[Show abstract][Hide abstract] ABSTRACT: : 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.
: 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.
: 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.
: 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.
Plastic and Reconstructive Surgery 03/2013; 131(3):487-97. DOI:10.1097/PRS.0b013e31827c6e9c · 2.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: L-selectin is thought to be a key molecule in neutrophil adhesion and recruitment to sites of ischemia-reperfusion injury (IRI). ADAM-17, a protease on the surface of neutrophils responsible for the cleavage of L-selectin during activation, directly modulates the adhesion of leukocytes to the blood vessel wall during an inflammatory response. This work aims to understand how hydrogen sulfide (HS), a gasotransmitter shown to mitigate IRI, affects neutrophil adhesion protein expression and tissue infiltration.
METHODS: In vitro: Neutrophils were isolated from human peripheral blood, extracted, washed and suspended in HBSS. Some neutrophils were incubated in 20μM NaHS or saline vehicle, labeled with monoclonal antibodies against PSGL-1, LFA-1, Mac-1 α, L-selectin and β2 integrin CBRM1/5 epitope, and analyzed by flow cytometry. Additional isolated neutrophils were treated with NaHS as above and briefly activated with IL-8, fMLP or TNF-α, labeled with anti-L-selectin monoclonal antibody, and analyzed using flow cytometry. In vivo: Cohorts of 3 C57BL/6 mice were treated with an intravenous dose of saline vehicle, 20μM NaHS, 50μg/mL TAPI-0 (a pharmacological ADAM-17 inhibitor), or 20μM NaHS and 50μg/mL TAPI-0. Mice subsequently underwent unilateral tourniquet induced hind-limb ischemia for 3 hours followed by 3 hours of reperfusion. Bilateral gastrocnemius muscles were harvested and processed for histology. Specimens were stained with hematoxylin and eosin, and neutrophil infiltration into the ischemic-reperfused tissue was quantified.
RESULTS: The administration of 20μM NaHS had no effect on adhesion protein expression in resting neutrophils. However, NaHS treatment significantly increased the extent of L-selectin shedding from cells by 1.8-fold and 2.5-fold following exogenous activation by fMLP and IL-8 (p<0.05). In addition, NaHS administration reduced neutrophil infiltration into tissue subjected to IRI by 4.6-fold (p<0.05). This effect was found to be ADAM-17-dependent, given that mice treated with both TAPI-0 and NaHS showed no significant reduction in neutrophil infiltration compared to the saline control (Figure).
CONCLUSIONS: NaHS administration results in the downregulation 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 NaHS seen in the setting of IRI. Furthermore, these are the first data to demonstrate changes in neutrophil protein expression induced by HS and may serve to guide potential therapeutic interventions in the future.
[Show abstract][Hide abstract] ABSTRACT: Neutrophils have recently been shown to release DNA-based extracellular traps that contribute to microbicidal killing and
have also been implicated in autoimmunity. The role of neutrophil extracellular trap (NET) formation in the host response
to nonbacterial pathogens has received much less attention. Here, we show that the protozoan pathogen Toxoplasma gondii elicits the production of NETs from human and mouse neutrophils. Tachyzoites of each of the three major parasite strain types
were efficiently entrapped within NETs, resulting in decreased parasite viability. We also show that Toxoplasma activates a MEK-extracellular signal-regulated kinase (ERK) pathway in neutrophils and that the inhibition of this pathway
leads to decreased NET formation. To determine if Toxoplasma induced NET formation in vivo, we employed a mouse intranasal infection model. We found that the administration of tachyzoites by this route induced a
rapid tissue recruitment of neutrophils with evidence of extracellular DNA release. Taken together, these data indicate a
role for NETs in the host innate response to protozoan infection. We propose that NET formation limits infection by direct
microbicidal effects on Toxoplasma as well as by interfering with the ability of the parasite to invade target host cells.
Infection and immunity 11/2011; 80(2):768-77. DOI:10.1128/IAI.05730-11 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: L-selectin is a key molecule that participates in neutrophil tethering and subsequent rolling. It is cleaved from the surface of neutrophils activated in the presence of lipopolysaccharides, N-formyl-methionine-leucine-phenylalanine (fMLP), or Interleukin-8 (IL-8). We previously showed that L-selectin is also shed from the neutrophil surface during rolling on sialyl Lewis-x coated surfaces in a force-, ADAM-17 sheddase-, and p38 MAP kinase-dependent manner under flow. c-Abl tyrosine kinase is phosphorylated when L-selectin on the surface of neutrophils is cross-linked with anti-L-selectin antibodies. Here, we study the effect of c-Abl inhibition on L-selectin shedding from primary human neutrophils in static conditions following exposure to fMLP, IL-8, and hypotonic buffer and under flow through sialyl Lewis-x coated microtubes. Results indicate that c-Abl inhibition by STI571 significantly affects neutrophil adhesion via L-selectin, by decreasing the average rolling velocity and increasing the flux of rolling cells. The change in surface receptor expression was verified by flow cytometry. Interestingly, other forms of L-selectin shedding induced by fMLP, IL-8 or osmotic swelling were unaffected by STI571 treatment. These findings implicate the c-Abl signaling molecule in regulating L-selectin mechanical shedding in response to shear stress, setting this type of signaling apart from those triggered by the presence of a hypotonic environment, fMLP, or IL-8. This study sheds light on the role of c-Abl in neutrophil adhesion not previously reported in the literature.