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doi:10.1006/cyto.2000.0851, available online at http://www.idealibrary.com on
EXPRESSION AND FUNCTIONAL ANALYSIS OFCHEMOKINE RECEPTORS IN HUMAN
PERIPHERAL BLOOD LEUKOCYTEPOPULATIONS
L. Patel, S. J. Charlton, J. K. Chambers, C. H. Macphee
Emerging evidence indicates that chemokine receptor expression patterns are critical indetermining the spectrum of action of the chemokines. We have analysed the expression patternsof 17 chemokine receptors and two orphan chemokine receptor-like genes in various freshlyprepared human peripheral blood leucocyte populations, including neutrophils, lymphocytes, andnaı̈ve and differentiated monocytes using real-time quantitative polymerase chain reaction(TaqMan�). This is the first comprehensive study of chemokine receptor expression in such awide variety of cell types. Human peripheral blood leukocyte populations were found to expressa wide range of chemokine receptors that varies depending on cell type and differentiation state.Novel expression patterns of certain chemokine receptors were seen during our analysis. Forexample, the orphan chemokine receptor HCR was expressed at very high levels by both primaryneutrophils and primary monocytes, and was further upregulated on neutrophil activation andduring monocyte to macrophage differentiation. When neutrophil calcium transients weremeasured in response to a panel of 30 different chemokines the results clearly correlated with thechemokine receptor expression profile. For example strong calcium responses were seen inneutrophils following stimulation with the CXCR1 and CXCR2 ligands, interleukin (IL-)8,GCP-2 and Gro-�. These data have implications for the study of the functional responses ofleukocytes to external stimuli and will aid in our understanding of general leukocyte biology.
� 2001 Academic Press
From the Department of Vascular Biology, SmithKline BeechamPharmaceuticals, Harlow, Essex CM19 5AW, UK
Correspondence to: Lisa Patel, Department of VascularBiology, SmithKline Beecham Pharmaceuticals, ColdharbourRoad, Harlow, Essex CM19 5AW, UK. E-mail: [email protected]
Received 15 August 2000; accepted for publication 20 December2000
� 2001 Academic Press1043–4666/01/070027+10 $35.00/0
KEY WORDS: monocyte/macrophage/neutrophil/lymphocyte/PCR
The chemokines are a superfamily of small pro-teins which play a critical role in immune and inflam-matory reactions. Most of the chemokines causechemotactic migration of leukocytes,1–5 in addition toother effects on such diverse processes as angiogenesis,6
embryogenesis,7,8 viral infection,9,10 and the prolifer-ation of haematopoietic precursors.11 Thirty-sixchemokines have been identified in humans so far, andare grouped into different families based on thearrangement of the amino-terminal two of their fourcysteine residues: CC, where the first two cysteineresidues are adjacent; CXC, where the first two cysteineresidues of the protein are separated by one aminoacid; CX3C and C. The CXC class includes interleukin
CYTOKINE, Vol. 14, No. 1 (7 April), 2001: pp 27–36
(IL)-8, IP-10 and SDF-1, and the CC class includeseotaxin, monocyte chemotactic protein (MCP1) andmacrophage inflammatory protein-1� and 1� (MIP-1�and -1�). Lymphotactin and fractalkine are the onlychemokines so far described with C and CX3C motifs,respectively.
Chemokines interact with G-protein-coupledreceptors possessing a seven transmembrane domain(7TM). Ten CC (CCR1–11), five CXC (CXCR1–5)and one CX3C (CX3CR1) receptors have been ident-ified, in addition to two orphan chemokine receptor-like genes, HCR12 and Bonzo/STRL33.13 Most ofthese receptors interact with several chemokines. Forexample, CCR3 has been identified as a receptor foreotaxin-1, eotaxin-2, RANTES (regulated on activa-tion, normal T cell expressed and secreted), MCP-2,MCP-3, MCP-4 and MIP-5, whilst CXCR2 binds IL-8,Gro-�, -� and -�, NAP-2 and ENA-78. Conversely,several of the chemokine receptors have been shownonly to bind one ligand to date. Amongst theseare CCR6, which binds Exodus-1,14 and CCR8which binds I-309.15 Interestingly, no chemokine yetidentified is uniquely active on any one leukocyte
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28 / Patel et al. CYTOKINE, Vol. 14, No. 1 (7 April, 2001: 27–36)
population, implying that chemokine receptor expres-sion patterns are critical in determining the spectrum ofaction of the chemokines.
