Reactive Oxidative Species–Modulated Ca2+ Release Regulates β2 Integrin Activation on CD4+ CD28null T Cells of Acute Coronary Syndrome Patients

Yvonne Samstag, Nicolai V. Bogert, Guido H. Wabnitz, Shabana Din, Markus Therre, Florian Leuschner, Hugo A. Katus and Mathias H. Konstandin
J Immunol October 15, 2020, 205 (8) 2276-2286; DOI: https://doi.org/10.4049/jimmunol.2000327

Key Points

  • CD28null T cells show high reactivity of β2 integrin upon chemokine stimulation.

  • ROS-mediated Ca2+ release is critical for β2 integrin activation in T cells.

  • Spontaneous β2 integrin activity of CD28null T cells in ACS is ROS dependent.

Abstract

The number and activity of T cell subsets in the atherosclerotic plaques are critical for the prognosis of patients with acute coronary syndrome. β2 Integrin activation is pivotal for T cell recruitment and correlates with future cardiac events. Despite this knowledge, differential regulation of adhesiveness in T cell subsets has not been explored yet. In this study, we show that in human T cells, SDF-1α–mediated β2 integrin activation is driven by a, so far, not-described reactive oxidative species (ROS)–regulated calcium influx. Furthermore, we show that CD4+CD28null T cells represent a highly reactive subset showing 25-fold stronger β2 integrin activation upon SDF-1α stimulation compared with CD28+ T cells. Interestingly, ROS-dependent Ca release was much more prevalent in the pathogenetically pivotal CD28null subset compared with the CD28+ T cells, whereas the established mediators of the classical pathways for β2 integrin activation (PKC, PI3K, and PLC) were similarly activated in both T cell subsets. Thus, interference with the calcium flux attenuates spontaneous adhesion of CD28null T cells from acute coronary syndrome patients, and calcium ionophores abolished the observed differences in the adhesion properties between CD28+ and CD28null T cells. Likewise, the adhesion of these T cell subsets was indistinguishable in the presence of exogenous ROS/H2O2. Together, these data provide a molecular explanation of the role of ROS in pathogenesis of plaque destabilization.

Introduction

Coronary artery disease is considered as a chronic inflammatory process within the arterial wall (1–6). Interference with the systemic inflammatory process can improve the outcome as shown in the recent Canakinumab Anti-inflammatory Thrombosis Outcome Study trial (7). The acute exacerbation of the smoldering inflammatory process in the coronary arteries clinically presents as acute coronary syndrome (ACS) (4). In patients with ACS, atherosclerotic plaques are destabilized, which then can rupture under shear stress. Thereby, subendothelial structures come into contact with the bloodstream to initiate a coagulation cascade and thrombus formation (4). Upstream in this process, various T cell populations are involved in this inflammatory response. Among those subsets, Th cells lacking the costimulatory molecule CD28 activate macrophages and vascular smooth muscle cells to release proinflammatory proteins and destabilizing enzymes. In peripheral blood, elevated levels of this T cell subset (CD3+CD4+CD28null) could be found in patients with unstable angina and has prognostic value for the recurrence of future cardiac events (8–10). In a previously described study, we showed that spontaneous β2 integrin activation on T cells precedes myocardial infarction and allows risk prediction independent of established risk markers, including high-sensitivity C-reactive protein (CRP) and cardiac troponin (cTnT) (11). Notably, integrin activation is pivotal for lymphocyte recruitment. These molecules are heterodimers, consisting of an α- and a β-subunit. Although highly expressed on leukocytes, integrins do not mediate adhesion until activated. Cell activation via chemokines leads to an increase in affinity and avidity of these receptors (12, 13). To date, little is known about the differential regulation of β2 integrin–mediated adhesion of T cell subsets, in particular the pathogenetically highly relevant CD3+CD4+CD28null T cell subset.

Atherosclerosis is linked to oxidative stress conditions in the vessel (5) (14–16). Thus, ApoE−/− mice lacking the NADPH oxidase are less susceptible toward atherosclerosis (17). Additionally, several epidemiologic studies found a link between intake of antioxidants and reduction of cardiovascular events (18, 19). It is well accepted that hydrogen peroxide acts as second messenger in different cellular systems for processes like proliferation, differentiation, cell survival, or death as well as adhesion (20–22).

