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BRIEF REPORT
From the Laboratory of Cellular and Molecular
Immunology, Institute of Hematology and Blood Transfusion; the
Belarussian Center for Pediatric Oncology and Hematology; the Institute
of Photobiology; the Belarussian State Medical University; and the
Belarussian State University, Minsk, Belarus; and the Fund
for Molecular Hematology and Immunology, Moscow, Russia.
CXC chemokines play a central role in regulation of neutrophil
activation and chemotaxis. Because the chemotactic responses of
neutrophils are impaired after phagocytosis, we explored the effect of
phagocytic stimuli on the expression of interleukin-8 (IL-8)
receptors, CXCR1 and CXCR2, in human neutrophils. After phagocytosis of
opsonized yeast, the expression of CXCR1 and CXCR2 was substantially
down-regulated and was accompanied by reduced Ca++
responses to corresponding ligands, IL-8 and neutrophil-activating peptide-2 (NAP-2). The levels of CXCR1 and CXCR2 mRNA were
constant during phagocytic stimulation of neutrophils. Confocal
microscopy revealed that CXCR reduction was not via internalization.
Metalloproteinase inhibitor, 1,10-phenantroline, prevented the
reduction of CXCRs induced by phagocytosis, indicating that proteolytic
degradation may be responsible for down-regulation. These observations
suggest that down-regulation of CXCR expression may substantially
reduce the responsiveness of phagocytosing neutrophils to CXC chemokines.
(Blood. 2002;100:2668-2671) Two 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
Neutrophils
Flow cytometry analysis
Confocal microscopy Smears were fixed in acetone, permeabilized with 0.1% Triton X-100 and stained with mAbs against CXCR1 and CXCR2 or IgG isotype control mAb at 20 µg/mL followed by incubation with rhodamine-conjugated bovine antimouse IgG antibody (Santa Cruz Biotechnology, CA). The stained cells were observed in confocal microscope (LSM 310; Zeiss, Oberkochen, Germany).Cytosolic-free Ca++ measurements Neutrophils at 107/mL were loaded with 2 µM Fura-2 AM (Molecular Probes, Eugene, OR) for 30 minutes at 37°C, washed, and resuspended in 20 mM HEPES buffer/1% bovine serum albumin/1 mM CaCl2 to a final concentration of 106/mL. To induce intracellular Ca++ accumulation via CXCR-mediated pathway, IL-8 (Peprotech, Rocky Hill, NJ) or NAP-2 (kindly provided by Dr Ernst Brandt, Forschungszentrum, Borstel, Germany), both at 75 ng/mL, were added to PMNs and monitored as described.11 The capacity of phagocytosing PMNs to induce Ca++ influx was tested with the use of recombinant complement fragment C5a at 100 ng/mL (kindly donated by Prof Otto Goetze, Goettingen, Germany) or 1 µM Ca-ionophore A23187 (Serva, Heidelberg, Germany).Reverse transcriptase-polymerase chain reaction analysis of purified neutrophil mRNA Total RNA from neutrophils was extracted by acidic guanidinum thiocyanate and reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Perkin Elmer, San Jose, CA) under the recommended conditions and amplified by PCR with the use of the CXCR1-, CXCR2-, or 2-microglobulin-specific oligo
primer pairs as described.6 A total of 10 µL 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). To
correct for any variation in RNA content and cDNA synthesis in the
different preparations, each sample was normalized on the basis of its
2-microglobulin content. Results were expressed as the
calculated ratio of CXCR mRNAs to 2-microglobulin
mRNA.6
Figure 1A-B shows the effect
of opsonized S cerevisiae (OSC) on the expression
of CXCR1 and CXCR2, compared with nonopsonized S
cerevisiae. PMNs showed no phagocytosis of
nonopsonized S cerevisiae, while about 80% of PMNs
contained intracellular OSC by 15 minutes of incubation (not shown).
The expression of CXCR1 in PMNs (mean fluorescence intensity [MFI])
was reduced by 46% ± 8% (P < .01; n = 16; data
from 16 independent donors), and the expression of CXCR2 was reduced by
75% ± 10% (P < .01; n = 16) after 15 minutes of
phagocytic stimulation with OSC (mean ± SD of percentage of CXCR
down-regulation in each experiment). The kinetics of CXCR2 reduction
was different from that of CXCR1: CXCR2 expression was decreased after
5 minutes of phagocytosis maximally (Figure 1D), whereas the expression
of CXCR1 gradually decreased during 40 minutes of observation. In
agreement with our data, it was previously shown that phagocytosis of
OSC by PMNs peaked at 5 minutes.8 Notably, the reduced
levels of expression of both receptors were maintained during
6 hours of PMN incubation with OSC (not shown). A 2:1 ratio of
OSC to PMN was sufficient for maximal down-regulation of CXCRs
(Figure 1C). Similar down-regulation of CXCR1 and CXCR2 expression was
observed after phagocytic stimulation with opsonized SRBCs or
zymosan particles (not shown).