Several lines of evidence indicate a certain dis-parity concerning the expression patterns of chemokinereceptors in various leukocyte subsets. For example,early reports using immunofluorescence to analysechemokine receptor expression on the cell surfaceindicated that freshly isolated neutrophils did notexpress CCR1.16 More recently, however, humanneutrophils have been shown to express functionalCCR1 receptors on their surface,17 which are able tomediate responses to both MIP-1 � and Lkn-1.18
Further evidence indicates that the regulation ofchemokine receptor expression during activation ordeactivation of monocytes is as important as regula-tion of chemokine production for tuning the chemo-kine system.19–22 Recent data show that monocytesexpress a wide variety of chemokine receptors, includ-ing CCR1, CCR2b, CCR3, CCR4, CCR5 andCXCR4.23 However, reports of chemokine receptorexpression patterns in differentiating monocytes andmacrophages have been conflicting. For example, someauthors23 report the downregulation of CCR2b duringhuman monocyte differentiation in culture, coupled toa loss of functional response of these cells to MCP1,one of the ligands for CCR2b. Other data, however,indicate that the levels of CCR2b do not significantlychange over a 10-day period of monocyte differenti-ation.24 Furthermore, there have been reports of T cellsubsets transiently acquiring responsiveness to chemo-kines,25 implying that the regulation of chemokinereceptor expression has a major effect on leukocyteresponsiveness and functionality.
In order to define the expression patterns ofchemokine receptors in different key leukocyte popu-lations, and to provide a detailed study of the potentialresponsiveness of leukocytes to various chemokinestimuli, we analysed the expression of 16 chemokinereceptors and two orphan chemokine receptor-likegenes using real-time quantitative TaqMan� poly-merase chain reaction in a variety of cells includingneutrophils, monocytes, macrophages and lympho-cytes. These expression data were then further corre-lated to functional responsiveness of cells to chemokineligands using fluorescence intensity plate reader(FLIPR) analysis to assay intracellular calcium tran-sients in neutrophils. Our results demonstrate thatdifferent leukocyte subsets express a surprisingly simi-lar array of chemokine receptors when resting. How-ever, this expression pattern changes significantly oncethe cells are activated, underlining the importance ofthe cellular environment on leukocyte responsivenessand function. We also show that TaqMan� is apowerful tool for the analysis of the expression of awide variety of genes in a large number of different
samples, and that receptor expression at the mRNAlevel correlates well with cellular responsiveness torelevant ligands.
RESULTS
Chemokine receptor expression and intracellularcalcium responses in neutrophils
Freshly isolated neutrophils were found to expressCC, CXC and CX3C chemokine receptors (Fig. 1A).Amongst these were CXCR1 and CXCR2, in agree-ment with published data,26 and CX3CR1. In addition,neutrophils were found clearly to express lower levelsof CCR1, CCR3, CCR5, CCR9, CXCR3, CXCR4 andHCR. Upon 3 h incubation of the neutrophils at 37�C,during which time the cells adhered to the bottom ofcell-culture flasks or 96-well plates as for the analysis ofcalcium transients, marked changes in the chemokinereceptor expression profile were observed (Fig. 1B).For example, levels of CXCR1 and CXCR2 increasedapproximately five-fold and ten-fold, respectively,whilst the levels of CCR1, CCR3, CCR9, CCR10,CXCR3, CXCR5 and Bonzo were also significantlyincreased (P<0.05). Interestingly, of all the chemokinereceptors tested in neutrophils, CX3CR1 was the onlyone found to be downregulated following incubation at37�C.
