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Departments of
* Respiratory Medicine,
Allergy and Rheumatology,
Bioregulatory Function,
Transfusion Medicine, and
|| Molecular Preventive Medicine and CREST, University of Tokyo Graduate School of Medicine, Japan
Correspondence: Koichi Hirai, Department of Bioregulatory Function, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: hiraiko-tky{at}umin.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/CXCL12 initiated Ca2+ mobilization and migratory responses in 20 h cultured PMNs. The surface CXCR4 expression was suppressed most potently by interferon-
(IFN-). IFN-, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF also inhibited spontaneous CXCR4 expression. Real-time, quantitative PCR experiments revealed that a spontaneous increase and an IFN--mediated decrease in surface CXCR4 paralleled changes in the CXCR4 mRNA level. These results on PMNs support the argument that the SDF-1 (CXCL12)/CXCR4 system is regulated by cell type-specific mechanisms.
Key Words: interferon- • granulocyte colony-stimulating factor • granulocyte-macrophage colony-stimulating factor • CXCR1 • CXCR2 • stromal cell-derived factor-1/CXCL12
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of SDF-1/CXCL12 on target cells are mediated specifically through a G protein-coupled seven-transmembrane receptor, designated CXC chemokine receptor 4 (CXCR4) [3 , 4 ]. CXCR4 mRNA is constitutively expressed in almost all types of leukocytes [2 , 5 ]. Although expression of SDF-1/CXCL12 is hardly regulated by exogenous stimuli, various cytokines have been shown to alter the surface CXCR4 expression level in various leukocytes, indicating that modulation of surface CXCR4 expression may represent an alternative mechanism of control of cell-specific, biological responses to SDF-1/CXCL12. To date, regulation of CXCR4 expression has been studied extensively in lymphocytes [6 7 8 9 ]. In addition, we have shown recently that eosinophil-surface CXCR4 expression is affected strongly by various cytokines [10 ]. Whereas regulation of CXCR4 expression by cytokines has been elucidated gradually, there has been no comprehensive investigation of the regulation of surface CXCR4 expression on polymorphonuclear neutrophils (PMNs). Given the potential importance of alteration of surface CXCR4 expression level in leukocytes, we explored the regulation of PMN CXCR4 expression by cytokines.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-), interleukin-10 (IL-10), granulocyte colony-stimulating factor (G-CSF), and IL-8/CXCL8 were purchased from Pepro Tech (London, UK).
Separation and culture of PMNs
Human neutrophils were separated from the blood of normal volunteers by density gradient centrifugation. In brief, buffy coat cells were obtained from venous blood by dextran T500 sedimentation, and eosinophils were eliminated by Percoll (1.088 g/ml; Pharmacia, Uppsala, Sweden) density centrifugation. The buoyant fraction was collected and overlaid on Ficoll-Paque (1.077 g/ml; Pharmacia) to eliminate mononuclear cells. The mean purity of PMNs was 98.4 ± 0.4%, and the viability was consistently >95%.
Neutrophils (0.5–1.0x106) were cultured in RPMI 1640 (Gibco-BRL, Grand Island, NY), supplemented with 10% fetal calf serum (Gibco-BRL) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) at 37°C in 5% CO2 in a total volume of 1 ml in flat-bottom, 48-well culture plates (Costar, Cambridge, MA) for the indicated times.
Flow cytometric analysis of surface CXCR expression on PMNs
Flow cytometric analysis of whole venous blood was performed as described previously [10
]. In brief, cells were stained with anti-CXCR4-fluorescein isothiocyanate (FITC; 12G5), anti-CXCR2-FITC (48311.211; DAKO, Kyoto, Japan), or anti-CXCR1-FITC (42705.111; DAKO) and anti-CD16-phycoerythrin (PE). Neutrophils were defined on the basis of their specific forward/side-scatter properties, and electronic gates were set on CD16-positive cells to identify PMNs. Mouse immunoglobulin (Ig)G2a-FITC with irrelevant specificity (20102.1) was used as a negative control.