In this study, we show that CD4+CD28null T cells represent a highly reactive subpopulation with increased β2 integrin–mediated adhesive capacity, which differs remarkably from CD28+ Th cells. Interestingly, no difference in the susceptibility regarding interference of already-known classical pathways toward integrin activation (PI3K, PLC, or PKC) could be found between both subsets (23). However, in this study, we introduce a hydrogen peroxide (H2O2)–calcium (Ca2+) signaling pathway that is crucially involved in SDF-1α–dependent, as well as spontaneous, adhesion of T cells from ACS patients. Notably, CD4+CD28null T cells were more dependent on this described H2O2–Ca2+ pathway, indicating differentially regulated signaling pathways for integrin-dependent adhesion in T cell subpopulations.

Materials and Methods

Cells

PBMCs were obtained by Ficoll-Hypaque (Linaris, Wertheim-Bettingen, Germany) density gradient centrifugation of heparinized blood from patients with unstable angina at the University Hospital Heidelberg after written informed consent under a protocol approved by the institutional review board in accordance with the Declaration of Helsinki. T lymphocytes were purified by MACS using the Pan T Cell Isolation Kit II from Miltenyi Biotec according to the manufacturer’s instructions. Cells were maintained in RPMI 1640 medium containing 10% FCS, 1% penicillin/streptomycin, and 2% HEPES (all from Life Technologies). For transfection into primary blood T cells, the Human T Cell Nucleofector Kit (Amaxa Biosystems) was used as described by the manufacturer.

Abs and reagents

AffiniPure FITC or 7-amino-4-methylcoumarin-3-acetic acid goat anti-human Fcγ fragment–specific IgG F(ab′)2 fragments were from Jackson ImmunoResearch Laboratories. The following Abs were from BD Pharmingen: CD3-APC-Cy7, CD28-PE, CD4-PerCp, CCR7-PE-Cy7, CD45RA-APC, CD18-FITC, CD28-APC, and CD4-AmCyan. FACS buffer contained PBS supplemented with 0.05% sodium azide, 0.5% BSA, and 5% FCS. The cDNA encoding for human ICAM-1–Fc was a kind gift from Dr. W. Kolanus (University of Bonn, Bonn, Germany). rICAM-1–Fc was prepared as described previously (24). SDF-1α and the CXCR4-FITC Ab were from R&D Systems (Wiesbaden, Germany). Phospholipase C inhibitor U73122, pertussis toxin (Ptx), PI3K inhibitor Ly-294002, and PKC inhibitor Ro-31-8220 were from Calbiochem Research Biochemicals. All other chemicals used were from Sigma-Aldrich (apocynin [Apo], H2O2, N-acetylcysteine [NAC], BAPTA-AM, diphenylene iodonium chloride [DPI]). In all experiments, cells were preincubated with the respective inhibitor for 60 min at 37°C. The oxidation reduction (redox)–sensitive GFP–Orp vector was used as described recently (25). The vector-encoding DS-Red (red fluorescence protein [RFP]) and GFP was purchased from Invitrogen, Karlsruhe, Germany. The vector-encoding catalase was a gift from Dr. K. Gülow, Deutsches Krebsforschungszentrum, Heidelberg, Germany. The peroxisome targeting sequence has been removed using the QuickChange Site-Directed Mutagenesis Kit from StrataGene, La Jolla, CA, following the manufacturer’s instructions.

Ligand complex–based adhesion assay

To quantify β2 integrin–mediated adhesiveness, we established a flow cytometry–based method (26, 27). This technique allows assessment of avidity as well as affinity-mediated changes in adhesiveness at the single-cell level. Briefly, to generate ICAM-1–Fc-F(ab′)2 complexes (scICAM-1), Ab [anti-human Fcγ-specific IgG F(ab′)2 fragments; in a 1:6.25 dilution], and ICAM-1–Fc (200 μg/ml) were incubated at 4°C overnight in PBS. Purified T cells were resuspended at 20 × 106 cells/ml in PBS containing 0.5% BSA, 2 mM Mg2+, and 1 mM Ca2+. To start the assay, 6.25 μl prepared scICAM-1, 12.5 μl PBS (with 0.5% BSA, 2 mM Mg2+, and 1 mM Ca2+) containing SDF (final concentration 50 ng/ml), or the solvent control was added to 31.25 μl of this cell suspension. To terminate the reaction, 500 μl of 4% paraformaldehyde under the same cationic conditions as during the stimulation were added. After 5 min, fixation was stopped by addition of ice-cold FACS buffer, and tubes were transferred on ice. When not indicated otherwise, SDF-1α stimulation (final concentration 50 ng/ml) was done for 1.5 min, Mg/EGTA (10 mM/1 mM), hydrogen peroxide (100 μM), and ionomycin (1 μg/ml) treatment was 30 min.

