We have previously reported (Badolato, R., J.M. Wang, W.J. Murphy, A.R. Lloyd, D.F.
Michiel, L.L. Bausserman, D.J. Kelvin, and J.J. Oppenheim. 1994. J. Exp. Med. 180:203; Xu,
L., R. Badolato, W.J. Murphy, D.L. Longo, M. Anver, S. Hale, J.J. Oppenheim, and J.M.
Wang. 1995. J. Immunol. 155:1184.) that the acute phase protein serum amyloid A (SAA) is a
potent chemoattractant for human leukocytes in vitro and mouse phagocytes in vivo. To identify the signaling mechanisms, we evaluated patterns of cross-desensitization between SAA and
other leukocyte chemoattrctants. We found that the chemotactic bacterial peptide, N-formyl-
methionyl-leucyl-phenylalanine (fMLP), was able to specifically attenuate Ca2+ mobilization in
human phagocytes induced by SAA, but only at very high concentrations, suggesting that SAA
uses a low affinity fMLP receptor. Here we demonstrate that SAA selectively induced Ca2+
mobilization and migration of HEK cells expressing FPRL1, a human seven-transmembrane
domain phagocyte receptor with low affinity for fMLP, and high affinity for lipoxin A4. Furthermore, radiolabeled SAA specifically bound to human phagocytes and FPRL1-transfected 293 cells. In contrast, SAA was not a ligand or agonist for FPR, the high affinity fMLP receptor. Thus, SAA is the first chemotactic ligand identified for FPRL1. Our results suggest that
FPRL1 mediates phagocyte migration in response to SAA.
Key words:
 |
Introduction |
Serum amyloid A (SAA),1 an acute phase protein, is normally present in serum at 0.1-µM levels, but increases
by 1,000-fold in systemic inflammatory conditions (1). It
has been proposed that SAA is mainly involved in lipid
transportation and metabolism (1). Chronic inflammatory conditions with elevated serum SAA may culminate in
amyloidosis, characterized by deposition of "amyloid" fibrils in tissues and associated with progressive destruction of organ function (2). Although a number of acute phase
proteins are known to modulate host immune responses,
we recently reported that recombinant human (rh)SAA exhibited considerable chemoattractant activity for human
monocytes, neutrophils, and T lymphocytes in vitro (5, 6).
rhSAA also induced infiltration of phagocytic cells and T
lymphocytes into injection sites in mice (5, 6), suggesting
that SAA, when present locally, may play a proinflammatory role by recruiting immune cells.
Since SAA induced significant Ca2+ mobilization in phagocytes (7), and both its chemotactic and Ca2+ mobilizing effects were inhibitable by pretreatment of the leukocytes with
pertussis toxin, we proposed that SAA may use seven-transmembrane, G protein-coupled receptor(s) (6, 7). In an effort
to identify the receptor(s) for SAA, we carefully evaluated
cross-desensitization of Ca2+ mobilization in monocytes and
neutrophils induced by SAA and other chemoattractants.
Among a number of chemoatrractants tested, only the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), when used at relatively high concentrations
(10 µM and more), was able to attenuate a subsequent cell
response to SAA, suggesting that SAA may use a receptor on
leukocytes for which fMLP has low affinity.
Two receptors that interact with fMLP have been identified and molecularly cloned (for review see references 8,
9). The prototype receptor FPR bound fMLP with high
affinity and was activated by low nanomolar concentrations
of fMLP. The other, a highly homologous variant of FPR,
named FPRL1 (also referred to as FPRH2 and LXA4R),
was originally cloned as an orphan receptor (10) but
was subsequently found to mediate Ca2+ mobilization in
response to high concentrations of fMLP (11, 13). Furthermore, a lipid metabolite, lipoxin A4 (LXA4), and its analogues were subsequently found to bind FPRL1 with high affinity and to increase arachidonic acid production and G
protein activation in FPRL1-transfected cells (15). LXA4
inhibits proinflammatory neutrophil responses (15) as
well as the release of the proinflammatory cytokine, IL-8,
by epithelial cells (20, 24). These effects of LXA4 have
been attributed to the activation of FPRL1 (or LXA4R) in
neutrophils and epithelial cells. Another lipid mediator receptor, the leukotriene B4 receptor, is structurally related
to FPRL1 (30.7% amino acid sequence identity; reference
25), and was also reported to be a fusion co-factor for HIV-1
(26), similar to various chemokine receptors (for review
see reference 27). This activity has not been reported for
FPRL1 (9, 27). Because SAA might use a low affinity
fMLP receptor on phagocytes, we further investigated
whether FPRL1 could be activated by SAA and demonstrate that SAA uses FPRL1 as a functional receptor.
| |
Materials and Methods |
Reagents and Cells.
rhSAA was purchased from Pepro Tech
Inc. with the sequence as follows: MRSFFSFLGEAFDGARDMWRAYSDM REANYIG SDKYFHAR GNYDAAKRGPGGV-WAAEAISNARENIQRFFGRGAEDSLADQAANEWGRSGK- DPNHFRPAGLPEKY.