Flow cytometric analysis of isolated PMNs was performed as described previously [11 ]. In brief, cells were preincubated with human IgG (5 mg/ml) followed by incubation with anti-CXCR4 mAb (12G5) or anti-CXCR2 mAb (48311.211). Mouse IgG2a with irrelevant specificity (UPC 10) was used as a negative control. After washing, the cells were stained with FITC-labeled goat F(ab')2 against mouse IgG. Stained PMNs were then analyzed using EPICS XL SYSTEM II (Coulter, Miami, FL). The median values of fluorescence intensity of human PMNs were converted to the numbers of molecules of equivalent soluble fluorochrome (MESF) units as described elsewhere [10 ]. Surface-receptor levels expressed in MESF units were calculated using the following formula: (MESF of PMNs stained by anti-CXCR4 or anti-CXCR2 mAb) - (MESF of PMNs stained by isotype-control mAb).
Apoptosis assay of cultured PMNs
Differential analysis of apoptotic and necrotic cells was performed using a MEBCYTO-Apoptosis Kit (Medical and Biological Lab., Nagoya, Japan), as described previously [10
]. Apoptotic cells were determined quantitatively by their ability to bind annexin V and exclude propidium iodide (PI). Cells stained with PI were considered to be necrotic cells. In some experiments, PMNs were double-stained with anti-CXCR4 mAb (12G5, 10 µg/ml) followed by a second-step reaction antibody [PE-conjugated goat F(ab')2 against mouse IgG Fc; Beckman Coulter, Tokyo, Japan] and FITC-conjugated annexin V.
Ca2+ mobilization assay of PMNs
Measurement of Ca2+ influx was performed as described previously [10
]. In brief, purified PMNs (purity: >99%) were resuspended in Hanks’ balanced saline solution with Ca2+ and Mg2+ (Gibco-BRL) and 2% bovine serum albumin at a cell density of 2.0 x 106/ml. Fura-2 AM (Dojindo, Tokyo, Japan) was added at a final concentration of 2 µM. After incubation for 20 min, excess dye was removed by centrifugation, and Ca2+ influx was measured using excitation at 340 and 380 nm on a Hitachi F-2500 fluorescence spectrometer (Hitachi Ltd., Tokyo, Japan). Calibration was performed using 0.1% Triton X for total Ca2+ release and 10 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid to chelate-free Ca2+.
Chemotaxis assay of PMNs
Migration of PMNs was measured using a 6.5 mm diameter, 3 µm pore polycarbonate TranswellTM culture insert (Costar), as described by Bleul et al. [12
]. In brief, 600 µl aliquots of samples to be tested were transferred to each well of a flat-bottom, 24-well plate, and 5 x 105 purified PMNs in 100 µl RPMI-1640 medium containing 0.25% human serum albumin were introduced into the top chamber. An equal number of purified PMNs were introduced into some lower wells without a top chamber to show the standard count of total cells. After incubation at 37°C, 5% CO2 for 90 min, the cells that had transmigrated into the lower well were suspended vigorously. Approximately 10,000 cells of standard sample were counted, and then other samples containing migrated PMNs were counted for the same duration using flow cytometry. The migration of PMNs was calculated as the percentage of total inoculated cells.
Measurement of cytokine generation by PMNs
Immunoreactive cytokines in the supernatant of cultured PMNs were quantitated using the following enzyme-linked immunosorbent assay (ELISA) kits: IFN- (Biosource International, Camarillo, CA), granulocyte-macrophage (GM)-CSF (Genzyme, Minneapolis, MN), and transforming growth factor-ß1 (TGF-ß1; R&D Systems, Minneapolis, MN). Prior to measurement of TGF-ß1, aliquots of supernatant were acidified transiently with HCl for 10 min at room temperature to activate latent TGF-ß1 to its immunoreactive form and then neutralized with NaOH before assay [13
]. The ELISA methods detected IFN-, GM-CSF, and TGF-ß1 concentrations of above 4 pg/ml, 2.8 pg/ml, and 7 pg/ml, respectively.