Using FLIPR analysis to assay intracellular Ca2+
transients in neutrophils that had been allowed to platedown for 3 h, we observed responses to several chemo-kines, including large responses to Gro-�, IL-8, GCP-2and MIP-2 (ligands for CXCR1 and CXCR2), as wellas responses to the CXCR4 ligands SDF-1� and �, anda weaker response to MIP-3. Responses were also seento the viral chemokine receptor, UL146. No responseswere seen to any of the other chemokines tested (seeFig. 2). Since a good correlation was seen betweenreceptor expression and chemokine responsiveness inneutrophils, we then went on to study chemokinereceptor expression solely at the mRNA level in humanprimary monocytes, macrophages and lymphocytes.Our decision to do this was also based on our obser-vations that it is difficult to induce lymphocyte andmonocyte/macrophage adherence to plates sufficientlystrongly to facilitate FLIPR analysis.
Chemokine receptor expression in monocytes andmacrophages
In agreement with previous results23,27,28 we wereable to detect CCR1, CCR2b, CCR5, CCR9, CX3CR1and CXCR4 in freshly isolated naı̈ve monocytes(Fig. 3A). In addition, we detected many of the remain-ing chemokine receptors including CCR10, CXCR1,CXCR2, CXCR3 and HCR in our samples. Of all ofthe chemokine receptors, HCR was expressed at the
Chemokine receptors and leukocytes / 29
highest levels in naı̈ve monocytes. During monocyte tomacrophage differentiation, significant changes wereseen in the chemokine receptor profile of the cells(Figs 3B and C). Several of the chemokine receptorswere significantly upregulated on differentiation(P<0.05), including CCR1, CCR3, CCR5, CCR10 andHCR, which were upregulated approximately six-, 20-,45-, two- and 18-fold respectively at their maximumlevels. Interestingly, whilst the expression levels ofCCR1, CCR2b, CCR3, CCR10 and HCR reached a
peak at day 1 of differentiation, levels of CCR5 con-tinued to rise to day 4. Concurrently, several of thechemokine receptors were downregulated during dif-ferentiation including CCR9, CXCR1, and CXCR2and CX3CR1 (with a P value of less than 0.05). ForCXCR1 CXCR2 and CX3CR1in particular, down-regulation of the mRNA was very rapid, often occur-ring within 1 h of cell adhesion (data not shown).These data were further supported by the observationthat these receptors were rapidly downregulated in theTHP-1 monocytic cell line upon the induction ofdifferentiation (data not shown). Of all the receptorsanalysed in monocytes and macrophages, HCR wasconsistently expressed at the highest levels, whilstCCR4, CCR6, CCR7, CCR8, CXCR5 and Bonzo wereexpressed at very low levels or not at all.
Chemokine receptor expression in lymphocytesChemokine receptor expression levels in lym-
phocytes were generally lower than those detected inboth monocytes and neutrophils. However, a widevariety of chemokine receptors were nonethelessdetected in these cells (Fig. 4), including CXCR2,CXCR3, CX3CR1, CCR3, CCR7, CCR1 and HCR.Of these, CXCR2 and CX3CR1 were expressed at thehighest levels (and at higher levels than those found inmonocytes). Low levels of CCR2b, CCR5, CCR9,CXCR1 and CXCR4 were also detected in lym-phocytes, whilst CCR6, CCR8, CCR4, CXCR5 andBonzo were not detected.
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Figure 1. Chemokine receptor expression in neutrophils.
Neutrophils were isolated from whole human blood by countercurrent centrifugal elutriation as described in Materials andMethods. Lysates were taken immediately after isolation and follow-ing a 1 h incubation at 37�C in neutrophil suspension buffer. Thedata represent the mean�SEM from three different humanneutrophil preparations. *P<0.05 and **P<0.005 when comparedwith freshly isolated neutrophils.
DISCUSSION
We have analysed the expression of 17 chemokinereceptors and two chemokine receptor-like genes in avariety of leukocytes using TaqMan� quantitativePCR analysis in order to further clarify the extent towhich the chemokine receptor profile of leukocytes ismodified on activation or differentiation. This is thefirst report on the expression patterns of the fullcomplement of chemokine receptors in leukocytes. Wefurther correlated this TaqMan� expression data witha study of the functional responsiveness of humanneutrophils to 30 different chemokine ligands, usingFLIPR analysis to assay calcium transients.
