In order to identify domains crucial for ligand recog-nition by FPRL1, we used chimeric receptors with segments in FPRL1 replaced by corresponding amino acid sequences derived from the p
Trang 1receptor-like 1 for agonist-induced cell chemotaxis
Yingying Le1,2, Richard D Ye3, Wanghua Gong4, Jianxiang Li1, Pablo Iribarren1and Ji Ming Wang1
1 Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, MD, USA
2 Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
3 Department of Pharmacology, College of Medicine, University of Illinois at Chicago, IL, USA
4 Basic Research Program, Center for Cancer Research, National Cancer Institute at Frederick, MD, USA
Leukocyte recruitment to sites of inflammation and
infection is dependent on the presence of a gradient of
locally produced chemotactic factors The bacterial
pep-tide N-formyl-methionyl-leucyl-phenylalanine (fMLF)
is one of the first identified and highly potent
leuko-cyte chemoattractants [1–3] fMLF interacts with at
least two human cell receptors formyl peptide receptor
(FPR) and its variant formyl peptide receptor-like 1
(FPRL1), both are members of the seven
transmem-brane domain, G protein-coupled receptor (GPCR)
family [4–6] FPR is activated by picomolar to low
nanomolar concentrations of fMLF and is defined as
the high-affinity fMLF receptor FPRL1 possesses 69% identity at the amino acid level to FPR and is defined as the low-affinity fMLF receptor based on is activation only by high concentrations of fMLF [4–6] Both FPR and FPRL1 are expressed by phagocytic leukocytes and have been detected in cells of nonhema-topoietic origin [5,6] However, compared with FPR, FPRL1 appears to be expressed in an even greater variety of cell types, including epithelial cells, resting
T lymphocytes, astrocytoma cells, neuroblastoma cells, and microvascular endothelial cells [5,6] Although the importance of FPR in host defense against bacterial
Keywords
chemotaxis; formyl peptide receptor; formyl
peptide receptor-like 1; structure–function
Correspondence
J M Wang, LMI, CCR, NCI-Frederick, Bldg.
560, Rm 31-40, Frederick, MD 21702, USA
E-mail: wangji@mail.ncifcrf.gov
(Received 19 August 2004, revised 23
November 2004, accepted 3 December
2004)
doi:10.1111/j.1742-4658.2004.04514.x
Formyl peptide receptor-like 1 (FPRL1) is a seven transmembrane domain,
G protein-coupled receptor that interacts with a variety of exogenous and host-derived agonists In order to identify domains crucial for ligand recog-nition by FPRL1, we used chimeric receptors with segments in FPRL1 replaced by corresponding amino acid sequences derived from the proto-type formyl peptide receptor FPR The chimeric receptors were stably transfected into human embryonic kidney epithelial cells and the capacity
of the cells to migrate in response to formyl peptide receptor agonists was evaluated Our results showed that multiple domains in FPRL1 are involved in the receptor response to chemotactic agonists with the sixth transmembrane domain and the third extracellular loop playing a promin-ent role Interestingly, the N-terminus and a segmpromin-ent between the fourth transmembrane domain and the third intracellular loop of FPRL1 are important for receptor interaction with a 42 amino acid amyloid b peptide (Ab42), an Alzheimer’s disease-associated FPRL1 agonist, but not with MMK-1, a synthetic FPRL1 agonist, suggesting that diverse agonists may use different domains in FPRL1 Considering the potential importance of FPRL1 in inflammation and neurodegenerative diseases, the identification
of functional domains in this receptor will provide valuable information for the design of specific receptor antagonists
Abbreviations
Ab 42 , 42 amino acid amyloid b peptide; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; fMLF, N-formyl-methionyl-leucyl-phenylalanine; FPR, formyl peptide receptor; FPRL1, formyl peptide receptor-like 1; HEK293 cells, human embryonic kidney epithelial 293 cells; LXA4, lipoxin A4; SAA, serum amyloid A; TM, transmembrane domain; W peptide, Trp-Lys-Tyr-Met-Val- D -Met.
