Novartis Horsham Research Centre, Horsham, West Sussex RH12 5AB, United Kingdom
Correspondence: Gabor Jarai, Ph.D., Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 5AB, United Kingdom. E-mail: gabor.jarai{at}pharma.novartis.com
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Key Words: chemokine receptors • neutrophils • eosinophils • degenerate PCR
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Chemoattractants and their receptors have been proposed as promising targets for the treatment of inflammatory and allergic diseases. Although there is an apparent promiscuity and redundancy in the system with regard to both chemoattractants and their receptors, the multistep navigation model implies that a migrating cell at any given step may depend on one single chemokine receptor. Consequently, blocking this receptor may result in disorientation of the cell, preventing it from reaching the final target. The function of chemokines, that is, to mediate the selective migration of leukocyte subsets to particular tissues, suggests that therapeutics based on chemokine receptor antagonism will be more selective and less compromising than the broad-range immunosuppressants commonly used today [12 , 13 ].
In this study we applied a degenerate-oligonucleotide-primer reverse transcriptase (RT)-PCR approach to study chemokine receptor expression in human neutrophils and to identify potential novel members of this family expressed on human peripheral blood neutrophils. We have also studied the regulation of RNA and protein levels for several of these receptors between resting and activated neutrophils, using Northern and Western blot analyses, RNase protection assays (RPAs), and fluorescein-activated cell sorter (FACS) analysis. Sequencing of cloned amplification products resulted in the identification of the known chemokine receptors CCR1, CCR4, CXCR1, CXCR2, CXCR4, human lipoxin A receptor 63, formyl peptide receptor 1, and a novel GPCR, EX33. Cloning and sequence analysis of a full-length cDNA clone for EX33 indicated that it shows only limited similarity to known receptors. A search of the mouse genomic sequence database using the nucleotide sequence of EX33 also allowed us to identify a putative murine homologue located in mouse chromosome 15. The protein sequences shared over 85% amino acid similarity across the translated region. Expression analysis indicated that human EX33 mRNA is abundant in bone marrow, lung, and peripheral blood leukocytes. Within the leukocyte population we were able to detect higher levels of EX33 transcripts in neutrophils and eosinophils than in T cells and B cells, indicating a restricted pattern of expression. We also present evidence suggesting that locally produced cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) not only regulate the level of cell activation within the inflammatory site for polymorphonuclear leukocytes but also appear to reduce cell surface expression of CXC chemokine receptors, possibly to retard their further movement away from the site of inflammation.
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(100 ng/mL) and
interleukin (IL)-1ß (100 ng/mL) for 48 h [15
].
Freshly isolated neutrophils were then stimulated by direct incubation
with the epithelial cultures or, as a control, in fresh medium for
5 h. After 5 h of incubation, neutrophils were carefully
washed off the epithelial monolayer and collected for analyses.
mRNA isolation and complementary DNA synthesis
Total RNA was extracted using TRIZOL reagent (Gibco-BRL,
Paisley, United Kingdom) as described by the manufacturer. One
milliliter of TRIZOL was used for lysis of every 5 million pelleted
neutrophils. mRNA was purified using the MESSAGEMAKER reagent assembly
kit (Gibco-BRL) under the conditions recommended by the manufacturer.
Three hundred nanograms of mRNA were used to synthesize complementary
DNA (cDNA), using the Superscript Choice system (Gibco-BRL).
Single-stranded and double-stranded cDNAs were made under the
conditions outlined by the manufacturer.
Degenerate-primer PCR and subcloning
Degenerate-oligonucleotide PCR was performed using primers and
conditions as described by Power et al. (1997) [9
].