For experiments using whole blood, 40 μl thereof was transferred to 37°C. Per sample, 2 μl anti–CCR7-Pe-Cy7 was added. Subsequently, 8 μl of the preformed ICAM-1 complexes and 14 μl PBS containing 5 mM Mg2+ were added. After 30 min, 10 volumes of prewarmed (37°C) FACS lysing solution (BD Biosciences) supplemented with 2 mM Mg2+/1 mM Ca2+ was added, and samples were vortexed and incubated for 5 min at 37°C. Fixation was stopped by adding ice-cold FACS buffer. After fixation, cells were pelleted and stained for surface markers as indicated by incubation on ice for 30 min in the dark. Cells were washed once and analyzed on an LSR II flow cytometer (BD Biosciences, Heidelberg, Germany). If data are presented as normalized mean fluorescence intensity (MFI), in each experiment, the respective positive control was set to one and all other depicted conditions are divided by the MFI of the positive control within each experiment. After normalization within each experiment, the average of three to four independent experiments was calculated and is depicted. In each experiment, appropriate solvent control and negative control were included but are not depicted for clarity of presentation purposes.

Quantification of H2O2 as second messenger

T cells expressing redox-sensitive GFP (roGFP)–Orp were stimulated at 1 mio/ml, and data were acquired online with an LSR II flow cytometer (BD Biosciences) using 405- and 488-nm laser lines for excitation and 500–550-nm as well as 515–545-nm filter sets for emission. Data were analyzed using the FlowJo software 8.8.6 (Tree Star).

Calcium flux measurements

To quantify calcium release, T cells were labeled with Fluo-4 at 1 mM in PBS 0.5% BSA for 30 min at room temperature. Cells were washed once, and MFI was quantified online by LSR II from BD Biosciences.

Patients

Patients presenting with unstable angina at the University Hospital Heidelberg were screened for participation in this study. Other inclusion criteria were age >18 and <80 y and written informed consent under a protocol approved by the institutional review board in accordance with the Declaration of Helsinki. Exclusion criteria were immunosuppressive medication, positive cardiac troponin T, acute infection, inflammatory comorbidity, other organic heart disease, and lack of coronary angiography to confirm coronary artery disease.

Statistics

For comparisons between two groups, the Student t test was used. Between ≥3 groups, ANOVA with Bonferroni post hoc analysis was applied using GraphPad Prism 4.0, version 2003 software. All p values <0.05 were considered statistically significant.