A wide variety of chemokine receptors weredetected in both freshly isolated neutrophils andneutrophils incubated for 3 h at 37�C, during whichtime the cells adhered to cell-culture flasks or plates.The rapid changes in chemokine receptor expressionprofiles in neutrophils observed during this 3 h incu-bation period are interesting, given that no otherstimulation was used to ‘‘activate’’ these cells, andindicates that neutrophils produce factors that are ableto influence their own gene expression profiles. This
30 / Patel et al. CYTOKINE, Vol. 14, No. 1 (7 April, 2001: 27–36)
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Figure 2. Fluorescence intensity plate reader (FLIPR) analysis of human primary neutrophils.
Neutrophils were isolated from whole human blood as described in Materials and Methods and allowed to plate for 3 h at 37�C before FLIPRanalysis was performed using a panel of chemokines in duplicate at a final concentration of 100 nM. Data shown (expressed in FluorescenceIntensity units, FIU) is from one experiment representative of three that were performed using neutrophils isolated from different individuals.
observation is of note when preparing neutrophils forgeneral experimental purposes, and may reflect a needfor caution when using neutrophils that have beenallowed to stand. We have shown that, in agreementwith published data,29 neutrophils express high levelsof CXCR1 and CXCR2.26,30 These levels are upregu-lated in neutrophils incubated at 37�C for 3 h, confirm-ing the importance of these receptors in neutrophilfunction. These data are further supported by thedemonstration of Ca2+ transients to the CXCR1 andCXCR2 ligands, Gro-�, IL-8, GCP-2 and MIP-2 andclearly shows a correlation between chemokine recep-tor expression at the mRNA level and functionalresponsiveness to chemokines. Many of the otherchemokine receptors including CCR1, CCR3, CCR5,CCR10, CXCR3, CXCR5, Bonzo and HCR were alsoupregulated in incubated neutrophils. Our findingssupport data that show the expression of functionalCCR1 molecules on the neutrophil surface18 since wewere able to demonstrate calcium transients inresponse to CK-b8 and MIP-3, both of which are ableto bind CCR1, in our samples.17 Other groups31,32
have reported that there is no CXCR3 or CXCR5mediated chemotaxis of human neutrophils. However,our finding that CXCR3 and CXCR5 mRNAs areupregulated in neutrophils after incubation for severalhours indicate that neutrophils may acquire respon-siveness to the ligands for these receptors after a briefperiod of incubation. Given that the original studiesused freshly isolated neutrophils we feel that it may beinteresting to return to these studies and repeat themwith neutrophils simply incubated at 37�C for several
hours, when the upregulation of receptor mRNAs maybe reflected at the protein level. We were further able todetect CX3CR1 mRNA in freshly isolated neutrophils.This expression was rapidly downregulated, uniquelyamong the chemokine receptors analysed in neutro-phils and may indicate a role for CX3CR1 in neutro-phils analogous to its role in monocytes, where itsexpression is also rapidly downregulated. Severalchemokines whose receptors were expressed in neutro-phils did not stimulate a calcium response. Amongstthese were MIP-1� MIP-1�, and eotaxin, which havepreviously been shown to induce little or no calciummobilisation in human neutrophils despite the presenceof their receptors.18,33 This may reflect the fact thatcertain chemokine receptors are able to induce differ-ent biological responses depending on the ligandbound to them. Our demonstration of a clear corre-lation between chemokine receptor expression patternat the mRNA level and functional responsiveness tochemokines is further supported by the fact that wewere unable to detect calcium transients in neutrophilsin response to many chemokines, including MCP-1,MCP-2, TARC, LARC and SLC none of whose recep-tors were detected at the mRNA level in these cells.This finding, and the difficulty in inducing lymphocyteand monocyte/macrophage adherence to plates suffi-ciently strongly to facilitate FLIPR analysis, led us tostudy chemokine receptor expression in monocytes,macrophages and lymphocytes solely through the useof TaqMan� analysis.
During our analysis we observed dramaticupregulations of chemokine receptors such as CCR1,
Chemokine receptors and leukocytes / 31
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Figure 4. Chemokine receptor expression in lymphocytes.
Lymphocytes were isolated from whole human blood by countercurrent centrifugal elutriation as described in Materials andMethods. Lysates were taken immediately after isolation. The datarepresent the mean�SEM from three different human lymphocytepreparations.
Figure 3. Chemokine receptor expression in monocytes andmacrophages.
