Circulating thioredoxin suppresses lipopolysaccharide-induced neutrophil chemotaxis

Abstract

Thioredoxin (Trx), a small (12-kDa), well characterized protein with a highly conserved active site (Cys-32–Gly–Pro–Cys-35), plays a variety of redox-related roles in organisms ranging from Escherichia coli to man (1). Intracellular Trx, together partly with peroxiredoxin (2), plays crucial roles in the scavenging of reactive oxygen species and the regulation of redox-sensitive transcription factors including activator protein-1 and nuclear factor-κB (3, 4). In addition, it plays key roles in the regulation of glucocorticoid receptor-mediated signal transduction and thus in the host defense against reactive oxygen species-mediated inflammation (5–7).

In addition to its intracellular functions, Trx is released by cells and has been shown to have several cytokine and chemokine-like activities. In fact, human Trx was cloned originally as an immunologically active “factor” released in vivo and in vitro by human T cell leukemia virus type I-transformed cells (8) and as an autocrine growth factor produced by Epstein–Barr virus (EBV)-transformed B cells (9). A series of studies demonstrate the cytokine-like activities of the intact Trx protein (10, 11) and, most recently, of the truncated form of Trx (Trx-80), which has been shown to activate monocytes and to be selectively mitogenic for lymphocytes (12, 13).

Trx is released from cells in response to oxidative stress (14, 15). In HIV disease, plasma Trx levels are chronically elevated in a subset of subjects (16) and, when elevated, are associated with poor prognosis in subjects with CD4 T cell counts below 200/μl of blood (15). Chronically elevated plasma Trx levels also have been detected in hepatitis C virus infection, where the elevated levels are associated with decreased responsiveness to IFN-α therapy (17). In contrast, beneficial effects of acute plasma Trx elevation caused by injection of recombinant human Trx have been demonstrated in ischemic reperfusion injury (18, 19) and other situations in which tissue damage results from neutrophil invasion and oxidant production.

Studies reported here, in which we document the role of circulating Trx in regulating the neutrophil extravasation into inflammatory sites, grow from the recent demonstration that locally administered Trx is a potent chemoattractant for neutrophils, monocytes, and lymphocytes in the mouse air pouch chemotaxis model (20). This chemokine-like activity of Trx suggests that circulating Trx might inhibit chemotaxis, because previous studies have demonstrated that pretreatment with chemokines blocks chemotaxis in in vitro assays (21, 22) and that, importantly, injection of IL-8 in rabbits (23, 24) also inhibits local chemotaxis induction. We confirm this hypothesis in a recent report that focuses primarily on Trx elevation in HIV disease but also includes our initial studies showing that i.v. injection of recombinant human Trx blocks lipopolysaccharide (LPS)-stimulated neutrophil chemotaxis in the mouse air pouch model (15).

Here we continue the characterization of this suppression and the mechanisms that mediate it. Thus, we show that (i) elevated plasma Trx levels raised either by i.v. injection of human recombinant Trx into wild-type mice or by stimulation of Trx release in mice that express a human Trx transgene inhibits neutrophil extravasation into the LPS-stimulated air pouch, (ii) Trx directly blocks the in vitro adhesion of LPS-stimulated neutrophils on endothelial cells, and (iii) circulating Trx suppresses the activation of p38 mitogen-activated protein kinase (MAPK) in LPS-stimulated neutrophils and prevents the normal down-regulation of CD62L on neutrophils that migrate into the LPS-stimulated air pouch.

We discuss these studies in the context of the beneficial effects mediated by acutely elevated plasma Trx in ischemia reperfusion injury and interstitial pneumonia and the potentially negative effects mediated by chronic elevation of plasma Trx in HIV and other diseases. In essence, we propose that in both cases the underlying mechanism traces to a physiologically significant inhibition of neutrophil chemotaxis by the circulating Trx.

HUVECs were precultured in the absence or presence of LPS and Trx for 3 h. Expressions of ICAM-1, VCAM-1, CD62P (P-selectin), and CD62E (E-selectin) were analyzed by flow cytometry. The mean fluorescence intensity of each is shown. Similar results were obtained in three different experiments.

Infiltrated cell numbers (× 10^{) were shown as the mean ± SD.}

Acknowledgments

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