Results

SDF-1α–induced β2 integrin activation is H2O2 dependent in T cells

H2O2 plays an important role as second messenger in different cellular systems (20–22). Its relevance as a mediator in T cell signaling toward β2 integrin activation has not been analyzed in detail. β2 Integrins are strongly activated by chemokine receptor triggering. Thus, we took advantage of a recently published hydrogen peroxide specific detection assay to measure H2O2 release after SDF-1α treatment (25). T cells were purified using Pan T Cell Isolation Kit II from Miltenyi Biotec and were transfected with an roGFP modified by ORP fusion, which makes the sensor specific for hydrogen peroxide. Purified T cells were stimulated with SDF-1α or treated with the solvent control [i.e., PBS (as indicated by the arrow in Fig. 1A) and the relative excitation by either 405 nm (oxidized sensor) or 488 nm (reduced sensor) was depicted online as a ratio (405/488 nm) by flow cytometry]. These experiments showed SDF-1α–induced H2O2 release in contrast to solvent control (Fig. 1A). To elucidate whether the produced H2O2 is involved in β2 integrin activation after chemokine stimulation, T cells were preincubated with two reducing substances, NAC and DTT. The SDF-1α–induced β2 integrin activation was quantified using the flow cytometry–based ligand complex–based adhesion assay (LC-AA) (26, 27), which enables the detection of both kinds of integrin-mediated adhesion processes (i.e., affinity and avidity changes). Soluble preformed multimeric ICAM-1 clusters conjugated with a fluorophore served as ligands for the analysis of β2-integrin activation of cells in solution. Treatment of purified T cells with 10 mM NAC inhibited chemokine-induced adhesiveness by 56.6 ± 0.03% (Fig. 1B, p < 0.01), and preincubation with 3 mM DTT diminished adhesiveness by 92.1 ± 0.01% (Fig. 1C, p < 0.001). In addition, T cells were cotransfected with catalase, truncated for the peroxisome localization sequence, to reduce intracellular H2O2 levels, and an RFP (DS-red), to detect transfected cells, and subjected to the adhesion assay after SDF-1α stimulation. RFP-positive cells were analyzed only. The SDF-1α–induced β2 integrin activation could be blocked in catalase-transfected T cells by 43.6 ± 0.1% (Fig. 1D, p < 0.05). One source of H2O2 in T cells are NADPH oxidases. To elucidate their role in integrin activation, T cells were preincubated with two different NADPH oxidase inhibitors, DPI or Apo. Both substances dose-dependently diminished integrin activation after SDF-1α treatment (Fig. 1E, 1F). Cell death has been excluded in these experiments by propidium iodide staining for all applied inhibitors at the used concentrations. To determine whether different concentrations of H2O2 activate β2 integrins, T cells were incubated with hydrogen peroxide. Fig. 1G clearly shows that H2O2-induced adhesiveness is already significantly detectable at a low concentration (5 μM) and becomes more prominent with increasing concentrations. Taken together, these data show that chemokine-induced reactive oxidative species (ROS) mediate β2 integrin activation in primary human T cells.

FIGURE 1.
FIGURE 1.

SDF-1α–induced β2 integrin activation is H2O2 dependent in T cells from healthy volunteers. (A) After transfection of T cells with the H2O2-specific ROS sensor, the fluorescence emission of roGFP was measured and depicted as ratio of excitation by either 405 or 488 nm. Higher values correspond to increased H2O2 production. The arrow indicates the addition of mock (PBS control) or SDF-1α stimulation. The figure shows one out of three independent experiments. (B and C) T cells were preincubated with NAC (B) and DTT (C) and were stimulated with SDF-1α for 1.5 min. β2 Integrin–dependent adhesion was quantified using the LC-AA (Materials and Methods). Each graph shows the mean of three independent experiments, whereas the values in every experiment were normalized to the SDF-1α–treated sample without inhibitor. (D) T cells were transfected with RFP alone (control) or RFP together with unlabeled catalase (catalase). SDF-1α–induced adhesiveness was analyzed gated on RFP-positive cells. The mean and SEM of three independent experiments is depicted normalized to the SDF-1α stimulation. (E and F) T cells were preincubated with DPI (E) or Apo (F) at the indicated concentrations. β2 Integrin–dependent adhesion was quantified as described in (A) (n = 3; mean and SEM). (G) T cells were incubated with H2O2 with the depicted concentrations, and adhesion was quantified using the LC-AA. *p < 0.05, **p < 0.01, ***p < 0.001.

H2O2- and SDF-1α–induced β2 integrin activation is Ca2+ dependent in T cells

ROS-mediated calcium release is known for many cell types, including T cells (28, 29). Also, significance of calcium signaling for integrin activation has been proven (30). The role of calcium after chemokine stimulation for β2 integrin activation has not been analyzed so far. Therefore, purified T cells were preincubated with the calcium chelator BAPTA-AM and SDF-1α–dependent adhesiveness has been analyzed (Fig. 2A). Pretreatment of T cells with BAPTA-AM inhibited integrin activation by 88 ± 0.02% (p < 0.001). Furthermore, T cells were preincubated with U73122 (PLC inhibitor), Ly-292004 (PI3K inhibitor), Ptx (G protein inhibitor), or Ro31-8220 (PKC inhibitor). As expected, interfering with the well-accepted signaling pathways (PLC, PI3K, G proteins, or PKC) toward β2 integrin activation inhibited SDF-1α–dependent adhesiveness (Fig. 2B–E) (23). As depicted in Fig. 3A, H2O2 also induced a strong calcium signal. Preincubation with inhibitors of PI3K (Ly-292004), PLC (U73122), or G protein (Ptx) had no influence on this calcium release. To demonstrate the dependence of the β2 integrin activation on H2O2-induced calcium release, T cells were again preincubated with the calcium chelator BAPTA-AM, and β2 integrin activation was quantified after H2O2 incubation (Fig. 3B). Indeed, 100 μM BAPTA-AM–diminished hydrogen peroxide evoked β2 integrin activation by 77.1 ± 0.05% (p < 0.001). In summary, chemokine stimulation induces ROS release, which leads to calcium-mediated β2 integrin activation in primary human T cells.

