L-Selectin and ADAM-17–Dependent Manner

Ball, Carissa J. M.S.; Reiffel, Alyssa J. M.D.; Chintalapani, Sathvika M.Eng.; Kim, Minsoo Ph.D.; Spector, Jason A. M.D.; King, Michael R. Ph.D.

Plastic and Reconstructive Surgery: March 2013 - Volume 131 - Issue 3 - p 487–497
doi: 10.1097/PRS.0b013e31827c6e9c
Experimental: Original Articles

Background: Reperfusion following ischemia leads to neutrophil recruitment into injured tissue. Selectins and β2-integrins regulate neutrophil interaction with the endothelium during neutrophil rolling and firm adhesion. Excessive neutrophil infiltration into tissue is thought to contribute to ischemia-reperfusion injury damage. Hydrogen sulfide mitigates the damage caused by ischemia-reperfusion injury. This study's objective was to determine the effect of hydrogen sulfide on neutrophil adhesion receptor expression.

Methods: Human neutrophils were either left untreated or incubated in 20 μM hydrogen sulfide and/or 50 μg/ml pharmacologic ADAM-17 inhibitor TAPI-0; activated by interleukin-8, fMLP, or TNF-α; and labeled against P-selectin glycoprotein ligand-1, leukocyte function associated antigen-1, Mac-1 α, L-selectin, and β2-integrin epitopes CBRM1/5 or KIM127 for flow cytometry. Cohorts of three C57BL/6 mice received an intravenous dose of saline vehicle or 20 μM hydrogen sulfide with or without 50 μg/ml TAPI-0 before unilateral tourniquet-induced hind-limb ischemia for 3 hours followed by 3 hours of reperfusion. Bilateral gastrocnemius muscles were processed for histology before neutrophil infiltration quantification.

Results: Hydrogen sulfide treatment significantly increased L-selectin shedding from human neutrophils following activation by fMLP and interleukin-8 in an ADAM-17–dependent manner. Mice treated with hydrogen sulfide to raise bloodstream concentration by 20 μM before ischemia or reperfusion showed a significant reduction in neutrophil recruitment into skeletal muscle tissue following tourniquet-induced hind-limb ischemia-reperfusion injury.

Conclusions: Hydrogen sulfide administration results in the down-regulation of L-selectin expression in activated human neutrophils. This leads to a reduction in neutrophil extravasation and tissue infiltration and may partially account for the protective effects of hydrogen sulfide seen in the setting of ischemia-reperfusion injury.


Ithaca, New York, and Rochester, N.Y.

From the Department of Biomedical Engineering, Cornell University; the Division of Plastic Surgery, Laboratory of Bioregenerative Medicine and Surgery, Weill Cornell Medical College; and the Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester.

Received for publication May 3, 2012; accepted August 31, 2012.

Presented at the Biomedical Engineering Society Annual Meeting, in Hartford, Connecticut, October 11 through 16, 2011; the 56th Annual Meeting of the Plastic Surgery Research Council, in Louisville, Kentucky, April 27 through 30, 2011; the 6th Annual Academic Surgical Congress, in Huntington Beach, California, February 1 through 3, 2011; and the 97th Annual American College of Surgeons Clinical Congress, in San Francisco, California, October 23 through 27, 2011.

Disclosure: The authors have no financial conflicts of interest to disclose.

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This work was supported by THE PLASTIC SURGERY FOUNDATION.

Michael R. King, Ph.D.; 526 Campus Road, 205 Weill Hall, Cornell University, Ithaca, N.Y. 14853,

©2013American Society of Plastic Surgeons