Human monocytes were differentiated in a 12-well microtitre plateformat as described in Materials and Methods. Medium was changedevery 48 h for a total of 4 days and lysates taken at days 1 and 4 formRNA quantification. The data represent the mean�SEM fromthree different human monocyte preparations. *P<0.05 and**P<0.005 when compared with naı̈ve monocytes. (a) Monocytes;(b) day 1 macrophage; (c) day 4 macrophages.
CCR2b and HCR during monocyte to macrophagedifferentiation whilst levels of CXCR1, CXCR2 andCX3CR1 were rapidly reduced. Similar alterations inchemokine receptor expression were seen on THP-1monocytic cell differentiation (data not shown). Thedemonstration of CXCR2 expression on naı̈ve mono-cytes supports previously published data demonstrat-ing a role of CXCR2 in monocyte adhesion androlling.34 However, using this system, we were barelyable to detect CCR8 on monocyte/macrophages. Thisis in conflict with previously published data showingthat CCR8 is expressed on naı̈ve monocytes.15 Ourfindings are, however, supported by the findings ofothers35 who report the absence of CCR8 transcript infreshly isolated monocytes, macrophages, lympho-cytes and neutrophils. In addition, others havereported a decrease in CCR2b expression duringmonocyte macrophage differentiation,23 and a concur-rent maintenance of CCR1 levels whilst we detected anupregulation both of CCR1 and CCR2b. However,
32 / Patel et al. CYTOKINE, Vol. 14, No. 1 (7 April, 2001: 27–36)
our data are consistent with the proposed role ofCCR2b in atherosclerosis36,37 and the observation thatCCR2b is expressed by adventitial macrophages in theatherosclerotic lesions of ApoE knockout mice,38 andwe suggest that the observed differences in gene expres-sion patterns may be due to the culturing conditionsused for the monocyte differentiation and the mono-cyte isolation procedure. Whilst we have preparedhigh-purity naı̈ve monocytes, several groups reportmonocyte isolation by plating from a mixed populationof mononuclear cells over a period of up to 24 h.Considering the fluid nature with which chemokinereceptors are modulated, this method may be harshand may produce results that do not reflect the situ-ation in naı̈ve monocytes. We have previously demon-strated that differentiation in the presence of humanserum leads to the formation of biologically relevantmacrophages, as judged by the expression of the mac-rophage markers, osteopontin and MMP9, and the cellsurface expression of CD36, indicating that our proto-col generates macrophages that appear reflective ofthose seen in vivo. However, whilst our monocyteswere differentiated into macrophages over a period of 4days in medium supplemented with human serum,other workers23 supplement their medium with foetalcalf serum, which we have found to produce somewhatdiffering effects on gene expression within humanmonocytes and macrophages (personal observations).
Lymphocytes were found to express a similarlywide variety of chemokine receptors. These includedCCR3, CCR7, CXCR2, CXCR3 and CX3CR1, all ofwhich have potential roles in lymphocyte biology.25,39–
45 In addition, we were able to detect low levels ofCCR9, the TECK receptor, as well as CCR2b andCXCR4 in lymphocytes. Previous studies have shownthe presence of CXCR4 in lymphocytes.46,47 However,this is the first report of CCR9 expression in freshlyisolated human lymphocytes, and our observation sup-ports previous findings that CCR9 is expressed inmature murine T-cells.48 A report49 has also shownthat peripheral blood lymphocytes migrate normally toa MCP-1 gradient. The detection of CCR2b expressionin lymphocytes supports this observation.
In conclusion our data support the widely rangingrole of chemokine receptors in the progression of bothchronic and acute inflammatory diseases. We haveshown that different peripheral blood leukocyte popu-lations express a varying range of chemokine receptors,and as well as showing novel expression patterns ofcertain receptors we have shown that there is a strongcorrelation between receptor expression at the mRNAlevel and functional responsiveness to a variety ofligands in neutrophils. These data will have implica-tions for the study of the functional responses ofleukocytes to external stimuli and will aid in ourunderstanding of general leukocyte biology.