Trang 2infections has been demonstrated by the increased
sus-ceptibility to Listeria monocytogene of mice depleted
of FPR1, the mouse homologue of human FPR [7],
the nonredundant biological role of FPRL1 has yet to
be clearly established
Recent studies have identified a variety of
host-derived, and structurally unrelated peptide agonists for
FPRL1, such as the 42 amino acid form of b amyloid
peptide (Ab42) associated with Alzheimer’s disease [8],
the acute phase protein serum amyloid A (SAA) [9], a
fragment of the neutrophil antibacterial granule
pro-tein cathelicidin LL37 [10], and a peptide derived from
human prion protein [11] The capacity of FPRL1 to
interact with diverse agonists suggests its potentially
broad role in the process of inflammation and
amy-loidogenic diseases Thus, elucidation of domains in
FPRL1 crucial for its function will not only shed light
on the structural basis for recognition by this receptor
of agonists associated with different pathophysiological
conditions, but also provide leads to the design of
receptor antagonists In this study, we evaluated the
function of various chimeric FPRL1 constructs by
replacing its segments with the corresponding
seq-uences derived from the prototype formyl peptide
receptor FPR We report that multiple domains are
involved in FPRL1 interaction with its specific peptide
agonists, with a prominent role for the sixth
trans-membrane domain (TM) and the third extracellular
loop in mediating the chemotactic function of this
receptor In addition, two defined FPRL1 agonists
Ab42and MMK-1 were found to use different domains
in the receptor
Results
Human FPR and FPRL1 are highly homologous and chimeric receptors based on their sequences may mini-mize possible global conformational changes (Fig 1)
To evaluate the relative contribution of several domains in FPRL1 to its capacity to interact with chemotactic agonists, we measured the migration of HEK293 cells stably expressing chimeric formyl pep-tide receptors Figure 2A,B illustrates the putative configuration of the wild-type FPR and its variant FPRL1 Eight chimeric receptor constructs are shown
in subsequent figures with open circles denoting seg-ments of FPR and filled circles FPRL1 Replacement
of the FPR segments with those from FPRL1 or vice versa was contiguous but not overlapping (except for chimera B, CH295-351) (Fig 3) This approach per-mits systematic evaluation of the impact of one swapped segment at a time on receptor response to the chemotactic agonists Levels of cell-surface expression
of the wide-type and chimeric formyl peptide receptors were examined by measuring the capacity of HEK293 cells transfected with the receptor cDNAs to bind a radioiodinated synthetic peptide, W peptide (125 I-labe-led Wpep), which has been shown to bind and activate both FPR and FPRL1 [14] The data confirmed that cells transfected with the receptors exhibited substan-tial and comparable binding sites for125I-labeled Wpep with similar estimated affinity (Table 1)
Four defined formyl peptide receptor agonists were used to evaluate the chemotactic responses of HEK293 cells expressing wild-type or chimeric receptors The
Fig 1 Alignment of the deduced amino acid sequences of human FPRL1 and FPR The entire FPRL1 sequence (upper) is shown and the amino acids are numbered above the sequence Residues of FPR that differ from FPRL1 are shown in the bottom (A) One-residue gap (*)
is introduced in the sequence of FPR to allow linear alignment with the FPRL1 The location of predicted transmembrane domains (TM) are underlined.