Analysis of intracellular CXCR4
Mononuclear cells were collected by density gradient centrifugation using Ficoll-Paque (1.077 g/ml). Neutrophils or mononuclear cells were fixed with 4% paraformaldehyde at 4°C for 30 min and resuspended in 0.1% Tween 20 at 4°C for 30 min for permeabilization. After fixation and permeabilization, intracellular CXCR4 was stained and analyzed by flow cytometry as described for surface staining of CXCR4. Lymphocytes were identified on the basis of their forward/side-scatter properties.
Real-time, quantitative polymerase chain reaction (PCR) analysis of CXCR4 mRNA
Total RNA was extracted from PMNs using a QIAGEN RNeasy Mini Kit (Qiagen, Hilden, Germany), and the first-strand cDNA was reverse-transcribed as described previously [14
]. cDNA was analyzed for human CXCR4 expression by real-time, quantitative PCR using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). The reaction master mix containing the cDNA sample generated from 10 ng RNA was prepared according to the manufacturer’s protocols to yield final concentrations of 1x Taqman Buffer A, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 400 µM dUTP, 3.5 mM MgCl2, 1.25 U AmpliTaq Gold DNA polymerase, 200 nM forward and reverse primers, and 100 nM Taqman probe. The CXCR4 gene-specific primers and probe were purchased from PE Applied Biosystems, and their nucleotide sequences were exactly the same as described previously [15
]. The thermal cycling conditions were as follows: cDNA denaturation, 10 min at 95°C; 48 cycles of denaturation (95°C for 15 s); and annealing/extension (60°C for 1 min). Input cDNA was normalized using a ß-actin primer/probe pair (PE Applied Biosystems) as an internal control gene.
The PCR products of CXCR4 and ß-actin were also analyzed by 2% agarose gel electrophoresis. After ethidium bromide staining, bands were visible only at the expected size for each target mRNA. The standard curve was constructed with serial dilutions of specific PCR products, as described previously [16 ]. In short, CXCR4 or ß-actin PCR products were obtained by amplifying unstimulated PMN cDNA with the same primers and probes as those used for real-time, quantitative PCR. The PCR products were electrophoresed through a 2% agarose gel and visualized with ethidium bromide followed by extraction using QIAEX II (Qiagen), according to the manufacturer’s instructions. The PCR products were quantified spectrophotometrically and stored at -20°C until use. Duplicate standards with copy number controls and no template controls were run in each optical 96-well plate. A negative-control reaction omitting the reverse transcription always showed the lack of a significant amplification signal as a result of contaminating chemokine-receptor DNA.
Statistics
Unless otherwise noted, all data are expressed as the mean ± SE, and differences between values were compared for significance by the paired t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/CXCL12 initiated Ca2+ mobilization and migratory responses in PMNs. Whereas SDF-1/CXCL12 was unable to induce an apparent Ca2+ influx in freshly isolated PMNs, it induced strong Ca2+ influx in 20 h cultured PMNs (Fig. 2A
). In addition, freshly isolated PMNs showed only marginal migration toward high concentrations of SDF-1/CXCL12, but PMNs cultured for 20 h showed enhanced migratory responses to SDF-1/CXCL12 (Fig. 2B)
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and IFN- to a lesser extent suppressed the levels of surface expression of CXCR4. In addition, PMN-directed hemopoietins, i.e., GM-CSF and G-CSF, also down-modulated the expression. Conversely, TGF-ß increased the expression slightly but significantly, whereas none of the other cytokines, including tumor necrosis factor (TNF-), IL-4, and IL-10, exerted significant effects. Time-kinetic studies demonstrated that spontaneous CXCR4 expression was apparent after 4 h of incubation and increased linearly thereafter up to 20 h (Fig. 4A
). IFN- suppressed the expression at all time points with statistical significance. In contrast, surface CXCR2 expression decreased drastically after culturing for 20 h, and IFN- exerted no significant effect on CXCR2 expression (Fig. 4A)
. Half-maximal inhibition of CXCR4 expression was observed at an IFN- concentration of approximately 0.1 ng/ml, a sensitivity compatible with that via interaction with specific IFN- receptors (Fig. 4B)
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and GM-CSF, decreased the surface expression of CXCR4 potently in PMNs, these effects were not mediated by indirect actions resulting from PMN-produced autocrine mechanisms. That is, PMNs cultured with IFN- or GM-CSF failed to release GM-CSF or IFN- in the supernatant, respectively (n=3; unpublished results). Similarly, significant production of TGF-ß was not detected in PMNs cultured with IFN- or GM-CSF.