FIGURE 2.
FIGURE 2.

SDF-1α–induced β2 integrin activation is calcium dependent in T cells. T cells from healthy volunteers were preincubated with inhibitors for calcium signaling [BAPTA-AM, (A)], PLC [U73122, (B)], PI3K [Ly-294002, (C)], G protein [Ptx, (D)], or PKC [Ro-31-8220, (E)] and stimulated with SDF-1α. The mean of three independent experiments and SEM is depicted (normalized to the stimulated sample without inhibitor). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.
FIGURE 3.

Hydrogen peroxide–induced β2 integrin activation is calcium dependent. (A) After labeling of T lymphocytes with Fluo-4, cells were preincubated with inhibitors for PI3K (Ly-294002), PLC (U73122), or G protein (Ptx) and incubated with hydrogen peroxide. The mean MFI and SEM of three independent experiments is depicted. (B) The H2O2-induced adhesiveness can be abolished by pretreatment with BAPTA-AM. Mean values of three independent experiments have been normalized to the H2O2-treated sample without inhibitor. T cells from healthy volunteers were analyzed. **p < 0.01, ***p < 0.001.

Human CD4+CD28null T cells show strong increment of β2 integrin–mediated adhesiveness after SDF-1α stimulation in patients with unstable angina

Th cells lacking the costimulatory molecule CD28 are strongly involved in plaque destabilization during ACS. The overall cell number of the CD28null subset is increased in ACS patients (9). Although the spontaneous β2 integrin activity of all T cell subsets is increased, the CD45RA+ effector memory cells lacking CD28 show the strongest spontaneous integrin activation (11). Because T cell recruitment precedes myocardial infarction, and β2 integrin activation is pivotal for this process, we sought to dissect the involved signaling pathway in more detail (11). To this end, we analyzed β2 integrin activation after chemokine stimulation on purified Th cell subpopulations of patients with ACS. T cell subsets were differentiated based upon the expression of CD45RA, CD28, and CCR7 (31, 32). Fig. 4A shows the distribution of the different subsets in one representative dot blot gated on CD3+CD4+ T cells. Starting in the upper right quadrant (CCR7+/CD45RA+), populations are numbered from I to IV clockwise. Subsets III (effector memory cells) and IV (CD45RA+ effector memory cells) can be divided further because of their expression of CD28 (Fig. 4B). In the subsequent experiments, the subset of CD28+ cells are marked with a, the CD28null cells with b. Without stimulation, there was no difference in adhesiveness between subsets with a mean scICAM-1 binding of ∼42 MFI in all subsets (Fig. 4C, 4D). However, as shown in Fig. 4C, 4D, after SDF-1α treatment, the MFI (scICAM-1 binding) showed statistically significant increases in CD28null cells (3122 in subset IIIb and to 2186 for IVb), but not in CD28+ cells, representing a roughly 25-fold increase when comparing CD28null with naive Th cells (107 in subset I). Stimulation with Mg2+/EGTA induced a rise of the MFI to 7104 in the subset Ia, further increasing to 24,022 MFI in subset IIIb and 21,143 MFI in subset IVb (Fig. 4C, 4E), representing a 3- to 4-fold increase. Mg2+ binds to the metal ion–dependent adhesion site (MIDAS) on the β2 integrin, forcing the protein into the high-affinity status, thereby inducing the binding of the scICAM-1 complexes. Differences between subsets may be explained by different expression of CD18 itself. Therefore, we also quantified the expression of the β2 chain of the integrin on the subsets (Fig. 5A). Expression of the β2 integrin CD18 ranged from 177 MFI on subset I, to 640 MFI on IIIb, and 637 MFI on VIb (Fig. 5B). Comparing CD28null with naive T cells (subset I), this represented a 3- to 4-fold increase, which correlated well with the Mg2+-induced raise in adhesiveness. Note that the pronounced difference of CD28null T cells versus naive T cells was not due to different kinetics in their adhesion behavior (Fig. 5C). Taken together, the strong SDF-1α–induced adhesiveness of CD28null T cells (factor 25 compared with naive T cells) could only partially be explained by a higher CD18 expression per se (factors 3–4 compared with naive T cells), suggesting additional functional differences within T cell subsets.