MATERIALS AND METHODS
Isolation of human neutrophilsNeutrophils were isolated from whole blood collected
from healthy donors using a counter current centrifugalelutriation, as previously described.50 Cell viability andpurity were assessed as for monocytes and lymphocytes.(Contamination of the neutrophil preparation with other celltypes was 0.4% lymphocytes and 0.1% monocytes as assessedfor three samples isolated from three different individuals.)Samples were lysed for RNA extraction using Trizol reagentboth immediately following isolation and after a 3 h incu-bation at 3 million cells/ml in RPMI at 37�C, during whichtime isolated neutrophils plated to the bottom of a cellculture flask or 96-well plate for FLIPR analysis. This timeperiod was used in order to see if neutrophils are able toinfluence their own functionality through the release ofself-stimulating factors.
Isolation of human monocytes and in vitromonocyte/macrophage differentiation
Human monocytes were isolated from buffy coat pre-parations of whole blood taken from healthy volunteers. Inbrief, the buffy coat was mixed with Optiprep� (RobbinsScientific Ltd) in a ratio of 2.5:1 and then overlaid with adiscontinuous Optiprep� gradient, prepared according to thereagent datasheet. Following centrifugation for 25 min at600�g the monocyte layer formed within the top 5–10 ml ofthe gradient was removed, washed with PBS and resuspendedin culture medium [RPMI 1640, supplemented with 2 mMglutamine and 2% human serum (type AB, Sigma)]. Cellviability was assessed by the ability to exclude trypan blueand was typically �95%. Monocyte purity was determinedby differential counts of DiffQuik (Porvair Sciences Ltd)stained cell preparations and was typically �97% (overallcontamination from three independent preparations fromthree different donors was 2.95% lymphocytes and 0.05%neutrophils). For monocyte–macrophage differentiation,monocytes isolated as above were resuspended in culturemedium at a density of 2.5�106/ml and seeded into 12-welltissue culture plates; medium was changed every 48 h. Underthese conditions, monocytes differentiate to macrophageswithout the application of any further stimulus. Cell lysatesfor RNA extraction were prepared by the addition of Trizolreagent (Gibco BRL) at 1 day and 4 days following plating.Lysates were similarly prepared from naı̈ve monocytes.
Isolation of human lymphocytesLymphocytes were isolated from human blood taken
from healthy volunteers using counter current centrifugalelutriation. Blood collected into EDTA anticoagulant wasspun through a Histopaque 1.077 gradient (Sigma) at 400�gfor 20 min. The resulting mononuclear layer was then washedwith elutriation buffer [PBS supplemented with 1% humanserum albumin (Sigma)] and loaded onto the counter currentcentrifugal elutriator (Beckman Instruments) at a flow-rate of10 ml/min. Lymphocytes typically eluted at 14–15 ml/min.Cell viability was assessed by the ability to exclude trypanblue and was typically �95%. Lymphocyte purity (typically100%) was determined by differential counts of DiffQuik
Chemokine receptors and leukocytes / 33
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34 / Patel et al. CYTOKINE, Vol. 14, No. 1 (7 April, 2001: 27–36)
(Porvair Sciences Ltd) stained cell preparations. Cell lysatesfor RNA extraction were prepared immediately following cellisolation using Trizol reagent (Gibco BRL).
RNA isolation and reverse transcriptionTotal RNA was extracted using Trizol reagent (Gibco
BRL) according to the manufacturer’s protocol. cDNA wasreverse transcribed from DNAse I (Gibco BRL) treated totalRNA using Superscript II reverse transcriptase (Gibco BRL)as per the manufacturer’s instructions and using randomhexamers as the primer. A negative control reaction omittingthe reverse transcriptase (RT) was also performed for eachDNase-treated RNA sample. Reactions were diluted 20-foldwith sterile water and stored at �20�C. All samples werereverse transcribed under the same conditions and from thesame reverse transcription master mix in order to minimisedifferences in reverse transcription efficiency.
Standard preparationA series of standards was prepared by performing
ten-fold serial dilutions of full-length chemokine receptorcDNAs in the range 20 million copies to two copies perTaqMan� reaction. Full-length chemokine receptor cloneswere isolated from different tissues by either library screeningor PCR amplification and DNA was prepared using theConcert Rapid MiniPrep Kit (Gibco BRL) according to themanufacturers instructions. All plasmid samples weretreated with RNase A prior to quantitation in order tominimise contamination with bacterial RNAs from theplasmid purification procedure and subsequently quantifiedusing a combination of absorbance at 260 nm and gelelectrophoresis.