Trang 3bacterial peptide fMLF at low nanomolar
concentra-tions induces potent chemotactic response mediated by
FPR but is a poor chemotactic factor for FPRL1 in
the same concentration range [12], whereas the
syn-thetic peptide MMK-1, which is identified from a
ran-dom peptide library [13,14], is active only on FPRL1
in a low nanomolar concentration range [14] Ab42, the
Alzheimer’s disease-associated FPRL1 agonist [8,15],
was also used to evaluate the contribution of FPRL1 domains to the receptor function By contrast, the synthetic peptide, W peptide, activates both FPR and FPRL1 at nanomolar concentrations with higher effic-acy on FPRL1 [14,16,17] This peptide did not induce significant chemotaxis of parental HEK293 cells [16] (and data not shown) The specificity of the peptide agonists on FPR and FPRL1 was tested on cells trans-fected with wild-type receptors HEK293 cells expres-sing FPR migrate in response to low concentrations
of fMLF with an EC50 of 0.1 nm and a maximal cell response at 10 nm of fMLF (Fig 2A) By contrast, fMLF at up to 100 nm did not induce significant migration of HEK293 cells expressing FPRL1 (FPRL1⁄ 293) (Fig 2B), although these cells migrated
in response to the peptide MMK-1 at picomolar and low nanomolar concentrations (Fig 2C) MMK-1 is not chemotactic for FPR⁄ 293 cells in a wide concen-tration range tested (Fig 2C) Consistent with previous results [8], Ab42 induced chemotactic response in cells expressing FPRL1 but not FPR (Fig 2C) As predic-ted, the bispecific chemotactic agonist W peptide induced significant migration of both FPR⁄ 293 and FPRL1⁄ 293 cells at low nanomolar concentrations with higher efficacy on FPRL1⁄ 293 cells (Fig 2A,B) These results demonstrate an effective functional expression of the formyl peptide receptors in HEK293 cells and confirmed the specificity and efficacy of their respective agonist peptides
We next investigated the chemotactic response of HEK293 cells transfected to express chimeric formyl peptide receptors Replacing the C-terminal half of the seventh TM and the cytoplasmic tail of FPR or the C-terminal third of the receptor with the correspond-ing sequence from FPRL1 yielded chimeric receptors A (CH295-351) (Fig 3A) and B (CH241-351) (Fig 3B) These two chimeras maintained responses to W pep-tide equivalent to wild-type FPR but to fMLF with
a lower efficacy (chimera A) or no response at all (chimera B) Although chimera A failed to mediate cell chemotaxis in response to MMK-1 at any concentra-tion tested, chimera B gained a significantly increased chemotactic response to MMK-1 albeit with lower potency and efficacy as compared with the wild-type FPRL1 (Figs 2C and 3C) These results suggest that replacing the C-terminus or a segment starting from the sixth TM region with the sequence from FPRL1 reduced the efficacy of FPR to respond to its cognate agonist fMLF but enabled the chimera to effectively interact with FPRL1 specific agonists Overall, FPR and FPRL1 share 68% identity at the amino acid level
in the region starting from the sixth TM to the cyto-plasmic tail and a higher degree of homology was
Fig 2 Putative structure and chemotactic responses mediated by
FPR and FPRL1 The putative FPR and FPRL1 are depicted by open
(A) and filled (B) circles, respectively FPR and FPRL1 were
trans-fected into HEK293 cells and tested for chemotactic response to
chemoattractants W peptide (W pep), fMLF (A and B), as well as
MMK-1 and Ab 42 (C) The results are presented as chemotaxis
index (CI) defining the fold increase of migrating cells in response
to receptor agonists over cell migration in the absence of agonists.
*P < 0.01; significantly increased cell migration as compared with
cell migration in the absence of stimulants W peptide (W pep) at
100 n M was used as a control in (C).
Trang 4found in the seventh TM and the proximal half of the
cytoplasmic tail connecting the seventh TM (Fig 1)
Thus, the sixth TM domain and the third extracellular
loop of FPRL1 are important in its interaction with
MMK-1
We also examined the chemotactic response of chi-mera B to Ab42 Chimera B (CH241-351) expressing HEK293 cells migrated to Ab42with potency and effic-acy similar to the wild-type FPRL1 (Figs 2C and 3C), suggesting that the sixth TM domain and the third
Fig 3 Schematic composition and function of FPR ⁄ FPRL1 chimeria A and B Chimeras A (A) and B (B) were constructed by replacing the C-terminal half of the seventh TM and the cytoplasmic tail of FPR, or its C-terminal third with the corresponding sequence from FPRL1 Each construct is named alphabetically followed by letters of the first and the last residues of the switched FPR (s) or FPRL1 (d) fragment in the chimera Chimeras were transfected into HEK293 cells and tested for chemotactic response to peptide agonists W peptide (W pep) and fMLF (A and B), as well as MMK-1 and Ab42(C) The results are presented as chemotaxis index (CI) defining the fold increase of migrating cells in response to receptor agonists over cell migration in the absence of agonists *P < 0.01; significantly increased cell migration as com-pared with medium W peptide (W pep) at 100 n M as used as a control in (C) The capacity of chimeras A and B to mediate Ca2+flux in response to peptide agonists were also measured (A–C).
