IL-6 Receptor Inhibition by Tocilizumab Attenuated Expression of C5a Receptor 1 and 2 in Non-ST-Elevation Myocardial Infarction

Abstract

Background: Elevated interleukin-6 (IL-6) and complement activation are associated with detrimental effects of inflammation in coronary artery disease (CAD). The complement anaphylatoxins C5a and C3a interact with their receptors; the highly inflammatory C5aR1, and the C5aR2 and C3aR. We evaluated the effect of the IL-6 receptor (IL-6R)-antagonist tocilizumab on the expression of the anaphylatoxin receptors in whole blood from non-ST-elevation myocardial infarction (NSTEMI) patients. Separately, anaphylatoxin receptor expression in peripheral blood mononuclear cells (PBMC) from patients with different entities of CAD was investigated. Materials and Methods: NSTEMI patients were randomized to one dose of tocilizumab (n = 28) or placebo (n = 32) and observed for 6 months. Whole blood samples drawn at inclusion, at day 2, 3 and after 6 months were used for mRNA isolation. Plasma was prepared for analysis of complement activation measured as sC5b-9 by ELISA. Furthermore, patients with different CAD entities comprising stable angina pectoris (SAP, n = 22), non-ST-elevation acute coronary syndrome (NSTE-ACS, n = 21) and ST-elevation myocardial infarction (STEMI, n = 20) were included. PBMC was isolated from blood samples obtained at admission to hospital and mRNA isolated. Anaphylatoxin-receptor-expression was analyzed with qPCR using mRNA from whole blood and PBMC, respectively. Results: Our main findings were (i) Tocilizumab decreased C5aR1 and C5aR2 mRNA expression significantly (p < 0.001) and substantially (>50%) at day 2 and 3, whereas C3aR expression was unaffected. (ii) Tocilizumab did not affect complement activation. (iii) In analyzes of different CAD entities, C5aR1 expression was significantly increased in all CAD subgroups compared to controls with the highest level in the STEMI patients (p < 0.001). For C5aR2 and C3aR the expression compared to controls were more moderate with increased expression of C5aR2 in the STEMI group (p < 0.05) and C3aR in the NSTE-ACS group (p < 0.05). Conclusion: Expression of C5aR1 and C5aR2 in whole blood was significantly attenuated by IL-6R-inhibition in NSTEMI patients. These receptors were significantly upregulated in PBMC CAD patients with particularly high levels of C5aR1 in STEMI patients.

Keywords: C3a receptor; C5a receptors; IL-6; complement; inflammation; myocardial infarction.

Figure 1

Flow chart showing the total number of patients and randomization to tocilizumab and placebo groups. (A) Number in each group with or without PCI. (B) Number in each group included early (≤2 days) or late (>2 days) from the onset of symptoms. NSTEMI, non-ST-elevated myocardial infarction.

Figure 2

Effect of tocilizumab on the expression of C5aR1, C5aR2, and C3aR in NSTEMI patients. The effect of tocilizumab, a monoclonal antibody inhibiting interleukin 6 receptor (IL-6R), on the expression of the three complement anaphylatoxin receptors [C5aR1 (A), C5aR2 (B), and C3aR (C)] was investigated in patients with non-ST-elevation myocardial infarction (NSTEMI). mRNA levels were quantified by qPCR and related to the reference gene beta-2-microglobulin. The tocilizumab group (gray bars, n = 28) and the placebo group (white bars, n = 32) are presented at four different time-points. Baseline levels show the receptor expression at inclusion, i.e., after hospital admission, before treatment was given. Follow-up time points were day 2 and 3, and 6 months. A group of healthy individuals (n = 15) were included as controls. The qPCR results were quantified using the 2^−ΔΔCT method, normalized to reference genes and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. *P < 0.05, ***P < 0.001 vs. healthy controls. ^†P < 0.05, ^†††P < 0.001 differences in change from baseline between tocilizumab and placebo. ^§p < 0.05, ^§§§p < 0.001 vs. baseline.

Figure 3

Effect of coronary intervention and time of inclusion on the expression of C5aR1, C5aR2, and C3aR in NSTEMI patients. Expression level of complement anaphylatoxin receptors C5aR1 (A,D), C5aR2 (B,E), and C3aR (C,F) in patients with non-ST-elevation myocardial infarction (NSTEMI) receiving placebo (n = 32) or tocilizumab (n = 28) divided into two groups according to percutaneous coronary intervention (PCI) (23 placebo and 21 tocilizumab, gray bars) or not (7 placebo and 9 tocilizumab, white bars) (A–C), and divided into two groups according to inclusion ≤2 days (22 placebo and 15 tocilizumab, gray bars) or >2 days (10 placebo and 13 tocilizumab, white bars) from symptom onset (D–F). Baseline levels show the receptor expression at inclusion, i.e., after hospital admission, before treatment was given. Follow-up time points were day 2 and 3, and 6 months. A group of healthy individuals (n = 15) were included as controls. The qPCR results were quantified using the 2^−ΔΔCT method, normalized to reference genes and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. *P < 0.05, **P < 0.01, ***P < 0.001 vs. healthy controls. ^†P < 0.05, ^†††P < 0.001 differences in change from baseline between tocilizumab and placebo. ^§p < 0.05, ^§§p < 0.01, ^§§§p < 0.001 vs. baseline.

Figure 4

Systemic complement activation in NSTEMI patients treated with tocilizumab or PCI. Complement activation, as evaluated by plasma sC5b-9, is shown for NSTEMI patients (total n = 117) treated with tocilizumab (n = 58; closed circles) or placebo (n = 59; open circles) (A). Complement activation, as evaluated by plasma sC5b-9, is shown for the same population of NSTEMI patients further divided into two groups according to percutaneous coronary intervention (47 placebo and 41 tocilizumab) or not (12 placebo and 17 tocilizumab) (B), and according to inclusion ≤2 days (36 placebo and 30 tocilizumab) or >2 days (23 placebo and 28 tocilizumab) from symptom onset to inclusion (C). The dotted line represents normal upper level of sC5b-9 in healthy blood donors. Data are given as mean and 95% CI.P < 0.01,P < 0.001 differences in change from baseline between tocilizumab and placebo. ^§p < 0.05, ^§§p < 0.01, ^§§§p < 0.001 vs. baseline.

Figure 5

Expression of C5aR1, C5aR2 and C3aR in patients with various entities of CAD. Expression of the C5aR1 (A), C5aR2 (B), and C3aR (C) in three different patients groups with verified coronary artery disease (CAD): stable angina pectoris (SAP, n = 22), non-ST-elevation acute coronary syndromes (NSTE-ACS, n = 21) and ST-elevation myocardial infarction (STEMI, n = 20). A group of healthy age and sex-matched individuals were included as controls (Ctrls, n = 29). mRNA levels were quantified by qPCR using the 2^−ΔΔCT method, normalized to reference genes (GAPDH) and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. Statistical significant differences are indicated between the patient populations and the healthy controls. *p < 0.05, **p < 0.01, ***p < 0.001 vs. healthy controls.