This rhSAA corresponds to SAA-1
, one of the major SAA
isoforms in the serum, except for the addition of a methionine at
the NH2 terminus as well as the substitution of aspartic acid for asparagine at position 60, which appears in the SAA2 isoform (for review see reference 28). rhSAA at concentrations used in the study was negative for endotoxin as assessed by Limulus amebocyte lysate assays (sensitivity: 0.06 IU/ml; BioWhittaker). High
density lipoprotein (HDL) was purchased from Sigma Chemical
Co. Human peripheral blood enriched in mononuclear cells or
neutrophils was obtained from normal donors by leukapheresis
(courtesy of the Transfusion Medicine Department, Clinical
Center, National Institutes of Health, Bethesda, MD). The blood
was centrifuged through Ficoll-Hypaque (Sigma Chemical Co.),
and PBMCs collected at the interphase were washed with PBS
and centrifuged through a 46% isoosmotic Percoll (Pharmacia,
Uppsala, Sweden) gradient followed by elutriation to yield
monocytes (purity: >90%). Neutrophils were purified by 3%
dextran/PBS sedimentation as described elsewhere (5) and were
>98% pure. The cells were resuspended in RPMI 1640 medium
containing 10% FCS (Hyclone) for future use. The molecular cloning of the receptors for fMLP has been described previously (10, 13, 29, 30). The cDNAs encoding classical formyl peptide
receptor FPR and its variant FPRL1 were stably transfected into
human embryonic kidney epithelial cell line 293, which was cultured in DMEM in the presence of 800 µg/ml geneticin (G418; GIBCO BRL) to maintain selection. A rat basophil leukemia cell line stably transfected with FPR (ETFR cells) was also used in the study (gift from Drs. H. Ali and R. Snyderman, Duke University Medical Center, Durham, NC).
Chemotaxis.
The migration of human 293 cells expressing
FPR (FPR/293) or FPRL1(FPRL1/293) as well as ETFR cells
was assessed by a 48-well microchemotaxis chamber technique
(31, 32). A 25-µl aliquot of rhSAA or other reagents diluted in
chemotaxis medium (RPMI 1640, 1% BSA, 25 mM Hepes) was
placed in the wells of the lower compartment, and 50 µl cell suspension (106 cell/ml in chemotaxis medium) were placed in the
wells of the upper compartment of the chamber (Neuroprobe,
Cabin John, MD). The two compartments were separated by a
polycarbonate filter (10 µm pore size, Neuroprobe) coated with
50 µg/ml collagen type I (GIBCO BRL) for 1 h at 37°C. The
chamber was incubated at 37°C for 5 h in humidified air with 5%
CO2. At the end of the incubation, the filter was removed, fixed
and stained with Diff-Quik (Harlew, Gibbstown, NJ). The number of migrated cells in three high-powered fields (400×) was
counted by light microscopy after coding the samples. Results are
expressed as the mean (± SD) value of the migration in triplicate
samples and are representative of at least five experiments performed. For better illustration, chemotaxis indices (CI) reflecting
the fold increase of cell migration in response to stimulant over
medium are used. Statistical significance of the difference between numbers of cells migrating in response to stimuli versus
baseline (migration toward control medium) was calculated with
Student's t test and the CI 
2 are statistically significant.
Calcium Mobilization.
Calcium mobilization was assayed by
incubating 107/ml of monocytes, neutrophils, or receptor cDNA
transfectants in loading buffer containing 138 mM NaCl, 6 mM
KCl, 1 mM CaCl2, 10 mM Hepes (pH 7.4), 5 mM glucose, and
0.1% BSA with 5 µM Fura-2 (Sigma Chemical Co.) at 37°C for
30 min. The dye-loaded cells were washed and resuspended in
fresh loading buffer. The cells were then transfered into quartz
cuvettes (106 cells in 2 ml) that were placed in a luminescence
spectrometer LS50 B (Perkin-Elmer Limited). Stimulants at different concentrations were added in a volume of 20 µl to the cuvettes at indicated time points. The ratio of fluorescence at 340 and 380 nm wavelengths was calculated using the FL WinLab
(Perkin Elmer) program. The assays were performed at least five
times and results from representative experiments are shown.