Briefly, 100 ng of single-stranded cDNA were used to seed a 100-µL
reaction mixture and was subjected to 40 cycles of PCR (95°C for 1
min, 37°C for 1 min, and 72°C for 1 min) using 3 µM
concentrations of each degenerate oligonucleotide primer (5' forward
GAY MGI TAY YTI GCI ATH GTX CA and 5' reverse RMR TAI ADI AII GGR TTI
AXR CA) in a Perkin-Elmer DNA thermal cycler (Perkin-Elmer, Norwalk,
CT). PCR reaction products were visualized on 1% agarose gels
containing 0.5 µg/mL of ethidium bromide. Approximately 10 ng of
amplified product were ligated to 25 ng of pCR2.1 vector (TA cloning
kit; Invitrogen, Groningen, The Netherlands), and the ligation was
introduced into 50 µL of One ShotTM competent cells (Invitrogen). The
libraries were plated onto agar plates containing 50 µg/mL of
carbenicillin, 100 µM isopropylthiogalactoside, and 50 µg/mL of
X-Gal. Plates were incubated at 37°C overnight and then briefly at
4°C to allow blue/white staining to be clearly distinguishable. White
colonies were picked into 200 µL of Luria-Bertani broth containing 50
µg/mL of carbenicillin and incubated overnight. Inserts from clones
were amplified from cultures using primers corresponding to the T7 and
Sp6 sites of the pCR2.1 vector and analyzed by electrophoresis on 1.5%
agarose gels. Amplified inserts which were of the predicted size
(500–550 bp) were digested with the frequent-cutting restriction
enzyme Alu, and products were analyzed after electrophoresis on 4%
agarose gels. Plasmids from selected clones were purified from 3-mL
overnight cultures using the Wizard Plus Minipreps DNA purification
system from Promega (Madison, WI).
DNA sequencing and analysis
Cycle sequencing of 300 ng of plasmid DNA was performed on an
automated ABI310 sequencer (Perkin-Elmer) with M13 reverse and forward
primers. Sequence similarity searches and alignments were performed
using the BLAST algorithm [16
], and the GCG (Madison,
WI) software package (version 9.1,), respectively. Multiple sequence
alignments and the generation of the dendogram were performed using the
MegAlign software of DNASTAR (LaserGene, Madison, WI).
Cloning of the full-length human EX33 cDNA by 3' and 5' rapid
amplification of cDNA ends
For the isolation of a full-length cDNA clone for EX33, 5' and
3' rapid amplification of cDNA ends (RACE) was performed using standard
methods. Briefly, cDNA prepared from peripheral blood leukocytes as
template and the gene-specific primers 5'-GCAAAGCAGAGGAACACAGCAAAC (5'
RACE) and 3'-GCCAAAGCCCAGCCAATTAAAG (3' RACE) were used for the
amplification of 5' and 3' gene fragments with the following PCR cycles
(referred to as "touch-down" PCR): 94°C for 30 s; five
cycles of 94°C for 5 s and 72°C for 4 min; five cycles of
94°C for 5 s and 70°C for 4 min; and finally 22 cycles of
94°C for 5 s and 68°C for 4 min. Amplification products were
subcloned and sequenced as described above.
Northern and "virtual Northern" analyses
For Northern blot analyses, 20 µg of total RNA were separated
by electrophoresis in 1% denaturing formaldehyde agarose gels for
4 h. The RNA was transferred to Hybond N membrane (Amersham
Pharmacia) by capillary transfer using 20x saline sodium citrate (SSC)
and UV cross-linked to the filter. Virtual Northern blots were prepared
by running 500 ng of amplicon cDNA in 1.5% agarose gels and blotting
using standard capillary transfer. To prepare probes, clone inserts
were isolated from 1% low-melting-point agarose gels following
digestion with EcoRI and labeled by random priming of 25 ng
of DNA in the presence of [-32P]dATP (Amersham) using
the Strip-EZ DNA random primed strippable DNA probe synthesis and
removal kit (Ambion, Austin, TX). Hybridizations were performed
overnight in ExpressHyb hybridization solution (Clontech) at 65°C.
Northern blots were washed in 2x SSC–0.05% sodium dodecyl sulfate
(SDS) for 20 min at room temperature, followed by two 20-min washes in
0.1x SSC–0.1% SDS at 50°C. For virtual Northern blots (Southern
blots), 500 ng of amplified cDNA products were loaded per gel and
blotted and probed as described above. After overnight hybridization,
blots were subjected to three consecutive 15-min washes at 65°C in
solutions containing 0.5x SSC–0.1% SDS followed by 0.2x SSC–0.1%
SDS and finally 0.1x SSC–0.1% SDS. Filters were exposed to
phosphorimager plates for between 2 h and 5 days and visualized
with a STORM 840 PhosphorImager (Molecular Dynamics, Little Chalfont,
Buckinghamshire, United Kingdom). Northern blots were hybridized with a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (Ambion) to
control for loading. Multiple-tissue Northern (MTNTM) blots as well as
human RNA Master BlotsTM were purchased from Clontech and hybridized
using conditions recommended by the manufacturer.
RNase protection assay.