Trang 5extracellular loop of FPRL1 also play an important
role in FPRL1 recognition of Ab42 in addition to
MMK-1 Consistent with the chemotaxis results,
chi-meras A and B showed differential patterns of Ca2+
mobilization responses to various formyl peptide
receptor agonists (Fig 3)
We further examined the chemotactic responses of
HEK293 cells transfected with chimeric receptors in
which regions between the N-terminus and the second
intracellular loop of FPR were substituted with
FPRL1 sequences Cells expressing chimeras C
(CH1-39) (Fig 4A), and F (CH106-145) (Fig 4D), which
contained the N-terminus or second intracellular loop
of FPRL1, respectively, failed to show any response
to the FPRL1 agonist MMK-1, suggesting that these
regions alone may not be sufficient for functional
interaction between FPRL1 and MMK-1 Chimera D
(CH40-86) (Fig 4B), which contains the C-terminal
half of the first TM, the first intracellular loop and
the second TM of FPRL1, and chimera E
(CH87-105) (Fig 4C), which contains the first extracellular loop
of FPRL1, exhibited significant response to MMK-1
compared with FPR-expressing cells (Fig 2C),
sug-gesting that these segments of FPRL1 contribute to
the MMK-1–FPRL1 interaction Ca2+ mobilization
experiments confirmed chimera responses to receptor
agonists in a manner comparable to chemotaxis
(Fig 4)
This notion was tested with the construction of
chimera G (RCH40-145) (Fig 5A) Compared with
FPRL1 (Fig 2A), chimera G lacks the FPRL1
segment starting from the C-terminal half of the first
TM through the second intracellular loop The potency
and efficacy of chemotaxis shown by chimera G in response to MMK-1 was markedly decreased (compare Fig 5C with Fig 2C), confirming the notion that these regions of FPRL1 play an important role in the FPRL1 response to MMK-1 Based on the deduced amino acid sequences of FPR and FPRL1, the seg-ment spanning the C-terminal half of the first TM to the N-terminal two-thirds of the second TM and the second intracellular loop are highly conserved except for several amino acids in the first extracellular loop and the flanking second and third TM (Fig 1) It is hypothesized that these nonconserved residues may determine the specificity and the capacity of the recep-tors to interact with their agonists
Chimera H (RCH140-145⁄ 241-351) (Fig 5B) con-tains the N-terminus and a segment between the fourth
TM and the third intracellular loop of FPRL1 This chimeric receptor exhibited considerably reduced potency in response to fMLF, but failed to exhibit a chemotactic response to MMK-1, therefore the seg-ment between the fourth TM and the third intracellu-lar loop of FPRL1 may not be important for functional interaction between FPRL1 and its specific ligand MMK-1 Compared with chimera G, chimera
H lacks the FPRL1 fragment from the sixth TM to C-terminus HEK293 cells expressing chimera H lost their chemotactic response to MMK-1 (Fig 5C), again confirming that regions from the sixth TM to the C-terminus of FPRL1 are important for the FPRL1– MMK-1 interaction
Surprisingly, Ab42induced a significant chemotactic response in cells expressing chimera C (CH1-39) and chimera H (RCH40-145⁄ 241-351), which were not responsive to MMK-1 (Fig 4A and Fig 5C) Thus, the N-terminus and a segment between the fourth TM and the third intracellular loop, including the second extracellular loop of FPRL1 participate in interaction between FPRL1 and Ab42 Compared with chimera H, chimera G contains extra FPRL1 sequences extending from sixth TM through the C-terminus and exhibits a more potent chemotaxis response to Ab42 than chi-mera H (Fig 5C), implying that this region in FPRL1
is crucial for the efficacy and potency of FPRL1 response to Ab42 These observations were corrobor-ated by Ca2+ mobilization responses of the chimeras
to the receptors agonists as shown in Fig 5
Discussion
Functional domains on the prototype formyl peptide receptor FPR have been extensively analyzed using receptor chimeras and site-directed mutations Chimeric receptors constructed between C5aR and FPR suggested
Table 1 Binding of chimeric formyl peptide receptor for 125 I-labeled
W pep HEK293 cells expressing wild-type or chimeric formyl
pep-tide receptors were incubated with a constant concentration of
125 I-labeled W pep in the presence of increasing concentrations of
unlabeled ligand After incubation cell pellets were collected and
measured for c emission The binding data were analyzed with the
program LIGAND for a Macintosh (P Munson, NIH, Bethesda, MD,
USA).
