Hydrogen sulfide reduces neutrophil recruitment in hind-limb ischemia-reperfusion injury in an L-selectin and ADAM-17-dependent manner

Abstract

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.

Figure 1. HS treatment modifies neutrophil tissue infiltration

Following injury, pro-inflammatory cytokines and chemokines released by damaged tissue attract neutrophils from the bloodstream. E- and P-selectin on the blood vessel wall interact with L-selectin to recruit neutrophils from the bloodstream, causing them to roll on the endothelium, shedding L-selectin as they roll. The neutrophil β₂-integrins form firm adhesions with endothelial ICAM-1, enabling the leukocytes to crawl and migrate into the injured tissue. The present work suggests that following treatment with HS, neutrophils roll on the endothelium, losing L-selectin too quickly to allow the formation of firm adhesions and infiltration into the injured tissue. This reduction in neutrophil infiltration may contribute to the protective effect observed when HS is administered to treat IRI.

Figure 2. HS treatment modifies L-selectin shedding from activated human neutrophils

NaHS treated and untreated neutrophils were incubated with either the ADAM-17 inhibitor TAPI-0 or p38 MAP kinase inhibitor SB203580. The cells were then activated with IL-8, fMLP, or TNF-α prior to flow cytometry quantification of L-selectin expression. pH = 7.4. n=5 human donors. Error bars are s.e.m..

Figure 3. Neutrophil infiltration into skeletal muscle when HS is administered prior to ischemia and reperfusion

Mice were treated with either saline (untreated) (A, B), NaHS (C, D), ADAM-17 inhibitor TAPI-0 (E, F), or NaHS and TAPI-0 (G, H) 20 minutes prior to the induction of unilateral hind-limb ischemia followed by 3 hours of reperfusion. H&E stained histology samples were obtained from both the non-ischemic (A, C, E, G) and ischemic hindlimbs (C, D, F, H). The number of neutrophils infiltrating the tissue was quantified based on nuclear shape and calculated as a fraction of the total number of nuclei present in each picture (I). P-values determined using a two-tailed t-test. * p<0.05, NS = not significant. Scale bar = 50 μm. n=3 mice per treatment group. Error bars are s.e.m..

Figure 4. ADAM-17 inhibition reverses the protective effect of HS in ischemic tissue

Mice were treated with either saline (untreated) (A, B), NaHS (C, D), ADAM-17 inhibitor TAPI-0 (E, F), or NaHS and TAPI-0 (G, H) 20 minutes prior to reperfusion, following 3 hours of unilateral hind-limb ischemia. Neutrophil infiltration into the tissue was quantified based on nuclear shape and calculated as a fraction of the total number of nuclei present in each picture (I). Samples were stained with H&E. P-values determined using a two-tailed t-test. * p<0.05, NS = not significant. Scale bar = 50 μm. n=3 mice per treatment group. Error bars are s.e.m..