Total RNA was extracted from neutrophils using RNAzol B (Life
Technologies, Paisley, United Kingdom). Multiprobe template sets hCR5
(containing DNA templates for CCR1, CCR3, CCR4, CCR5, CCR8, CCR2a+b,
CCR2a, CCR2b, and GAPDH) and hCR6 [containing DNA template for CXCR1,
CXCR2, CXCR3, CXCR4, Burkitt’s lymphoma receptor (BLR)-1, BLR-2/CCR7,
V28, and GAPDH] were purchased from PharMingen, San Diego, CA. The DNA
template mixture was used to synthesize [-32P]uridine
triphosphate (3,000 Ci/mmol; Amersham Life Science, Buckinghamshire,
United Kingdom)-labeled probes in the presence of a GACU pool using a
T7 RNA polymerase (Promega). Hybridization with 5–15 µg of each
target RNA was performed overnight followed by digestion with RNase A
(Roche Diagnostics, Lewes, East Sussex, United Kingdom) and T1
(Calbiochem, La Jolla, CA) according to the PharMingen standard
protocol. The samples were treated with proteinase K (Roche
Diagnostics), extracted with chloroform, and precipitated in the
presence of ammonium acetate. The samples were loaded on an
acrylamide-urea sequencing gel (Novax) next to the labeled probes and
run at a constant 1,200 V for 20 min with 1x QP running buffer
(Novax). The gel was visualized using a STORM 840 PhosphorImager after
2–24 h of exposure.
Generation of anti-EX33 polyclonal antibodies and Western blot
analyses
A synthetic oligopeptide, corresponding to the first 16 amino
acids at the N terminus of the EX33 protein within a predicted
extracellular region of the protein, was used to immunize two New
Zealand White female rabbits. After the third test bleed, the animals
were sacrificed, and the polyclonal antibodies were affinity purified
using the antigenic peptide and stored in phosphate-buffered saline
(PBS). Human neutrophils were isolated from blood and incubated with or
without GM-CSF for 5 and/or 16 h as described above.Cells were
then washed twice with ice-cold PBS, and lysed in RIPA buffer (1x PBS,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented
with an antiprotease cocktail (Roche Diagnostics) and
phenylmethylsulfonyl fluoride (100µM). Cell lysates were centrifuged
for 30 min at high speed, and the solublized protein was stored at
-20°C. Protein concentration was measured using the BCA kit (Perbio,
Knutpunkten, Sweden) according to the manufacturer’s recommendation,
and 40–80 µg of protein were equally diluted 1:1 with sample buffer
(250 mM Tris-HCl, pH 6.8, 20% glycerol, and 10% 2-mercaptoethanol)
and boiled for 5 min before loading onto an SDS-4–14% gradient
polyacrylamide gel (Novax). Proteins were separated electrophoretically
by size and transferred onto nitrocellulose membrane (Novax). Membrane
containing the proteins was blocked overnight at 4°C in PBS
containing 0.1%Tween-20 (PBST) and 5% nonfat dry milk (Blotto; Santa
Cruz Biotechnology, Santa Cruz, CA). The membrane was sequentially
incubated overnight at 4°C with the affinity-purified rabbit
anti-EX33 antibody (1:200) in PBST plus Blotto and then with an
alkaline phosphate-conjugated goat anti-rabbit antibody (1:10,000) in
PBST plus Blotto (Santa Cruz). The membrane was washed repeatedly in
PBST between incubations. Blots were developed using an excitation
chemifluorescence kit from Amersham.
FACS® analysis.
Cell staining was performed using primary
phosphatidylethanolamine (PE)-conjugated monoclonal antibodies against
known CXCR chemokine receptors (R&D Systems) and rabbit anti-EX33
antibody followed by fluorescein isothiocyanate-conjugated,
affinity-purified, anti-rabbit antibodies (PharMinogen). The following
mouse antibodies were used: anti-CXCR1, anti-CXCR2, and anti-CXCR4.
Mouse immunoglobulin G2a-PE conjugated antibody was used as control
(R&D Systems) The samples were analyzed on an FACS
Calibur® (Becton Dickinson, Mountain View, CA).
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 View larger version (50K): [in a new window] |
Figure 1. Isolation and restriction enzyme analysis of
chemokine receptor-like gene fragments from neutrophils.
