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Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway

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Abstract

Intravascular hemolysis, a major manifestation of sickle cell disease (SCD) and other diseases, incurs the release of hemoglobin and heme from red blood cells, in turn triggering inflammatory processes. This study investigated the in vitro effects of heme, a major inflammatory DAMP, on the adhesive properties of isolated human neutrophils. Heme (20 and 50 µM) significantly increased the adhesion of neutrophils to fibronectin and to recombinant ICAM-1, under static conditions, even more efficiently than the potent pro-inflammatory cytokine, tumor necrosis factor-α (TNF); a microfluidic assay confirmed that heme stimulated neutrophil adhesion under conditions of shear stress. Heme-induced neutrophil adhesion was associated with the increased activities, but not expressions, of the Mac-1 and LFA-1 integrin subunits, CD11b and CD11a, on the cell surface. Notably, heme (50 µM) significantly induced NFκB translocation in neutrophils, and inhibition of NFκB activity with the BAY11-7082 molecule abolished heme-induced cell adhesion to fibronectin and significantly decreased CD11a activity. Flow cytometric analysis demonstrated major reactive oxygen species (ROS) generation in neutrophils following heme stimulation that could be inhibited by the antioxidant, α-tocopherol, and by BAY11-7082. Furthermore, co-incubation with α-tocopherol abrogated both heme-stimulated neutrophil adhesion and CD11a/CD11b activation. Thus, our data indicate that heme, at clinically relevant concentrations, is a potent activator of neutrophil adhesion, increasing the ligand affinity of the β2 integrins via a mechanism that may be partially mediated by an NFkB-dependent pathway and the generation of ROS. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in SCD vaso-occlusion and other vascular inflammatory processes, our findings provide further evidence that cell-free heme is a major therapeutic target in the hemolytic diseases.

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Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

This study was supported by a fellowship from CAPES to LIMT and by a research Grant from São Paulo Research Foundation (FAPESP, Grant Number: 2018/08010–9).

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Authors and Affiliations

  1. Hematology and Transfusion Center, Cidade Universitária, Rua Carlos Chagas, 480, Barão Geraldo, Campinas, 13083-970-SP, Brazil

    Lediana I. Miguel, Flávia C. Leonardo, Lidiane S. Torres, Flávia Garcia, Rafaela Mendonça, Wilson A. Ferreira Jr, Érica M. F. Gotardo, Fernanda C. Z. Fabris, Pamela L. Brito, Fernando F. Costa & Nicola Conran

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  1. Lediana I. Miguel
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  2. Flávia C. Leonardo
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Contributions

LIMT, NC, FCL and FFC designed the study. LIMT, FCL, LST, FG, RM, WAM, EMFG, FCZF and PLB performed data acquisition. LIMT, FG, FCL, MFG and NC contributed to data and statistical analysis. LIMT and NC prepared the manuscript. All the authors approved the final version.

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Correspondence to Nicola Conran.

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The authors declare that they have no conflicts of interest relevant to this study.

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This study was approved by the Ethics Committee of the University of Campinas, Brazil (under protocol number CAAE10550012.3.0000.5404), and conducted in accordance with national guidelines for human research and with the Declaration of Helsinki.

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Informed written consent was obtained from all study participants.

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11010_2021_4210_MOESM1_ESM.docx

.Imaging cytometry analysis of NFκB nuclear translocation from the cytoplasm to the neutrophil nucleus. Neutrophils were labelled with anti-CD66b PE, permeabilized and stained with anti-NFκB Alexa Fluor® 488 antibody (in green) and the 7AAD nuclear dye (in red). Representative images of untranslocated (UT) and translocated (T) NFκB in (A; basal) unstimulated neutrophils, (B) neutrophils stimulated with TNF (200 ng/mL), (C) heme (50 μM), or (D) heme (50 μM) and Bay 11-7082 (20 μM) (37°C, 5% CO2; 30 min). Representative similarity histograms for each group, calculating the percentage of neutrophils displaying NFκB translocation are presented in Figure 3A (docx 101 KB)

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Miguel, L.I., Leonardo, F.C., Torres, L.S. et al. Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway. Mol Cell Biochem 476, 3963–3974 (2021). https://doi.org/10.1007/s11010-021-04210-5

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