FIGURE 4.
FIGURE 4.

Human CD4+CD28null T cells show strong increment of β2 integrin–mediated adhesiveness after SDF-1α stimulation in patients with unstable angina. (A) Distribution of the different subsets according their expression of CD45RA and CCR7 in one exemplary dot blot gated on CD3+/CD4+ double-positive cells. Starting in the upper right quadrant (CD45RA+/CCR7+), populations are numbered from I to IV clockwise. (B) The expression of CD28 on these four subsets is depicted. The subset of CD28+ cells are marked with a, the CD28null cells with b. (C) The figure shows representative dot blots of unstimulated or SDF-1α- or Mg/EGTA-treated samples gated on the CD45RA−/CCR7– effector memory Th cell subset [III, compare to (A)]. (D and E) The MFI of three independent experiments is summarized for β2 integrin–dependent adhesion (scICAM-1 binding according to LC-AA) after SDF-1α (D) or Mg/EGTA (E) stimulation. The Roman numbers correspond to the T cell subsets, as explained in (A) and (B). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.
FIGURE 5.

Differences in β2 integrin–dependent adhesion of CD4+CD28null T cells from ACS patients cannot be explained by CD18 expression or varying kinetics. (A and B) The expression of the β2 integrin CD18 on the T cell subsets is depicted for one representative example [(A), gated on subset III; compare to Fig. 4] or summarized for three independent experiments (B) (n = 3; mean and SEM). (C) Kinetics of β2 integrin activation after SDF-1α stimulation for three independent experiments (±SEM) is summarized differentiated for CD28+ and CD28null Th cells. *p < 0.05, **p < 0.01, ***p < 0.001.

The H2O2–Ca2+–β2 integrin pathway is pivotal in CD4+CD28null T cells

To address the question which of the known pathways caused the functional difference regarding β2 integrin activation, T cells purified from ACS patients were preincubated with increasing concentrations of NAC (reducing agent), BAPTA-AM (calcium chelator), U73122 (PLC inhibitor), Ro31-8220 (PKC inhibitor), and Ly-292004 (PI3K inhibitor). Interestingly, CD28+ and CD28null Th cells significantly differed regarding their sensitivity toward NAC (Fig. 6A) as well as BAPTA-AM (Fig. 6B). Inhibition of PLC (Fig. 6C), PI3K (Fig. 6D), and PKC (Fig. 6E) showed no difference between the subsets. This unequal susceptibility of the T cell subsets to reducing agents and calcium chelators suggested that these cells might release more calcium and ROS after SDF-1α stimulation and/or are more dependent on these in regard to integrin adhesiveness. We measured calcium levels in the two subsets. Indeed, CD28null Th cells released more calcium after SDF-1α treatment than CD28-expressing cells (Fig. 6F). Unfortunately, transfection of CD28null cells with the roGFP-redox sensor was ineffective, and nongenetic ROS sensors were not sensitive enough in these primary cells. Therefore, direct visualization of hydrogen peroxide release in both subsets was methodologically not possible.

FIGURE 6.
FIGURE 6.

The H2O2–Ca2+–β2 integrin pathway is pivotal in CD4+CD28null T cells of patients with ACS. (A–E) T cells were preincubated with the respective inhibitors [NAC (A), BAPTA-AM (B), U73122 (C), Ly-292004 (D), and Ro-31-8220 (E)] and stimulated with SDF-1α. CD4+CD28+ and CD4+CD28null Th cells were analyzed. Values are normalized to chemokine stimulated sample without inhibitor. (F) One representative experiment out of three for online calcium flux quantification is depicted for CD28+ and CD28null Th cells. The first arrow indicates addition of SDF-1α, the second the stimulation with ionomycin. *p < 0.05, **p < 0.01, ***p < 0.001.