Mechanisms of CXCR4 expression in PMNs
It has been demonstrated that lymphocytes possess a large cytoplasmic CXCR4 store, which is transported rapidly to the cell surface in response to exogenous stimuli [5
]. To determine whether similar mechanisms explain spontaneous CXCR4 expression in PMNs, we analyzed intracellular CXCR4 in PMNs and lymphocytes. In contrast to lymphocytes, only marginal levels of intracellular CXCR4 were detected in freshly isolated PMNs (Fig. 5
). Accordingly, we studied the change in PMN CXCR4 expression at the mRNA level (Fig. 6A
and 6B
). Real-time, quantitative PCR experiments showed that the surface CXCR4 expression was regulated at a level of pretranslation: The spontaneous increase in the surface CXCR4 protein was accompanied with increase in the CXCR4 mRNA expression (Fig. 6B)
. Conversely, the CXCR4 mRNA expression in IFN--treated PMNs remained low, at levels equivalent to those in freshly isolated PMNs. To elucidate the mechanism of regulation of the CXCR4 mRNA level, we estimated the half-life of CXCR4 mRNA further. It was shown that CXCR4 mRNA has a relatively short half-life of about 2 h in human umbilical vein endothelial cells [17
] and 
1 h in monocytes [18
]. In the case of PMNs, we found that the half-life of CXCR4 mRNA was even shorter, and the half-life was shortened further in IFN--treated PMNs: The half-life was 56 min and 42 min (Exp. 1) and 43 min and 31 min (Exp. 2) for untreated and IFN--treated PMNs, respectively (Fig. 7
). Thus, the reduced levels of CXCR4 mRNA in IFN--treated PMNs were mediated, at least in part by the shortened half-life of CXCR4 mRNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/CXCL12 induced an apparent chemotactic response in 20 h cultured cells (Fig. 2B)
. So far, studies of the effects of SDF-1/CXCL12 on PMNs have yielded conflicting results. Although some groups observed chemotaxis or Ca2+ influx upon stimulation with SDF-1/CXCL12 [4
, 19
], another group was unable to induce significant chemotaxis [12
]. The latter group used simple, one-step Ficoll-Hypaque centrifugation [12
, 20
], whereas the former groups used more complicated and time-consuming methods using glass-bead treatment [4
, 21
] or analyzed PMNs isolated from the leukocytes in transfusion blood [19
, 22
]. Our present results strongly suggest that the conflicting observations concerning the expression and function of PMN CXCR4 may be explained as consequences of different separation conditions, such as the length of isolation procedures.
In contrast to IL-8/CXCL8, which is up-regulated transcriptionally by inflammatory and immunological stimuli (reviewed in ref. [23
]), SDF-1/CXCL12 is expressed ubiquitously in almost all tissues, with little evidence of modulation of expression by exogenous stimulation [2
]. These facts indicate that SDF-1/CXCL12 plays a role in baseline trafficking of PMNs into extravascular tissues rather than in recruiting PMNs to inflammatory sites. Although expression of SDF-1/CXCL12 was hardly modulated by exogenous stimuli, various cytokines were able to modulate surface CXCR4 expression in leukocytes, indicating that the biological action is probably regulated at the level of CXCR4 receptor expression. Of particular importance is that surface CXCR4 expression is regulated in a stimulus- and cell-specific way. For example, IL-4 up-regulates [6
7
8
], and IFN- down-regulates [9
] CXCR4 expression in T cells, and both cytokines exerted exactly the opposite effects on eosinophil CXCR4 expression [10
]. In the present study, we investigated the effects of cytokines on CXCR4 expression in PMNs and found that the profile for cytokine-mediated regulation of PMN CXCR4, such as the effect of IFN-, is different from the profiles in other types of leukocytes such as eosinophils [10
]. These findings extend our previous knowledge that the SDF-1 (CXCL12)/CXCR4 system is regulated by cell-type-specific mechanisms, which may be important in predicting the fate of each cell type during inflammatory processes of different etiology.
