Phagocytosing neutrophils down-regulate the expression of chemokine receptors CXCR1 and CXCR2

Abstract

IntroductionTwo major responses are central to neutrophil leukocyte (polymorphonuclear neutrophil [PMN]) functioning: chemotaxis and phagocytosis. The migration of leukocytes is governed by chemotactic cytokines called chemokines. Chemokines are a large family of small chemotactic proteins divided into 4 subfamilies according to the positioning of cysteines in their primary sequences. Those whose first 2 cysteines are separated by one amino acid belong to the CXC (or ␣) subfamily and regulate the responses of PMNs. 1,2 Human CXC chemokines interact with the specific G-proteincoupled receptors CXCR1 and CXCR2. CXCR1 has high affinity for interleukin-8 (IL-8) and low affinity for other ␣-chemokines, neutrophil-activating peptide-2 (NAP-2), melanoma growthstimulatory activity (growth-regulated oncogene), and others, whereas CXCR2 shows a high affinity for all CXC chemokines that contain an N-terminal Glu-Leu-Arg motif. 3 Because it has long been shown that after phagocytosis the chemotactic responses of neutrophils are impaired, 4 we investigated the effect of phagocytic stimulation of human neutrophils on the expression of IL-8 receptors CXCR1 and CXCR2. Study design NeutrophilsNeutrophils were isolated from heparinized blood of healthy volunteers by dextran sedimentation of erythrocytes followed by centrifugation on Lymphoprep (Nycomed, Oslo, Norway) and lysis of residual erythrocytes with distilled water. 5,6 Approval was obtained from the institutional review board at the Institute of Hematology and Blood Transfusion, Minsk, Belarus, for these studies. Informed volunteer consent was obtained according to the Declaration of Helsinki. Neutrophils were 95% to 96% pure and more than 98% viable. Purified neutrophils were incubated at 37°C in RPMI containing 2% fetal calf serum (Hyclone, Logan, UT) at 2 to 3 ϫ 10 6 cells per milliliter in polypropylene tubes (Corning, NY) as described. 6 The cells were stimulated with heat-killed and opsonized 7 Saccharomyces cerevisiae (OSC; OSC/PMN ratio, 2:1), opsonized sheep red blood cells (SRBCs; 5:1), or opsonized zymosan particles (30 mg/mL; Sigma, St Louis, MO). All reagents and solutions were controlled for endotoxin contamination by means of Limulus amebocyte assay (E-toxate; Sigma). Phagocytic activity of PMNs was determined as described previously 8 in cell smears by means of Wright stain (Accustain; Sigma). Flow cytometry analysisNeutrophils were stained as described 6 with the use of our mouse monoclonal antibody (mAb) 6 E3 recognizing N-terminal amino acids 10 through 12 (Trp-Asp-Phe) of human CXCR1, mAb R115 to CXCR2, 9 kindly provided by Dr Ernst Brandt (Forschungszentrum, Borstel, Germany), mAb to receptor for complement fragment C5a (C5aR, CD88 10 ; all mAbs were immunoglobulin G1 [IgG1]), kindly donated by Prof Otto Goetze (Georg-August-University, Goettingen, Germany), or isotype control mAb and were analyzed by FACScan (Becton Dickinson, Mountain View, CA) by means of Lysis II software (Becton Dickinson). Confocal microscopySmears were fixed in acetone, permeab...

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“…2 and 4). Inasmuch as previous studies indicate that phagocytosis per se reduces surface expression of both receptors (20), it is likely that down-regulation of CXCR1 and CXCR2 early after phagocytosis of pathogens (within 90 min) is not regulated at the level of gene expression, an idea supported by our findings (see gene expression at 90 min). However, significantly reduced expression of genes encoding CXCR1 and CXCR2 at much later times after phagocytosis (e.g., at 3 and 6 h) likely contributes to the continued absence of expression of these receptors on the surface of human PMNs (Fig.…”

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“…Thus, it is logical to suppose that TLRs might down-modulate neutrophil trafficking to keep these cells at the site of infection. Indeed, it has been shown that bacteria phagocytosis results in decreased surface expression of CXCR1 and CXCR2 in human neutrophils (21,22). Extrapolating to a systemic infection context with high blood levels of TLR agonists, the down-regulation of chemokine receptors in peripheral circulating neutrophils (''wrong compartment'') may explain the drastic reduction of these cells in the infection focus.…”