Receptor constructs Binding sites per cell Estimated Kd(n M )
Trang 6Fig 4 Construction and chemotactic responses of FPR ⁄ FPRL1 chimeric receptors with substitutions between the N-terminus and the sec-ond intracellular loop Chimeras C, D, E and F were constructed by substituting the segments in FPR with following correspsec-onding parts in FPRL1: the N-terminus and N-terminal half of the first TM (A: chimera C), the C-terminal half of the first TM through the second TM (B: chi-mera D), the first extracellular loop (C: chichi-mera E), the C-terminal two-thirds of the third TM and the second intracellular loop (D: chichi-mera F) The chemotactic and Ca 2+ flux responses of each chimera to different chemoattractants were measured *P < 0.01; significantly increased cell migration compared with medium control.
Trang 7the involvement of multiple domains of FPR in
recogni-tion of the agonist fMLF, including the first, second,
and third extracellular loops The TMs in FPR are also
implicated in contributing to the formation of a ligand
binding structure [18] Studies with chimeric receptors
composed of segments from FPR and FPRL1 suggested that the first and third extracellular loops with adjacent
TM in FPR were essential for its high-affinity binding for fMLF [12] In addition, three noncontiguous clusters
of amino acid residues in the first extracellular loop and
Fig 5 Contribution of the N-terminus and the second extracelluar loop with adjacent regions in FPRL1 to agonist induced cell migration Chi-mera G was constructed by substituting the segment from C-terminal half of the first TM domain through the second intracellular loop of FPRL1 with the corresponding part of FPR (A) Chimera H contains the N-terminus and a segment between the fourth TM and the third intracellular loop of FPRL1 in the backbone of FPR (B) The chemotactic and Ca 2+ flux responses of chimera G and H to different chemo-attractants were measured (A, B and C) *P < 0.01; significantly increased cell migration compared with medium W peptide (W pep) at
100 n M was used as a control in (C).
