Hemoglobin increases leukocyte adhesion and initiates lung microvascular endothelial activation via Toll-like receptor 4 signaling

Abstract

Cell-free hemoglobin is a pathophysiological driver of endothelial injury during sepsis and acute respiratory distress syndrome (ARDS), but the precise mechanisms are not fully understood. We hypothesized that hemoglobin (Hb) increases leukocyte adhesion and endothelial activation in human lung microvascular endothelial cells (HLMVEC). We stimulated primary HLMVEC, or leukocytes isolated from healthy human donors, with Hb (0.5 mg/mL) and found that leukocyte adhesion to lung endothelium in response to Hb is an endothelial-dependent process. Next, we stimulated HLMVEC with Hb over time (1, 3, 6, and 24 h) and found increased transcription and release of inflammatory cytokines (IL-1β, IL-8, and IL-6). In addition, Hb exposure variably upregulated transcription, total protein expression, and cell-surface localization of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Since VCAM-1 was most upregulated by Hb, we further tested mechanisms for Hb-mediated upregulation of VCAM-1 in HLMVEC. Although upregulation of VCAM-1 was not prevented by hemoglobin scavenger haptoglobin, heme scavenger hemopexin, or inhibition of nod-like receptor protein 3 (NLRP3) signaling, blocking Toll-like receptor 4 (TLR4) with small molecule inhibitor TAK-242 (1 µM) prevented upregulation of VCAM-1 in response to Hb. Consistently, Hb increased nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS), which were both prevented by TLR4 inhibition. Together, these data demonstrate that Hb increases leukocyte-endothelial adhesion and activates HLMVEC through TLR4 signaling, indicating a potential mechanism for Hb-mediated pulmonary vascular injury during inflammatory and hemolytic conditions.

Keywords: Toll-like receptor; endothelial activation; hemoglobin; leukocyte adhesion; pulmonary endothelium.

Graphical abstract

Figure 1.

Hemoglobin (Hb) increases leukocyte adhesion to human lung microvascular endothelial cells (HLMVEC). Hb was used to stimulate either endothelial cells (EC) or leukocytes (polymorphonuclear neutrophils, PMN) alone, or both EC and PMN in combination, before exposing HLMVEC to PMN and assessing leukocyte adhesion. Representative images (Blue = nuclei, Red = EC, Green = PMN; scale bar = 2 mm) (A) and quantification of immunofluorescence staining of PMN (GFP) (B) after adhesion assay (n = 12 replicates) indicate that Hb-mediated PMN adhesion to HLMVEC is dependent on EC. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons. Graph represents means ± SE.

Figure 2.

Hemoglobin (Hb) increases transcription and cell surface expression of E-selectin in human lung microvascular endothelial cells (HLMVEC). HLMVEC were stimulated with Hb over 1, 3, 6, and 24 h. A: E-selectin mRNA (n = 7–9 replicates) increased significantly at 24 h. Representative Western blot (B) and quantification of E-selectin (n = 5 replicates) (C) indicate no change of E-selectin protein expression over time. Representative images [Blue = nuclei, Red = E-selectin; scale bar = 300 µm] (D) and quantification of E-selectin immunofluorescence (n = 9 replicates) (E) show increased cell surface expression of E-selectin at 3 and 6 h. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons. Graphs represent means ± SE.

Figure 3.

Hemoglobin (Hb) does not increase transcription, protein, or cell surface expression of P-selectin in human lung microvascular endothelial cells (HLMVEC). HLMVEC were stimulated with Hb over 1, 3, 6, and 24 h. A: P-selectin mRNA (n = 7–9 replicates) did not change over time. Representative Western blot (B) and quantification of P-selectin (C) (n = 4 replicates) indicate no change of P-selectin protein expression over time. Representative images [Blue = nuclei, Red = P-selectin; scale bar = 300 µm] (D) and quantification of P-selectin immunofluorescence (n = 9 replicates) (E) show no changes in P-selectin cell surface expression. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons. Graphs represent means ± SE.

Figure 4.

Hemoglobin (Hb) increases transcription and cell surface expression of intercellular adhesion molecule-1 (ICAM-1) in human lung microvascular endothelial cells (HLMVEC). HLMVEC were stimulated with Hb over 1, 3, 6, and 24 h. A: ICAM-1 mRNA (n = 7–9 replicates) increased significantly at 3 h. Representative Western blot (B) and quantification of ICAM-1 (n = 8 replicates) (C) indicate no change of ICAM-1 protein expression over time. Representative images [Blue = nuclei, Red = ICAM-1; scale bar = 300 µm] (D) and quantification of ICAM-1 immunofluorescence (n = 9 replicates) (E) indicate increased cell surface expression of ICAM-1 at 24 h. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons. Graphs represent means ± SE.

Figure 5.

Hemoglobin (Hb) increases transcription, protein, and cell surface expression of vascular cell adhesion molecule-1 (VCAM-1) in human lung microvascular endothelial cells (HLMVEC). HLMVEC were stimulated with Hb over 1, 3, 6, and 24 h. A: VCAM-1 mRNA (n = 7–9 replicates) increased significantly at 3 and 6 h. Representative Western blot (B) and quantification of VCAM-1 (n = 7 replicates) (C) indicate significantly increased total VCAM-1 protein expression at 3 and 6 h. Representative images [Blue = nuclei, Red = VCAM-1; scale bar = 300 µm] (D) and quantification of VCAM-1 immunofluorescence (n = 9 replicates) (E) shows increased cell surface expression of VCAM-1 at 3, 6, and 24 h. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons. Graphs represent means ± SE.

Figure 6.

Inhibition of Toll-like receptor 4 (TLR4) with TAK-242 (TAK) prevents Hb-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1), NF-κB activation, and reactive oxygen species (ROS) generation. HLMVEC were stimulated with Hb for 3 h with or without 1-h pretreatment with TAK-242 (1 µM). A: increased VCAM-1 mRNA (n = 3 or 4 replicates) in response to Hb was prevented by TLR4 inhibition. Representative Western blot (B) and quantification of VCAM-1 (n = 4 or 5 replicates) (C) and phospho-NF-κB (n = 3 or 4 replicates) (D) indicate VCAM-1 protein expression and NF-κB activation were prevented by TLR4 inhibition. Representative images [blue = nuclei, red = CellRox; scale bar = 300 µm] (E) and quantification of CellRox (AF647; MFI/cell, n = 5 replicates) (F) indicate Hb-mediated increase in ROS was prevented by TLR4 inhibition. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons. Graphs represent means ± SE.