However, importantly, the difference between CD28+ and CD28null Th cells after chemokine stimulation regarding β2 integrin activation could be abrogated by costimulation with hydrogen peroxide. Thus, additional hydrogen peroxide induced a further increment in β2 integrin activation in CD28+ T cells after SDF-1α treatment compared with chemokine stimulation alone (Fig. 7A, p < 0.01). In contrast, no further significant increment could be seen in CD28null cells by additional hydrogen peroxide stimulation. Similarly, the functional difference regarding β2-integrin activation between CD28+ and CD28null cells could be abolished if T cells were costimulated with SDF-1α and ionomycin (Fig. 7B). This demonstrates that weaker β2 integrin activation in naive T cells is due to the lack of hydrogen peroxide or calcium. In contrast to hydrogen peroxide costimulation, ionomycin treatment also had a significant additive effect in CD28null Th cells together with SDF-1α (Fig. 7B, p < 0.01).

FIGURE 7.
FIGURE 7.

Costimulation with H2O2 or ionomycin additionally to SDF-1α treatment mimics reactivity of the CD28null phenotype. (A and B) T cells from ACS patients were stimulated with PBS (black columns) or SDF-1α (white columns). Analysis was done separately for CD28+ and CD28null Th cells as depicted. Additionally, cells were costimulated with hydrogen peroxide (A) or ionomycin (B). All values are normalized for SDF-1α–stimulated CD28null Th cells (n = 3; mean and SEM). (C) Whole blood of ACS patients was preincubated with NAC or BAPTA-AM. Spontaneous β2 integrin activation has been quantified for CD28+ and CD28null Th cells. Values were normalized for the sample without inhibitor. The mean of three independent experiments and SEM are depicted. *p < 0.05, **p < 0.01, ***p < 0.001.

Spontaneous β2 integrin activation on T cells of ACS patients depends on H2O2–Ca2+–β2 integrin pathway

Recently, we have shown that spontaneous β2 integrin activation on Th cells precedes myocardial infarction and that, to our knowledge, this novel functional biomarker has prognostic value regarding incidence of future major cardiovascular events (11). To further test the relevance of the described H2O2–Ca2+ pathway for spontaneous β2 integrin activation of T cells in patients presenting with ACS, the LC-AA has been repeated in whole blood as applied before. As shown in Fig. 7C, treatment with NAC inhibited spontaneous β2 integrin activation by 21.64 ± 0.02% in CD28+ Th cells (Fig. 7C, p < 0.001) and 39.13 ± 0.07% in CD28null Th cells (Fig. 7C, p < 0.01). Thus, inhibition of the latter subset was significantly stronger. Preincubation with BAPTA-AM reduced scICAM-1 binding by 12.3 ± 0.02% in the CD28+ subset (Fig. 7C, p < 0.05), whereas in the CD28null subset of Th cells, integrin activation was reduced by 33.08 ± 0.08% (Fig. 7C, p < 0.05). Also, in this study, this inhibition after BAPTA-AM treatment was stronger in the CD28null subset.

In a last set of experiments, we also asked the question whether SDF-induced β2 integrin activation is differentially regulated in peripheral blood T cells of healthy donors compared with ACS patients. Furthermore, we complemented our surface staining with Abs against CCR6 and CXCR3 to identify Th1, Th2, and Th17 cell subsets. As shown in Supplemental Fig. 1, CD28null subsets were highly reactive compared with the other subsets, as seen before in both groups (healthy volunteers and ACS patients), whereas there was no significant difference between these particular Th subsets (Th1, Th2, and Th17). However, interestingly, upon SDF stimulation, CD28null T cells from healthy controls showed increased β2 integrin activity compared with the ACS patients (n = 4).

Discussion

It is now well accepted that atherosclerosis is a chronic inflammatory disease in which ROS play an important role for initiation through cholesterol oxidation (oxidized low-density lipoprotein) and progression of the pathology (5, 14–16, 31). Moreover, several epidemiologic studies provide evidence for protective effects by antioxidant intake in patients with cardiovascular disease regarding mortality and morbidity (19, 33). In ACS patients, Th cells play a central role for plaque destabilization and consecutive thromboembolism with myocardial infarction (2, 5). Before fulfilling their potentially lethal function within the arterial wall, T cells have to be recruited in a β2 integrin–dependent fashion. Interestingly, hydrogen peroxide was found to be a major component in wound-to-leukocyte signaling in zebra fish (34). In this study, we show that hydrogen peroxide mediates chemokine-induced β2 integrin activation in primary human T cells by calcium release. Furthermore, we demonstrate that CD4+ T cells lacking the costimulatory molecule CD28 (CD28null Th cells), for which the pathogenetic significance in plaque destabilization is known, represent a highly reactive subset with regard to ROS-mediated signaling. Importantly, we provide evidence that this H2O2-Ca2+ cascade is spontaneously activated in T cells of patients with ACS. Because this activation precedes myocardial infarction (11), interference with this pathway may be a valuable target for future therapeutic intervention.