Trang 8the adjacent TM domains in FPR were identified as
important for its high-affinity interaction with fMLF
[19] Consistent with these results, in this study,
HEK293 cells expressing chimeric receptors (B, D, E, F,
G and H) in which one or more of above domains of
FPR were replaced by the sequences from FPRL1 either
did not migrate or showed greatly reduced efficacy in
response to fMLF
It is intriguing that W peptide (WKYMVm), a
hexa-peptide that uses both FPR and FPRL1 to stimulate
phagocytes with a certain degree of preference for
FPRL1 [16,17], induced migration of HEK293 cells
expressing all chimeric receptors, including chimeras B
and E (Figs 3B and 4C) which failed to show any
chemotactic response to fMLF and responded to
MMK-1 only at concentrations > 100 nm These
results, in addition to the binding data obtained with
125I-labeled W peptide, indicate that the chimeric
recep-tors are indeed expressed on the surface of HEK293 cells
and are capable of coupling to signaling pathways
required for proper function In addition, unlike fMLF
and MMK-1, W peptide appears to interact with diverse
FPR and FPRL1 domains regardless of their specificity
for agonists of an individual receptor
In order to further investigate the capacity of FPRL1
to interact with a lipid metabolite lipoxin A4 (LXA4),
chimeric receptors were generated with sequences from
FPRL1 (also termed LXA4 receptor) and LTB4
recep-tor [20] It was shown that N-glycosylation of FPRL1 is
essential for its recognition of peptide ligands, but not
LXA4 Moreover, the seventh TM segment and
adja-cent regions in FPRL1 are essential for LXA4
recogni-tion, but more regions in the receptor are required for
its high-affinity interaction with peptide agonists [20]
Our analysis of eight formyl peptide receptor chimeras
also suggests the requirement of multiple domains in
FPRL1 for its interaction with peptide agonists, and
fur-ther indicates that the sixth TM and third extracellular
loop are major determinants for agonist recognition
Recently, a number of novel peptide agonists has
been identified that selectively activate FPRL1 [6]
These agonists include peptide domains derived from
the envelope proteins of human immunodeficiency virus
type 1 (HIV-1) [6] and at least three amyloidogenic
polypeptides, SAA [9], Ab42 [8] and a 21 amino acid
fragment of human prion (PrP 106–126) [11]
Further-more, a cleavage fragment of neutrophil granule-derived
bactericidal cathelicidin, LL-37, is also a chemotactic
agonist for FPRL1 [10] It is intriguing that FPRL1
recognizes such a diverse array of ligands that have no
homology at the amino acid level Our study shows that
MMK-1 and Ab42 do not share identical domains in
FPRL1, supporting the hypothesis that FPRL1 may use
different structural determinants to recognize diverse agonists Thus, our results provide a structural basis for FPRL1 interaction with both synthetic and host-derived peptide agonists and will facilitate further identi-fication of key functional amino acid residues in FPRL1 and the design of receptor antagonists
Experimental procedures
Materials
The bacterial chemotactic peptide fMLF was purchased from Sigma (St Louis, MO, USA) W peptide (Trp-Lys-Tyr-Met-Val-d-Met), which activates both formyl peptide receptors FPR and FPRL1 [16,17], and MMK-1 (LESIFRSLLFRVM) [13,14], which specifically activates FPRL1, were synthesized and purified by the Department of Biochemistry, Colorado State University (Fort Collins, CO, USA), based on the pub-lished sequences The 42 amino acid form of amyloid b,
Ab42, peptide was purchased from California Peptide Research Inc (Napa, CA, USA)
Construction of chimeric formyl peptide receptor
cDNAs that encode FPR and FPRL1 were obtained from
a human HL-60 granulocyte cDNA library [21,22] Chi-meric receptor genes were constructed by exchange of DNA fragments between FPR and FPRL1 as previously described [12] to yield chimeras containing reciprocal seg-ments selected from these receptors The restriction sites AvaI (the first transmembrane domain, TM-1), NcoI (the first extracellular loop), and PvuII (TM-7) contained in the cDNAs of both receptors were used to generate the chi-meric constructs (Fig 1) The restriction sites for SalI (TM-3), BclI (the second intracellular loop), and MluI (the third intracellular loop) were created in both cDNAs by PCR using oligonucleotide primers that contained point mutations (Fig 1) The restriction sites generated did not cause changes in the putative amino acid sequences with one exception: the introduction of BclI site converted Val147 to a conserved residue Ile147 in the second intracel-lular loop of FPRL1 In addition, two putative amino acids (Met85 and Ala86) in FPRL1 gene were removed from one construct CH40-86 due to the existence of a second NcoI site The PCR-amplified cDNA was digested with appropri-ate endonucleases and ligappropri-ated in-frame to creappropri-ate chimeric FPR⁄ FPRL1 genes The correct sequence of each construct was confirmed by DNA sequencing
