Immune activation in the peripheral blood of patients with acute ischemic stroke

Abstract

Lymphocytes, neutrophils and macrophages are found in the brain in areas of acute ischaemic stroke. There is also evidence of modulation of systemic immune function after stroke, with post-stroke immunosuppression being observed. Because lymphocytes are activated in the peripheral immune compartment, before entry to the target organ, we reasoned that activated lymphocytes would be present in the circulation, prior to entering the brain, in patients after stroke. Because immune responses are controlled by regulatory mechanisms, we also reasoned that the post-stroke immunosuppression would involve T regulatory cells. The aim of the study was to look for evidence of immune activation and alterations in regulatory T cells in the peripheral blood of patients after acute ischaemic stroke, in comparison to age-matched healthy controls and patients with other neurological diseases (OND), and to determine the phenotype of the activated cells. The percentages of total and activated T cells, B cells, monocyte/ macrophages, and NK/NK-T cells were determined by labelling peripheral blood leukocytes with specific cell surface markers and analysis with 4-colour flow cytometry. The percentages of activated T cells and regulatory T cells were significantly increased in patients with ischemic stroke compared to healthy subjects and patients with OND. There was also an increase in the percentage of CCR7+ T cells. There were no significant differences in the activation of other cell types. In conclusion, there is evidence of immune activation and Treg cells in acute ischaemic stroke.

Introduction

In stroke, there is activation of microglia, endothelial activation, breakdown of the blood brain barrier and recruitment of leukocytes to the site of injury (Arumugam et al., 2005, Garcia, 1975, Huang et al., 2006, Nilupul et al., 2006). In experimental stroke, the cells in the area of injury include neutrophils and T cells. T cells are found in the ischaemic lesion from day 1, and reach a peak at day 7 (Schroeter et al., 1994). This local inflammatory response contributes to tissue injury, because defects in immunity, such as genetic deficiency of adhesion molecules (Arumugam et al., 2005), disrupt the response to ischaemia. Furthermore, mice that are immunodeficient have less injury than normal mice in experimental stroke (Yilmaz et al., 2006). However, although immune responses in stroke contribute to tissue damage, attempts at improving stroke outcome by immunosuppression have been unsuccessful, and indeed produced a worsening of outcome (Enlimomab acute stroke trial investigators, 2001).

Because T cells are activated in the peripheral immune compartment, the cells that enter the brain are likely to be present in peripheral blood. In an experimental model of stroke in mice there is evidence of a substantial activation of the peripheral immune system, characterized by massive activation of spleen cells to produce cytokines and chemokines (Offner et al., 2006a). There have been few studies of peripheral blood cells after human stroke. There is evidence of increased T cell reactivity, measured as response to PPD, in stroke patients (Tarkowski et al., 1991). Increased production of cytokines from peripheral blood cells after lipopolysaccharide (LPS) stimulation has been demonstrated after stroke (Ferrarese et al., 1999) and there is up-regulation of CD154 (CD40 ligand) and CD40 in human peripheral blood after transient ischaemic attack and stroke (Garlichs et al., 2003). As well as evidence of immune activation, there is also evidence of post-stroke immune suppression. This is thought to contribute to the occurrence of infection after stroke. Recently, reduced numbers of lymphocytes were found in the blood of patients shortly after stroke (Vogelgesang et al., 2008). Immunosuppression occurs after other CNS damage (Meisel et al., 2005)as well as stroke and also occurs after tissue injury in other organ systems (Klava et al., 1997) where it is thought to be due to increased immunoregulatory activity. We suggest that post-stroke immunosuppression is also likely to be due to increased immunoregulatory activity, possibly by activation of Treg cells. Therefore, to obtain further information about peripheral immune activation in human stoke, the present study was performed to investigate the numbers of circulating T cells, B cells and macrophages after stroke, particularly to look for evidence of T cell activation and also for evidence of increased numbers of regulatory T cells (Treg cells), which play a part in regulating immune responses.