Proteolytic cleavage by metalloproteinases represents the main mechanism of reducing CXCR expression induced by tumor necrosis factor (TNF) or lipopolysaccharide.12,13 To investigate the mechanism of CXCR down-regulation by phagocytic stimuli, we explored the effect of the metalloproteinase inhibitor 1,10-phenantroline. Figure 1E-F shows that phenantroline at 2 mM prevented reduction of CXCRs. The levels of protection varied from 75% to 82% for CXCR1 and from 60% to 83% for CXCR2 (data of 4 separate experiments). It should be mentioned that 1,10-phenantroline itself did not reduce the phagocytic activity of PMNs (a reduction of less than 10%) and exerted no effect on expression of CXCRs (not shown). To explore mechanisms of down-regulation of the expression of CXCRs by phagocytosis, we assessed the levels of CXCRs mRNA in PMNs during incubation with OSC. Figure 1G demonstrates no mRNA down-regulation after 15 minutes or 1.5 hour of phagocytosis. Exogenous and endogenous cytokines, mainly IL-8 and TNF, were shown to reduce the expression of CXCRs on PMNs.12-14 Because phagocytosis induces the synthesis of IL-8 and TNF in PMNs,7,15,16 we tested the effect of anti-TNF antibody 5N and anti-IL-8 antibody WS-4 at a neutralizing concentration of 20 µg/mL.6 However, the antibodies did not prevent phagocytosis-induced reduction of CXCR expression (not shown). Besides, when supernatants aspirated from neutrophils phagocytosing OSC for 60 minutes were immediately added to a fresh portion of the same PMNs for 60 minutes at 37°C, no reduction of CXCR1 and CXCR2 expression was detected (not shown). These data imply that down-regulation of CXCRs in the phagocytosing neutrophils is not mediated by soluble factors. Internalization represents a second important mechanism of membrane-receptor regulation.17,18 Because inhibitors of internalization, cytochalasin B and vinblastine,18 also arrested the phagocytosis (not shown), we explored the distribution of CXCRs in phagocytosing PMNs using confocal microscopy. Figure 1H (i,ii; upper section) demonstrates that PMNs incubated alone expressed high levels of the membrane CXCRs as revealed by FACScan analysis and confocal microscopy. Exogenous IL-8 caused a dramatic drop of the CXCR expression on the surface of PMNs revealed by FACScan analysis, whereas the CXCR fluorescence on confocal microscopy was not reduced and was redistributed in the area of cell membrane and cytoplasm (panels Hi,ii; lower section), representing a picture of IL-8-induced CXCR internalization.12 Both FACScan analysis and confocal microscopy showed a substantial reduction of CXCR expression in phagocytosing PMNs (Figure 1Hiii-iv). On the confocal microscopy, there was no redistribution but there was significant reduction of fluorescence of the CXCRs in the phagocytosing PMNs containing intracellular OSC (pN), thus indicating that the mechanism of down-regulation of CXCRs by phagocytosis is not via receptor internalization. Notably, in the same cell suspension, nonphagocytosing PMNs that contained no intracellular OSC (N) showed normal CXCR fluorescence pattern (Figure 1Hiii-iv), confirming that soluble factors play no role in the down-regulation of CXCRs induced by phagocytosis. To reveal functional consequences of CXCR1 and CXCR2 reduction caused
by phagocytosis, we explored the effect of corresponding ligands, IL-8
and NAP-2, on intracellular Ca++ signaling in phagocytosing
PMNs. Figure 2A,B,D demonstrates that the
Ca++ accumulation induced by IL-8 and NAP-2 (CXCR2
ligand)3 was suppressed in PMNs after 20 minutes of
incubation with OSC, compared with PMNs incubated alone. Similar
reduction of Ca++ accumulation in phagocytosing PMNs was
observed at various IL-8 concentrations, ranging from 10 ng/mL to 1000 ng/mL (not shown). Nonopsonized S cerevisiae did not affect
Ca++ signaling (not shown). In spite of transitory
elevation of intracellular Ca++ by phagocytosis itself
(Figure 2A-C), the responsiveness of OSC-treated PMNs to complement
fragment C5a19 at 100 ng/mL (Figure 2C-D) and
Ca-ionophore20 at 1 µM (not shown) was not suppressed,
indicating that the capacity of phagocytosing neutrophils to increase
cytoplasmic calcium was not impaired by phagocytosis itself.