Expression of wild-type and chimeric formyl peptide receptors in HEK293 cells
The entire coding regions including the 5¢-end translation initiation sequences of the receptor constructs were
Trang 9subcloned into the expression vector SFFVneo and
trans-fected into human embryonic kidney (HEK) epithelial 293
cells (American Type Culture Collection, Manassas, VA,
USA) using SuperFect reagent (Qiagen, Valencia, CA,
USA) Stable receptor-expressing cell lines were established
by their resistance to G418 (800 lgÆmL)1, GibcoBRL,
Rockville, MD, USA), and maintained in Dulbecco’s
modi-fied Eagle’s medium (DMEM; BioWhittaker) with 10%
fetal bovine serum, 100 unitsÆmL)1 penicillin, 100 lgÆmL)1
streptomycin, 2 mm l-glutamine and 800 lgÆmL)1G418
To examine the cell-surface expression of wild-type and
chimeric formyl peptide receptors in transfected HEK293
cells, radioiodinated W peptide (125I-labeled W pep, NEN
Bio Life Sciences, Boston, MA, USA) was used The cells
(2· 106
cells in 200 lL RPMI-1640 containing 1% bovine
serum albumin [BSA], 25 mm Hepes, and 0.05% NaN3)
were incubated with a constant dose of125I-labeled W pep,
in the presence of increasing concentrations of unlabeled
lig-and After incubation and rotation at room temperature for
1 h, the cells were pelleted through a 10% sucrose⁄ NaCl ⁄ Pi
cushion for 1 min at 10 000 g The supernatant was
removed and the radioactivity associated with cell pellets
was measured in a gamma counter (CliniGamma,
Pharma-cia Biotech Inc.) The binding data were analyzed with a
Macintosh computer-aided program ligand (P Munson,
Division of Computer Research and Technology, NIH,
Bethesda, MD, USA)
Calcium mobilization
Calcium mobilization was assayed by incubating 107mL)1
of monocytes, neutrophils, FPRL1 or FPR transfectants in
loading buffer containing 138 mm NaCl, 6 mm KCl, 1 mm
CaCl2, 10 mm Hepes (pH 7.4), 5 mm glucose, 0.1% BSA
with 5 lm Fura-2 (Sigma) at 37C for 30 min The
dye-loaded cells were washed and resuspended in fresh loading
buffer The cells were then transferred into quartz cuvettes
(106 cells in 2 mL), which were placed in a luminescence
spectrometer LS50 B (PerkinElmer Ltd, Beaconsfield, UK)
Stimulants at different concentrations were added in a
vol-ume of 20 lL to the cuvettes at indicated time points The
ratio of fluorescence at 340 and 380 nm wavelength was
calculated using the fl winlab program (PerkinElmer)
Chemotaxis assays
Cell migration was assessed using a 48-well microchemotaxis
chamber technique [11,14] Different concentrations of
chemoattractants were placed in wells of the lower
compart-ment of the chamber (Neuro Probe, Cabin John, MA,
USA) Cell suspension (50 lL, 1· 106cellsÆmL)1in DMEM
containing 1% BSA) was seeded into wells of the upper
compartment which was separated from the lower
compart-ment by a 10 lm pore-size polycarbonate filter (Osmonics,
Livermore, CA, USA) precoated with 50 lgÆmL)1collagen
type I (Collaborative Biomedical Products, Bedford, MA, USA) After incubation at 37C for 300 min, the filters were removed, stained and the number of cells migrating across the filters was counted by light microscopy after cod-ing the samples All experiments were performed for at least three times with comparable results Results are originally calculated as the means of cell numbers (± SD) counted in three high-powered fields in three replicate samples The data was then converted to chemotaxis index representing the fold increase of cell responses to stimulants over the response to control medium The results shown are from representative experiments The statistical significance of the difference between cell migration in response to chemo-attractants versus control medium was determined by unpaired Student’s t-test
Acknowledgements
The authors thank Dr J J Oppenheim for reviewing the manuscript; N Dunlop for technical support; and
C Fogle and C Nolan for secretarial assistance The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorse-ment by the US Governendorse-ment The publisher or recipi-ent acknowledges right of the US Governmrecipi-ent to retain a nonexclusive, royalty-free license in and to any copyright covering this article This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No N01-C0-12400
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