Functional status of peripheral blood T-cells in ischemic stroke patients

Abstract

Stroke is a major cause of disability and leading cause of death in the northern hemisphere. Only recently it became evident that cerebral ischemia not only leads to brain tissue damage and subsequent local inflammation but also to a dramatic loss of peripheral blood T-cells with subsequent infections. However, only scarce information is available on the activation status of surviving T cells. This study therefore addressed the functional consequences of immunological changes induced by stroke in humans. For this purpose peripheral blood T-cells were isolated from 93 stroke patients and the expression of activation makers was determined. In addition ex vivo stimulation assays were applied to asses the functionality of T cells derived from blood of stroke patients. Compared to healthy controls, stroke patients demonstrated an enhanced surface expression of HLA-DR (p<0.0001) and CD25 (p = 0.02) on T cells, revealing that stroke leads to T cell activation, while CTLA-4 remained undetectable. In vitro studies revealed that catecholamines inhibit CTLA-4 upregulation in activated T cells. Ex vivo, T cells of stroke patients proliferated unimpaired and released increased amounts of the proinflammatory cytokine TNF-alpha (p<0.01) and IL-6 (p<0.05). Also, in sera of stroke patients HMGB1 concentrations were increased (p = 0.0002). The data demonstrate that surviving T cells in stroke patients remain fully functional and are primed towards a TH1 response, in addition we provide evidence that catecholamine mediated inhibition of CTLA-4 expression and serum HMGB1 release are possible mediators in stroke induced activation of T cells.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Th1-type cytokines are downregulated in stroke patients.

IL-2, IL-12p70, IFN-γ and TNF-α serum cytokine levels are significantly reduced in sera of stroke patients (grey bars) compared to healthy controls (white bars) in the first two weeks following stroke. * p(IL-2, IL-12p70, TNF-α) = 16; 20; 20; 21; 13; n(IFN-γ) = 16; 20; 20; 19; 12 (control; day 0; day 1; day 7; day14).

Figure 2. HMGB1 is upregulated in serum of stroke patients.

HMGB1 is upregulated in serum of stroke patients (grey bars) compared to healthy controls (white bars) in the first two weeks following stroke while its decoy receptors sRAGE and esRAGE remain indistinguishable within 48h following stroke. ** p(HMGB1) = 16; 87; 84; 46; 24; n(sRAGE,) 12; 32; 29, esRAGE) = 10; 37; 34, (control; day 0; day 1; day 7; day14).

Figure 3. CD4+ T-cells in the peripheral blood of stroke patients are activated.

Comparison of expression of activation markers on lymphocytes between healthy controls (white bars) and stroke patients (grey bars) in the first two weeks following stroke reveal that CD4+ T-cells in the peripheral blood are activated. * p<0.05; ** p<0.01; *** p<0.001. Medians and interquartile ranges. n(CD4 + CD25 + , CD25 + %CD4 + ) = 14; 33; 32; 24; 14; n(CD4 + HLA-DR + ) = 12; 64; 66; 42; 22; n(HLA-DR + %CD4 + ) = 12; 63; 66; 43; 22; (control; day 0; day 1; day 7; day14).

Figure 4. Lack of CTLA-4 expression and inhibition by catecholamines.

A) The percentage of PBMC expressing CTLA-4 is undistinguishable between healthy controls (white bars) and stroke patients (grey bars) in the first two weeks following stroke. Medians and interquartile ranges. n = 9; 31; 33; 27; 20; (control; day 0; day 1; day 7; day14). B) Percentage of CTLA-4 expression on PBMC decreases after in vitro treatment with PHA+epinephrine or PHA+norepinephrine compared to treatment with PHA alone. *** p<0.001. Medians and interquartile ranges. .n = 13; 6; 9; (PHA; PHA+Epi; PHA+Norepi).

Figure 5. Surviving T lymphocytes are functional.

A) The proliferation expressed as stimulation index of T cells after ex vivo stimulation with PHA reveals normal proliferative responses of stroke patients (grey bars) lymphocytes compared to healthy controls (white bars) in the first week following stroke. Medians and interquartile ranges. n = 7; 36; 35; 32; (control; day 0; day 1; day 7). B–E) Exvivo stimulation of T cells with PHA leads to increased proinflammatory cytokine release in supernatants of stroke patients cells (grey bars) compared to healthy controls (white bars) within the first week following stroke. * p<0.05; ** p<0.01. Medians and interquartile ranges. n(IL-1β, IL-6, TNFα, TNF-β) = 10; 37; 36; 28; (control; day 0; day 1; day 7).

Figure 6. Relative proportion of activated T cells were undistinguishable between patient subgroups.

Activation markers on lymphocytes from stroke patients without subsequent infection (white bars) remained undistinguishable from those of stroke patients with subsequent infection (grey bars) within the relative proportion of lymphocytes in the first two weeks following stroke. * p(CD4 + CD25 + , CD25 + %CD4 + ) = 5; 7; 7; 7; 7; 6; 6; 3; n(CD4 + HLA-DR + , HLA-DR + %CD4 + ) = 14; 12; 16; 11; 12; 9; 8; 5 (no infection day 0; subsequent infection day 0; no infection day 1; subsequent infection day 1; no infection day 7; subsequent infection day 7; no infection day 14; subsequent infection day 14).

Figure 7. Increased metanephrine and cortisol serum levels in patients with subsequent infection.

Metanephrine and cortisol serum levels are increased in serum of stroke patients with subsequent infection (grey bars) compared to stroke patients without subsequent infection (white bars) within 24h following stroke. * p(Metanephrine) = 8; 8; 8; 7; n(Cortisol) = 10; 8; 9; 9; (no infection day 0; subsequent infection day 0; no infection day 1; subsequent infection day 1).