Interestingly, the expression of C5a receptors (CD88)10
was reduced in phagocytosing PMNs by 38% ± 17% (not shown, data of
7 experiments).
It has long been known that the chemotactic responses of neutrophils are impaired after phagocytosis.4,21 The data presented here demonstrate phagocytosis-induced down-regulation of the expression of chemokine receptors CXCR1 and CXCR2 and reduced Ca++ mobilization in response to corresponding CXCR ligands. Since CXC chemokines regulate various functions of PMNs via Ca-mediated pathway, the data suggest that phagocytosis may inhibit the capacity of CXC chemokines to modulate these functions. The phenomenon described may be relevant to a recent demonstration of the substantial reduction of CXCR expression on PMNs in septic conditions,22 since phagocytosis of micro-organisms by circulating PMNs is a common event in bacteremia.
The authors are grateful to Dr Joost Oppenheim, Laboratory of Molecular Immunoregulation, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD, for his support of a present research project and review of the manuscript; to Svetlana Akalovich, Institute of Hematology and Blood Transfusion, Minsk, Belarus, for kind help with leukocyte FACScan staining; to Prof Otto Goetze, Georg-August-University, Goettingen, Germany, for the gift of recombinant human C5a and monoclonal antibody to CD88; and to Dr Ernst Brandt, Forschungszentrum, Borstel, Germany, for kind donation of NAP-2 and monoclonal antibody R115 to human CXCR2.
Submitted September 6, 2001; accepted May 22, 2002.
Supported by research funding from the Office of International Affairs, Department of Health and Human Services, National Cancer Institute, National Institutes of Health, Bethesda, MD; grants from INTAS, Brussels, Belgium; research funding from the Belarussian Ministry of Health; and grant B00-147 from the Belarussian Foundation for Fundamental Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nikolai N. Voitenok, Institute of Hematology and Blood Transfusion, Dolginovski Tract 160, Minsk, 223059, Republic of Belarus; e-mail: nvoitenok{at}infonet.by.
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© 2002 by The American Society of Hematology.
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  B. Schmausser, C. Josenhans, S. Endrich, S. Suerbaum, C. Sitaru, M. Andrulis, S. Brandlein, P. Rieckmann, H. K. Muller-Hermelink, and M. Eck Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori: a New Pathomechanism in H. pylori Infection? Infect. Immun., December 1, 2004; 72(12): 6773 - 6779. [Abstract] [Full Text] [PDF]
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 S. D. Kobayashi, K. R. Braughton, A. R. Whitney, J. M. Voyich, T. G. Schwan, J. M. Musser, and F. R. DeLeo From the Cover: Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils PNAS, September 16, 2003; 100(19): 10948 - 10953. [Abstract] [Full Text] [PDF]
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 H. J.J.M. Rennen, O. C. Boerman, W. J.G. Oyen, and F. H.M. Corstens Kinetics of 99mTc-Labeled Interleukin-8 in Experimental Inflammation and Infection J. Nucl. Med., September 1, 2003; 44(9): 1502 - 1509. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) subfamily and regulate the responses of PMNs.
) and CXCR2
(
) in phagocytosing neutrophils (closed symbols) and in control
cells (open symbols; mean ± SD, n = 3). (E,F) Effect of
phenantroline at 2 mM (line 3) on the modulation of CXCR1 (E) and CXCR2
(F) expression after OSC (2), compared with untreated PMNs (1). The
expression levels in panels A, B, E, F, and H are displayed as mean
fluorescence intensity (MFI) of duplicate samples; the
difference between double FACScan estimates was in the range of 2% to
10%. Each PMN sample was stained with irrelevant monoclonal antibody
of IgG1 isotype, and the resulting MFI was subtracted from MFI with
specific mAb. To test a possible effect of the OSCs themselves on the
PMN-staining procedure, OSC suspension was added to control PMNs at the
end of incubation, along with sodium azide, following by a full
staining procedure: no effect of OSC was detected (not shown). Results
similar to those of panels A and B were obtained in 16 separate
experiments; dose dependence similar to that of panel C was obtained in
3 experiments; time dependence similar to that of panel D, in 5 experiments; and the effect of phenantroline was reproduced in 4 separate experiments. (G) CXCR1 (closed bars) and CXCR2 (open bars)
mRNA expression in human neutrophils after treatment of PMNs with OSC.
Results are expressed as a ratio of CXCR mRNA to
