NF-κB Is Activated in CD4+ iNKT Cells by Sickle Cell Disease and Mediates Rapid Induction of Adenosine A2A Receptors

Gene Lin,Joshua J Field,Joel Linden,Jennifer C Yu,Ruey Ken,Donna Neuberg,David G Nathan,Arun Rishi

doi:10.1371/journal.pone.0074664

Gene Lin, Joshua J Field + Show 6 more

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https://doi.org/10.1371/journal.pone.0074664

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Abstract

Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11–7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists.

Highlights

Reperfusion injury following tissue ischemia is initiated by the activation of invariant natural killer T (iNKT) cells [1,2,3]
The findings demonstrate that in 8 of 8 patients examined acute painful vasoocclusive crises (pVOC) was accompanied by a rapid increase in the percentage of iNKT cells in the circulation that are activated to produce cytokines
We found that p-NF-kB and A2A receptors (A2ARs) immunoreactivity are both elevated in iNKT cells of SCD patients during acute pVOC (Figure 2, C and D)

Summary

Introduction

Reperfusion injury following tissue ischemia is initiated by the activation of iNKT cells [1,2,3]. Disseminated ischemiareperfusion injury is a manifestation of HbSS sickle cell disease that is caused by a homozygous point mutation in the ß-globin gene. The mutation promotes deoxyhemoglobin polymerization, formation of rigid sickled RBCs and production of large numbers of adhesive reticulocytes [4]. Tissue damaging vaso-occlusion in SCD has been viewed as resulting from obstruction of small blood vessels by sickled RBCs [5]. A modified paradigm has emerged suggesting that the clinical manifestations of SCD occur in part as a consequence of white cell activation [6]. As in ischemia-reperfusion injury, in NY1DD mice with SCD the activation of iNKT cells in response to tissue ischemia initiates an inflammatory cascade [7]. Poor lung function in SCD mice is ameliorated by iNKT cell depletion, by blockade of CD1drestricted signaling [7], or by stimulation of anti-inflammatory A2AR receptors that are induced in SCD mice and that inhibit iNKT cell activation [8]

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