IL-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome

Abstract

Cytokine release syndrome (CRS) is a life-threatening complication induced by systemic inflammatory responses to infections, including bacteria and chimeric antigen receptor T cell therapy. There are currently no immunotherapies with proven clinical efficacy and understanding of the molecular mechanisms of CRS pathogenesis is limited. Here, we found that patients diagnosed with CRS from sepsis, acute respiratory distress syndrome (ARDS), or burns showed common manifestations: strikingly elevated levels of the four proinflammatory cytokines interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and IL-10 and the coagulation cascade activator plasminogen activator inhibitor-1 (PAI-1). Our in vitro data indicate that endothelial IL-6 trans-signaling formed an inflammation circuit for robust IL-6, IL-8, and MCP-1 production and promoted PAI-1 production; additionally, an IL-6 signaling blockade by the human monoclonal antibody tocilizumab blunted endothelial cell activation. Plasma from severe COVID-19 patients similarly exhibited increased IL-6, IL-10, and MCP-1 levels, but these levels were not as high as those in patients with CRS from other causes. In contrast, the PAI-1 levels in COVID-19 patients were as highly elevated as those in patients with bacterial sepsis or ARDS. Tocilizumab treatment decreased the PAI-1 levels and alleviated critical illness in severe COVID-19 patients. Our findings suggest that distinct levels of cytokine production are associated with CRS induced by bacterial infection and COVID-19, but both CRS types are accompanied by endotheliopathy through IL-6 trans-signaling. Thus, the present study highlights the crucial role of IL-6 signaling in endothelial dysfunction during bacterial infection and COVID-19.

Keywords: COVID-19; IL-6; cytokine release syndrome; endothelial cell; tocilizumab.

Fig. 1.

Cytokine and chemokine profiles of CRS patients. (A‒D) Cytokine, chemokine (A‒C), and PAI-1 (D) levels were measured in the sera of patients with CRS (sepsis, n = 37; ARDS, n = 19; burns, n = 35). Comparisons were made by using a Mann‒Whitney U test (A‒C) or Kruskal‒Wallis test for multiple-comparisons (D). Statistically significant differences are indicated; n.s.: nonsignificant; n.d.: nondetectable. Bar indicates the median. Blue graph indicates healthy control (HC, n = 36). Other colors are indicated in the graph.

Fig. 2.

Correlation between IL-6 levels and cytokines and chemokines in patients with CRS. (A–C) Pearson’s correlations between cytokine and PAI-1 levels in each cohort of CRS patients: sepsis (A), ARDS (B), and burns (C). The r coefficient of correlation (from Pearson’s correlation of determination) and the respective P values are shown.

Fig. 3.

Effect of IL-6R trans-signaling inhibition on cytokine release from endothelial cells. (A, C, D, and F) HUVECs were treated with LPS (100 ng/mL) or IL-6 (20 ng/mL) in the presence of sIL-6R (+: 50 ng/mL, ++: 100 ng/mL) in vitro. IL-6, IL-8, MCP-1, and PAI-1 levels in the culture supernatants after 72 h are shown. (B and E) HUVECs were treated with IL-6 in the presence of sIL-6R. IL-6 mRNA levels after 6 h are shown. Antibodies (20 μg/mL) were applied at the same time as treatment with LPS or IL-6. Statistical comparisons are indicated; n.s.: nonsignificant. The P values were determined using an unpaired two-tailed Student’s t test (D–F) or one-way ANOVA (A–C). Data are representative of three independent experimental replicates and presented as means ± SD. n = 3 samples per group in A–F.

Fig. 4.

Effect of disrupting IL-6 signaling on PAI-1 production in severe COVID-19 patients. (A and B) Levels of 11 cytokine and chemokines (A) and PAI-1 (B) in the sera of severe COVID-19 patients. Blue indicates healthy control (HC, n = 10). Red indicates severe COVID-19 (n = 7). (C) Changes in cytokines, CRP, and PAI-1 levels in seven patients before (pre) and after (post) treatment with tocilizumab. Comparisons were made by applying a Mann‒Whitney U test followed by correction (A and B) or a paired two-tailed t test (C). Statistical comparisons are indicated; n.s.: nonsignificant.