PMN CXCR4 expression was down-regulated by G-CSF and GM-CSF. In vitro studies have demonstrated that G-CSF (reviewed in ref. [24 ]) and GM-CSF (reviewed in ref. [25 ]) stimulate mature PMN functions, including degranulation, phagocytosis, adhesion, and chemokinesis. Furthermore, G-CSF may modulate PMN function in vivo, because G-CSF levels are often elevated in the serum or inflammatory sites in septic patients [26 ]. It is interesting that G-CSF was shown to up-regulate the expression of CXCR1/CXCR2 and intensify the chemotaxis of PMNs toward IL-8/CXCL8 [27 ]. Thus, under G-CSF-rich conditions, such as septic states, G-CSF may render circulating PMNs more sensitive to IL-8/CXCL8, thereby facilitating PMN migration from the circulation into affected tissues. Our present results that G-CSF and GM-CSF down-regulated surface CXCR4 expression in PMNs are also in line with this assumption. The decrease in CXCR4 expression caused by G-CSF and GM-CSF may decelerate the movement of PMNs from the circulation to noninflamed tissues expressing SDF-1/CXCL12, which in turn shifts the distribution of PMNs in favor of inflamed tissues.
Surface expression of CXCR4 on PMNs was most potently suppressed by IFN-. Bonecchi et al. [28
] demonstrated that IFN- induces the expression of CC chemokine receptor 1 (CCR1) and CCR3 in PMNs. They also described that IFN- inhibited CXCR4 mRNA accumulation in some donors, albeit they have not studied the change in surface expression [28
]. Although it has been well-documented that IFN- can activate multiple PMN functions (reviewed in ref. [29
]), a role for IFN- in PMN distribution is totally speculative. The fact that PMNs are a cellular source of IL-12 [30
] may indicate a possible role of PMNs under T-helper cell type 1 (Th1)-dominant conditions, and a Th1-dominant situation may lead to an increase in the PMN influx to inflammatory sites via down-regulation of CXCR4 by IFN-. The present data showing down-regulation of CXCR4 in PMNs may suggest an additional aspect of Th1 response induced by IFN-.
In summary, we have demonstrated that functional expression of CXCR4 is inducible in PMNs and that SDF-1/CXCL12 elicits apparent migration in CXCR4-expressed PMNs. IFN-, G-CSF, and GM-CSF inhibited CXCR4 expression. Because alteration of surface CXCR4 expression may be implicated in the in vivo distribution of leukocytes, a more comprehensive understanding of the role of CXCR4/SDF-1 (CXCL12) in normal and disordered conditions should be pursued.
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ACKNOWLEDGEMENTS |
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Received September 26, 2000; revised November 29, 2001; accepted December 19, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rn represents the normalized reporter signal (Rn) minus the baseline signal. (A, Lower) Standard curve-plotting log starting copy number versus threshold cycle (Ct). (
) Standard samples (Std) plotted in duplicate. The parameter Ct represents the cycle at which the amplification plot passed a fixed threshold. CXCR4 mRNA quantification was linear over a range of 104 copies. The data are representative of three independent experiments, all showing similar results. (B) Relative expression of CXCR4 mRNA. The data are expressed as a ratio: copy number of CXCR4 gene:copy number of ß-actin gene (mean±SE, n=3). **, P < 0.01 between the indicated experimental settings.