Decreased CXCR1 and CXCR2 expression on neutrophils in anti-neutrophil cytoplasmic autoantibody-associated vasculitides potentially increases neutrophil adhesion and impairs migration

Abstract

Introduction: In anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), persistent inflammation within the vessel wall suggests perturbed neutrophil trafficking leading to accumulation of activated neutrophils in the microvascular compartment. CXCR1 and CXCR2, being major chemokine receptors on neutrophils, are largely responsible for neutrophil recruitment. We speculate that down-regulated expression of CXCR1/2 retains neutrophils within the vessel wall and, consequently, leads to vessel damage.

Methods: Membrane expression of CXCR1/2 on neutrophils was assessed by flow cytometry. Serum levels of interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), angiopoietin 1 and angiopoietin 2 from quiescent and active AAV patients and healthy controls (HC) were quantified by ELISA. Adhesion and transendothelial migration of isolated neutrophils were analyzed using adhesion assays and Transwell systems, respectively.

Results: Expression of CXCR1 and CXCR2 on neutrophils was significantly decreased in AAV patients compared to HC. Levels of IL-8, which, as TNFα, dose-dependently down-regulated CXCR1 and CXCR2 expression on neutrophils in vitro, were significantly increased in the serum of patients with active AAV and correlated negatively with CXCR1/CXCR2 expression on neutrophils, even in quiescent patients. Blocking CXCR1 and CXCR2 with repertaxin increased neutrophil adhesion and inhibited migration through a glomerular endothelial cell layer.

Conclusions: Expression of CXCR1 and CXCR2 is decreased in AAV, potentially induced by circulating proinflammatory cytokines such as IL-8. Down-regulation of these chemokine receptors could increase neutrophil adhesion and impair its migration through the glomerular endothelium, contributing to neutrophil accumulation and, in concert with ANCA, persistent inflammation within the vessel wall.

Figure 1

Down-regulated expression of CXCR1 and CXCR2 on neutrophils in AAV. Levels of CXCR1 (A) and CXCR2 (B) expression on the membrane of neutrophils were measured by flow cytometry and compared between AAV patients in remission (n = 37), AAV patients with active disease (n = 5) and healthy controls (HC, n = 30). Expression levels are presented as MFI corrected for nonspecific binding of isotype control antibodies on neutrophils. Horizontal lines represent medians. *, P < 0.05; **, P < 0.01 ***, P < 0.001. Correlation between CXCR1 and CXCR2 expression (C) on neutrophils was analyzed in patients in remission and with active disease (n = 42, Spearman r = 0.87, P < 0.0001). Dotted line represents the best-fit correlation between CXCR1 and CXCR2 expression. AAV, ANCA-associated vasculitides; ANCA, anti-neutrophil cytoplasmic autoantibody; MFI, mean fluorescence intensity.

Figure 2

Down-regulation of CXCR1 and CXCR2 on neutrophils by IL-8 and TNFα. Isolated neutrophils from HC were pre-incubated with serial doses of IL-8 (A, left figure), TNFα (B, left figure), ANGPT-1 (C) or ANGPT-2 (D). Isolated neutrophils from AAV patients were pre-incubated with serial doses of IL-8 (A, right figure) and TNF α (B, right figure). Membrane expression of CXCR1 (black dots) and CXCR2 (black diamonds) was measured by flow cytometry. Expression of CD177 (open dots) was measured in parallel as control for stimulation. Results are presented as percentages compared to control incubation with normal medium (ctrl). CXCR1 and CXCR2 are equally down-regulated by TNFα (overlapping graphs in panel B). Results are the mean (± SD) of three experiments, *, P < 0.05; **, P < 0.01 (CXCR1 and CD177). #, P < 0.01 (CXCR2). AAV, ANCA-associated vasculitides; ANCA, anti-neutrophil cytoplasmic autoantibody; ANGPT, angiopoietin; IL; interleukin; TNF, tumor necrosis factor.

Figure 3

CXCR1/CXCR2 expression correlates with IL-8 levels in quiescent AAV. Expression of CXCR1, CXCR2 and levels of IL-8 were measured in parallel in patients in remission (n = 37). Membrane expression of CXCR1 and CXCR2 is presented as MFI corrected for nonspecific binding of isotype control antibodies on neutrophils. IL-8 levels are presented as concentration in serum. A. Correlation between CXCR1 expression on neutrophils and serum levels of IL-8 (Spearman r = -0.34, P = 0.04). B. Correlation between CXCR2 expression on neutrophils and serum levels of IL-8 (Spearman r = -0.41, P = 0.01). Dotted lines represent the best-fit correlation between expression of CXCR1/2 and IL-8 levels. AAV, ANCA-associated vasculitides; ANCA, anti-neutrophil cytoplasmic autoantibody; IL, interleukin.

Figure 4

Levels of IL-8, ANGPT-1 and ANGPT-2 in sera from patients with AAV. Levels of IL-8 (A), ANGPT-1 (B) and ANGPT-2 (C) in sera of AAV patients in remission (n = 37) and active disease (n = 18) were measured by ELISA and compared to HC (n = 30). Results are presented as the concentration of each cytokine in serum. Bars denote medians, *, P < 0.05; ***, P < 0.001. AAV, ANCA-associated vasculitides; ANCA, anti-neutrophil cytoplasmic autoantibody; ANGPT, angiopoietin; IL; interleukin.

Figure 5

CXCR1/CXCR2 blockade enhances neutrophil adhesion and inhibits neutrophil transmigration through an endothelial cell monolayer. Isolated neutrophils from HC were pre-incubated with serial doses of repertaxin, IL-8 (10 ng/ml) or TNFα (2 ng/ml) before being loaded onto a glomerular endothelial monolayer or a transwell insert. After co-incubation of pretreated neutrophils with glomerular endothelial cells for 30 minutes for the adhesion assay (A, n = 5) or with transwell inserts coated with endothelial cells for 2 hours (B, n = 6), the number of adherent or migrated neutrophils were quantified by MPO activity assay. Results are presented as the percentages of adherent or migrated neutrophils after stimulation compared to control incubation. *, P < 0.05. HC, healthy control; IL; interleukin; MPO, myeloperoxidase; TNF, tumor necrosis factor.