Hydrogen peroxide can induce oxidative stress that leads to T cell hyporesponsiveness (35) or death (36). However, hydrogen peroxide is not, per se, harmful. The effects of hydrogen peroxide are highly dependent on its concentration as well as on the duration of its presence and play an important role in T cell activation. TCR stimulation induces production of ROS (25, 37) with subsequent calcium release (38), resulting in T cell activation (39). However, there are conflicting data, which suppose ROS production downstream of the calcium release (40) after TCR stimulation. The role of ROS and calcium in chemokine stimulation of T cells has not been investigated yet. For monocytes and neutrophils, hydrogen peroxide is a known second messenger after chemokine stimulation (41, 42). With this study, we clearly show that chemokine stimulation of untransformed human T cells induces the production of hydrogen peroxide. Furthermore, ROS-induced β2 integrin activation in T cells could be inhibited by calcium chelation. Therefore, in the context of chemokine stimulation, ROS seems to be upstream of calcium release mediating β2 integrin activation in primary human T cells.

T cells represent a highly heterogeneous cell population. The introduction of multicolor flow cytometry now allows further phenotyping based on the maturation status because of the expression of CD45RA, CCR7, and CD28. Thus, Th cells can be classified in six functionally distinct subsets according to their proliferation, cytokine synthesis, and Ag-specific response (31). So far, little was known about the regulation of β2 integrin–mediated adhesiveness in these subpopulations, which is pivotal for cell recruitment and trafficking. Th cells lacking the costimulatory molecule CD28 play an important role in different chronic inflammatory pathologies like rheumatoid arthritis, granulomatosis with polyangiitis, or atherosclerosis. They accumulate in the respective affected organs (9, 43, 44). In this study, we show that CD4+CD28null T cells represent a highly reactive subpopulation with increased β2 integrin–mediated adhesive capacity, which remarkably differs from CD28+ Th cells. Interestingly, for the already-known classical pathways toward integrin activation (PI3K, PLC, or PKC), no difference in the susceptibility regarding preincubation with specific inhibitors thereof could be found. However, the described H2O2–Ca2+ pathway was differentially regulated. On the molecular level, enzymes and proteins responsible for the different redox status in these subsets are not yet known. They may represent interesting therapeutic targets, potentially allowing subset specific modulation of the immune response.

As we have shown recently, β2 integrin activation on T cell subsets is an independent predictor for major cardiovascular events in patients with ACS independent of established risk factors (11). In principle, integrin activation of T cells can be induced either by TCR or G protein–coupled receptors. Our finding that preincubation of whole blood of patients with NAC or BAPTA-AM inhibited the spontaneous integrin activation clearly evidences the relevance of the H2O2 calcium cascade in these patients. However, the stimulus activating this pathway in this collective cannot be determined based on the presented data, albeit different Ags as well as chemokines are known to be relevant in the pathogenesis of atherosclerosis (43–49).

In summary, hydrogen peroxide modulates β2 integrin activation in T cells after chemokine stimulation by calcium release. Interestingly, the pathogenetically pivotal CD28null T cells represent a highly reactive subset regarding integrin activation through H2O2 and calcium. Most importantly, in patients with ACS activation of this H2O2, calcium cascade is involved in spontaneous integrin activation, which is known to precede myocardial infarction (11).

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ACS
    acute coronary syndrome
    Apo
    apocynin
    DPI
    diphenylene iodonium chloride
    LC-AA
    ligand complex–based adhesion assay
    MFI
    mean fluorescence intensity
    NAC
    N-acetylcysteine
    Ptx
    pertussis toxin
    redox
    oxidation reduction
    RFP
    red fluorescence protein
    roGFP
    redox-sensitive GFP
    ROS
    reactive oxidative species.

  • Received March 28, 2020.
  • Accepted August 17, 2020.

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