Degenerate-primer RT-PCR was performed on cDNA of resting (N) and
activated (N+) neutrophils (a). Amplification products were
cloned and their insert sizes determined by 1.5% agaorose gel analysis
(b). Fragments with the expected size were further analyzed by
AluI digestion and 4% agarose gel analysis to identify
unique clones. Restriction patterns of the 38 unique clones are shown.
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Figure 2. Sequence analysis of EX33. The EX33 cDNA and deduced
amino acid sequence are shown (GenBank no. AF282693) (a). Numbers on
the left refer to nucleotide positions. The transmembrane domains
predicted by Kyte-Doolittle hydrophobicity analysis are underlined and
labeled (I–VII). The GRY motif is highlighted, and asterisks label the
three conserved cysteine amino acid residues. The arrowhead indicates
the position of the exon-exon boundary as determined by comparison with
genomic sequence (GenBank no. AC020641). Alignment of the human EX33
protein sequence (top) with that of the mouse predicted sequence
(bottom) was performed using BestFit (GCG) (b). Three conserved
cysteine amino acids and the GRY motif common to both human and mouse
EX33 sequences are boxed. Amino acid similarity and identity across
this region are shown by colons and vertical lines, respectively.
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Figure 3. Sequence similarity relationship of EX33 to
chemotactic receptors. A dendogram, which was generated using the
deduced amino acid sequence of EX33 and of known receptors, is shown.
Multiple alignments were done using the Jotun Hein method.
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Figure 4. mRNA expression analysis of chemokine receptor-like
genes in resting and activated human peripheral blood neutrophils, and
cellular distribution of EX33 among leukocytes. cDNA clones identified
by degenerate-primer RT-PCR are shown along with their expression as
determined by Northern (*) and virtual Northern (unmarked) analysis in
resting (N) and GM-CSF-stimulated (N+) neutrophils (a).
Transcriptional response of various chemokine receptor genes to GM-CSF
stimulation was determined by RPA using resting (N) and
GM-CSF-activated (N+) neutrophils (b). EX33 expression in
unstimulated and stimulated peripheral blood leukocytes was measured by
RT-PCR analysis (c). Leukocytes and stimulants are shown above each
lane. GAPDH was used as a control.
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45 kDa that was in good agreement with
the calculated value based on predicted amino acid sequences. The EX33
protein showed decreased abundance in neutrophils stimulated with
GM-CSF as compared with unstimulated neutrophils (Fig. 5b
); the origin of the second band is not known but is most likely
to be the result of cross-reaction of the antibody. Western blot
analysis of the protein expression pattern of the known chemokine
receptors CXCR1, CXCR2, and CXCR4 also indicated somewhat decreased
protein expression after GM-CSF stimulation (data not shown); however,
the extent of down-regulation appeared much more pronounced at the mRNA
level. To confirm these results and examine receptor expression on the
cell surface, FACS analysis was performed using the same antibodies.
The results of this analysis are presented in Figure 5a
. Constant
levels of cell surface expression were detectable for CXCR1, 2, and 4
and EX33 in freshly isolated cells throughout the 5-h culture period;
however, neutrophils stimulated with GM-CSF significantly decreased
expression levels of these four receptors at 5 and 16 h after
stimulation. This pattern of down-regulation of receptor expression on
the surface of neutrophils was consistent for all CXCRs tested as well
as for EX33, although the signal intensity as well as the extent of
down-regulation appeared to be smaller for EX33. This might in fact
have reflected lower expression levels or, alternatively, might have
been attributed to the potentially lower affinity of the polyclonal
antibody.
 View larger version (19K): [in a new window] |
Figure 5. Protein expression analysis of known chemokine
receptors and of EX33. A time-course analysis of cell surface
expression of CXCR1, CXCR2, CXCR4, and EX33 in resting and GM-CSF
stimulated neutrophils was performed (a). Numbers indicate hours in
culture after isolation and + or - indicate activated or
resting conditions, respectively. Western blot analysis of EX33 was
performed on resting and GM-CSF activated neutrophils with -tubulin
as control for loading (b). _art>
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Using a degenerate PCR approach, we identified several known receptors, among them known chemokine receptors in peripheral blood neutrophils. These included CXCR1 and CXCR2, the two main receptors involved in neutrophil chemotaxis. We also detected expression of CXCR4, the function of which in neutrophils is less clear but appears to be distinct from other chemokine receptors [reviewed in ref. 22 23 ]. Of the CC chemokine receptors we identified CCR1 and CCR4. CCR1 appeared to be expressed in a number of different cell types including neutrophils [22 , 23 ]. CCR4, however, has been shown to be primarily expressed in thymocytes, natural killer cells, and T cells and specifically in the T helper 2 cell subset [22 , 23 ], and we believe that this is the first report on its expression in peripheral blood neutrophils. Its function in these cells, however, is unclear and awaits further studies.