Relative quantitation of mRNAs by real-timequantitative RT-PCR using the fluorescentTaqMan� 5� nuclease assay
TaqMan� is a recently developed technique, in whichthe release of a fluorescent reporter dye from a hybridisationprobe in real-time during PCR is proportional to theaccumulation of the PCR product.51–54 Quantification isbased on the early, linear part of the reaction, and bydetermining the threshold cycle (Ct), at which fluorescenceabove background is first detected. The copy number ofRNA can be calculated by comparison with a standard curveof cloned DNA. Previous studies have shown a strongcorrelation between TaqMan� data and more conventionalassays using direct measures of RNA, such as the RNaseprotection assay.55 However, TaqMan� PCR requires lessRNA and is significantly less time consuming than othermethods, which is of particular consideration when availablecell numbers are limited.
Five microlitres of each cDNA sample, as well asstandard curve samples, were analysed for chemokine recep-tor expression by real-time quantitative RT-PCR using thefluorescent TaqMan� 5� nuclease assay. TaqMan� assayoligonucleotide primers and probes were designed usingPrimer Express� software version 1.0 (PE Biosystems, War-rington) (see Table 1 for sequences), and were all shown toamplify from both genomic DNA and cDNA samples. EachTaqMan� hydrolysis probe consisted of the fluorescent
reporter dye FAM (6-carboxyfluorescein) covalently linkedto the 5�-end of the oligonucleotide and the quencher dyeTAMRA (6-carboxytetramethylrhodamine) attached to the3�-end via a linker group (PE Biosystems, Warrington).
5�– and 3� nuclease assay PCRs were performed inMicroAmp� Optical 96-well Reaction Plate and OpticalCaps (PE Biosystems, Warrington) using the ABI PRISM�
7700 Sequence Detection System for thermal cyclingand real-time fluorescence measurements (PE Biosystems,Warrington). Each 25 �l reaction consisted of 1X TaqMan�
Universal PCR Master Mix (PE Biosystems, Warrington)[10 mm Tris–HCl (pH 8.3), 50 mM KCl, 10 mM EDTA,60 nM passive reference dye 1 (ROX (6-carboxy-X-rhodamine)), 0.2 mM dATP, 0.2 mM dCTP, 0.2 mMdGTP, 0.4 mM dUTP, 5.5 mM MgCl2, 8% glycerol,0.625 U AmpliTaq Gold� DNA polymerase, and 0.25 UAmpErase� uracil N-glycosylase (UNG)], 300 nM forwardprimer, 300 nM reverse primer, 100 nM TaqMan� quan-titation probe and 5 �l template. Reaction conditions were asfollows: 50�C for 2 min, 95�C for 10 min then 40 cycles of95�C for 15 s and 60�C for 1 min. Emitted fluorescence foreach reaction well was measured every cycle during both thedenaturation and annealing/extension phases, and amplifica-tion plots were constructed using the ABI PRISM� 7700Sequence Detection System (SDS) software version 1.6 (PEBiosystems, Warrington).
Subsequent analysis was performed on the data outputfrom the Sequence detector software using Microsoft Excel.Quantity values generated for the expression of each chemo-kine receptor by Sequence Detector (values generated bycomparison of the fluorescence generated by each samplewith standard curves of known quantities) were dividedby the quantity of total RNA present in each TaqMan�
reaction (assuming that cDNA is generated from RNA in aratio of 1:1). This gave a normalised value for the expressionlevel of each chemokine receptor in each sample.
Statistical analysisDifferences were assessed using a two-tailed Student’s
t-test.
Assessment of intracellular calcium transients inhuman neutrophils
Intracellular Ca2+ responses were assayed using a fluor-escence intensity plate reader (FLIPR) (Molecular Devices)essentially, as previously described,56 with minor modifica-tions as follows. Briefly, cells were prepared by countercur-rent centrifugal elutriation and seeded at 300 000 cells perwell in the presence of RPMI supplemented with 1% BSA.Plates were left at 37�C for 3 h to allow cells to attach priorto loading with dye. Cells were washed free of extracellulardye very gently by hand in order to minimize cell detachment.
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