Down-regulation of CXCR1 and CXCR2 expression on human neutrophils upon activation of whole blood byS. aureusis mediated by TNF-α

Abstract

SUMMARYIt was suggested that bacterial products can inhibit the expression of leucocyte chemokine receptors during sepsis and affect leucocyte functions in septic syndrome. Superantigens and toxins produced by Staphylococcus aureus are capable of activating leucocytes via binding to MHC-II antigens on monocytes and T-cell receptor molecules on T lymphocytes. It was recently shown that staphylococcal enterotoxins directly down-regulate the expression of CC chemokine receptors on monocytes through binding to MHC class II molecules. We studied the effects of killed S. aureus on the expression of interleukin-8 receptors, CXCR1 and CXCR2, on polymorphonuclear leucocytes (PMN), which are known to lack the expression of MHC-II antigens. It was shown that S. aureus down-regulated the cellsurface expression of CXCR1 and CXCR2 on PMN in the whole blood and total blood leucocyte fraction containing PMN and monocytes, but did not modulate IL-8 receptor expression in purified PMN suspension. Antibody to TNF-a abrogated down-regulation of IL-8 receptors induced by S. aureus. In contrast, LPS reduced CXCR1 and CXCR2 expression in purified PMN and whole blood in a TNF-aindependent manner. We further showed that TNF-a-induced decrease of CXCR1 and CXCR2 expression was associated with lower IL-8 binding and lower CXCR1 and CXCR2 mRNA levels, and was abrogated by protease inhibitors. We suggest that during septicemia, S. aureus may inhibit neutrophil responsiveness to IL-8 and other CXC chemokines via TNF-a-mediated down-regulation of CXCR1 and CXCR2.

Impaired neutrophil chemotaxis in sepsis associates with GRK expression and inhibition of actin assembly and tyrosine phosphorylation

“…The chemokine receptors CXCR1 and CXCR2 are central for many PMN functions. 29 Although some reports have shown that in vitro treatment with cytokines and/or bacterial products could downregulate CXCR1 and CXCR2 expression on human neutrophils, 23,30 studies with patients with sepsis, confirming our data, showed that expression of CXCR1, the high-affinity receptor for IL-8, were not altered in sepsis. 31,32 Most neutrophil chemoattractants recognize membrane GPCR on the leukocytes and, as a result of receptor activation by the chemoattractant, a complex sequence of signaling events are triggered, inducing profound alterations in cytoskeleton dynamics and activating signaling pathway, allowing neutrophils to develop motile, adhesive, phagocytic, and antimicrobial responses.…”

“…23 Fluorescence microscopy for F-actin, phosphotyrosine or GRK The contents of F-actin, GRK2, GRK5, and tyrosine-phosphorylated residues were determined in neutrophils stimulated with IL-8, LTB 4 , or zymosan by fluorescence microscopy. After treatment, neutrophil slides were prepared by cytospin centrifuge, and F-actin was stained with TRITC-labeled phalloidin (Sigma, St Louis, MO).…”

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

“…6 A total of 10 L each final PCR product was fractionated in 1.5% agarose gel in the presence of ethidium bromide. Quantitation of fluorescence of cellular product bands was performed with Scion Image Software (Frederick, MD).…”

“…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.…”

“…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).…”