Together with the known receptors, we also identified and cloned the gene for a new receptor, EX33. DNA and amino acid sequences as well as predicted domain structure analyses clearly demonstrated that EX33 is a novel GPCR. Primary-amino-acid-sequence comparisons also indicated that EX33 is a distinctly new member of the family, because its sequence similarity to known receptors was limited. The in silico isolation of the putative mouse homologue allowed us to compare the DNA and amino acid sequences of EX33 between the two species. We observed a high degree of similarity manifested in various features, e.g., intron-exon structure, length of the protein-coding region, conservation of the somewhat unusual GRY motif, and lack of the fourth extracellular cysteine. The conservation of these characteristics between the two species may suggest their functional importance.
Analysis of the expression of EX33 genes in a variety of cells and tissues revealed that they most probably encode a receptor with granulocyte-specific expression. Northern blot and RT-PCR analysis of EX33 provided evidence of its abundant expression in neutrophils and eosinophils, with considerably lower levels present in other cell types and tissues.
The natural ligand(s) of EX33 is yet to be identified, thus classifying EX33 as a novel orphan receptor. Our preliminary analysis using several known chemotactic factors such as IL-8, epithelial-cell-derived neutrophil-activating factor-78, N-formyl-methionyl-leucyl-phenylalanine, and transfectant cell lines expressing EX33 suggested that these are not ligands for EX33 (data not shown). Experiments are currently under way to identify the EX33 ligand(s) that, in turn, will help to determine whether EX33 is involved in chemotaxis and, in general, to elucidate its role in granulocyte function.
The expression of several known chemokine receptors and of EX33 was studied at the transcriptional level as well as the level of their abundance on the cell surface in freshly isolated and ex vivo-stimulated human peripheral blood neutrophils. In general, our data indicated that RNA levels as well as cell surface protein expression levels for CXCRs such as CXCR1, 2, and 4 were reduced, whereas for CCR1 and CCR4, mRNA and protein levels (data not shown) increased after stimulation of neutrophils. Furthermore, expression of EX33 was similar to that of CXCRs, was also decreased after 5 h of coculture stimulation with activated HBECs at the mRNA level, and was also confirmed at the protein level by FACS analysis and Western blotting. This down-regulation could have been due to the presence of IL-8 in the supernatant of the HBECs, which may cause an agonist-dependent internalization/down-regulation of CXCR1 [24 ]. A ligand of CXCR4 could have the same effect on that receptor. However, when we used solely GM-CSF, a main component of the supernatant that is secreted by activated HBECs (data not shown), we observed an identical regulatory trend. Furthermore, the up-regulation of the mRNA for the CC receptors also appeared identical when using GM-CSF or HBEC coculture stimulation, indicating a major role of GM-CSF in these phenomena.
It has been demonstrated that GPCRs and among them chemokine receptors
such as CXCR1 and CXCR2 are regulated by agonist-dependent mechanisms
[25
]. Our present work suggests that GM-CSF
down-modulates the intact cell surface expression of CXCR1 and CXCR2 by
a mechanism that is independent from agonist-mediated internalization.
Previous data on chemokine receptor regulation provide support for the
existence of such mechanisms. It has been shown that certain stimulants
including lipopolysacharide are able to down-modulate CXCR1 and 2
through a unique agonist-independent, tyrosine kinase-dependent
mechanism [26
]. Similarly, tumor necrosis factor- was
shown to down-regulate the expression of CXCR2 receptor on human
polymorphonuclear leukocytes, possibly through receptor shedding
[27
].
The present results have several biological implications. The finding that inflammatory cytokines such as GM-CSF can induce chemokine receptors to undergo significant and prolonged reduction in cell surface expression has mechanistic implications for understanding granulocyte trafficking in vivo. It is accepted that at inflammatory sites GM-CSF is involved in neutrophil activation, has a priming effect, and is antiapoptotic. Our data suggest, in addition, that GM-CSF might be involved in the retardation of the polymorphonuclear neutrophil at the site of inflammation by down-regulating cell surface expression of chemokine receptors.
Received November 5, 2000; revised January 16, 2001; accepted January 19, 2001.
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