Ligand Binding Assays.
rhSAA (20 µg) was radio-iodinated on
tyrosine residues with the chloramine T method and the specific activity of the labeled SAA was 5.8 mCi/mg (courtesy of J. Dobbs,
SAIC Frederick, NCI-FCRDC, Frederick, MD). A constant concentration of 16 nM 125I-labeled SAA was incubated for 20 min at
37°C with human monocytes or 293 cells transfected with
chemoattractant receptor cDNAs (1.5-2 × 106/sample, in 200 µl
RPMI 1640, 1% BSA, 0.05% NaN3) in the presence of increasing
concentrations of unlabeled SAA. After incubation, the cells were
washed once with ice-cold PBS then were layered onto a 10% sucrose/PBS cushion in Eppendorf tubes. The cells were centrifuged
at 10,000 g for 1 min and the tips of the tubes containing cell pellets
were cut and measured for radioactivity in a gamma counter. The
binding data were analyzed and plotted with a computer-aided program LIGAND (P. Munson, Division of Computer Research and
Technology, NIH, Bethesda, MD). The level of specific binding
was determined by subtraction of nonspecific binding (cpm on cells
in the presence of 1 µM unlabeled SAA) from the total binding
(cpm on cells in the absence of unlabeled SAA). Experiments were
performed at least five times, yielding similar results each time.
| |
Results |
Assays of Ca2+ mobilization have provided a useful approach to identify ligands for chemoattractant receptors. In
primary cells, cross-desensitization of Ca2+ transients is often due to two agonists acting at the same receptor (33).
Since SAA induced Ca2+ mobilization in phagocytes (7),
we used cross-desensitization to characterize the molecular
nature of SAA receptor(s). In a series of cross-desensitization experiments, SAA at 1 µM did not desensitize the
Ca2+ flux in monocytes or neutrophils induced by chemokines such as monocyte chemotactic protein (MCP)-1,
RANTES, MCP-3, macrophage inflammatory protein
(MIP)-1, IL-8, and stromal cell-derived factor (SDF)-1
(data not shown). Therefore, SAA is unlikely to share a receptor with any of the chemokines tested. SAA also did not
attenuate the cell response to the bacterial chemotactic
N-formylated peptide fMLP when fMLP was used at 100 nM
(10
7 M) (Fig. 1 A). However, in reciprocal tests, fMLP
at 100 nM showed a partial desensitizing effect on SAA-
induced Ca2+ mobilization in monocytes (Fig. 1 B). Furthermore, the cell response to SAA was completely desensitized
by higher concentrations of fMLP (103 M = 1 mM, Fig. 1
C), suggesting that SAA might use a receptor(s) for which
fMLP has low affinity.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Cross-desensitization of Ca2+ mobilization in human monocytes between SAA and fMLP. Fura-2-loaded monocytes were sequentially stimulated with SAA and fMLP (A) or vice versa (B and C), and the
ratio of fluorescence at 340 and 380 nm wavelengths was recorded and
calculated with the FLWinLab program.
|
|
Since fMLP is known to induce Ca2+ mobilization in
phagocytes through at least two seven-transmembrane, G
protein-coupled receptors, FPR and FPRL1 (10, 11, 13,
29), we tested the effect of SAA using cells transfected to
express these receptors that originally were not responsive
to fMLP stimulation. fMLP in a wide range of concentrations induced Ca2+ mobilization in FPR-transfected rat basophil leukemia cell line (ETFR cells), with an EC50 of 10 pM (data not shown). In contrast, the EC50 for fMLP
to induce Ca2+ mobilization in FPRL1 transfected cells
(FPRL1/293 cells) was much higher at 10 µM (Fig. 2 A).
These results confirmed the previous observation that FPR
is a high affinity receptor for fMLP, whereas FPRL1 has a
much lower affinity (10, 11, 13, 29). rhSAA induced Ca2+
mobilization in cells transfected with FPRL1 (FPRL1/293
cells; Fig. 2 B), but not in FPR-expressing cells or mock-transfected 293 cells (Fig. 2, C and D). The EC50 of rhSAA
on FPRL1 transfected cells was 250 nM, suggesting that SAA
activates FPRL1 with higher efficacy than fMLP. This was
supported by studies of cross-desensitization of Ca2+ flux between SAA and fMLP in FPRL1/293 cells. As shown in Fig. 2 E, although sequential stimulation of FPRL1/293 cells with
SAA and fMLP resulted in bidirectional desensitization, SAA
was able to desensitize the cell response to a 100-fold excess
of fMLP. In contrast, fMLP at 100-fold excess of SAA only
partially desensitized the effect of SAA (Fig. 2 E).
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Calcium mobilization in
FPRL1-transfected HEK 293 cells.
FPRL1/293 cells were loaded with
Fura-2 and were stimulated with various
concentrations of fMLP (A) or SAA (B).
SAA does not induce Ca2+ mobilization
in FPR-expressing 293 cells (C) or
mock-transfected 293 cells (D). E shows
the sequential stimulation of FPRL1/293
cells with SAA and fMLP or vice versa.
|
|
Leukocyte infiltration in vivo is considered to be based
on migration of cells toward a gradient of locally produced
chemoattractant(s). This process can be emulated by in
vitro assays of chemotaxis, which provides a very sensitive
and biologically relevant means of evaluating the function
of cloned chemoattractant receptors (32). Since SAA
has been shown in our previous studies to induce leukocyte
infiltration in vivo and chemotaxis in vitro (5, 6), we next
investigated whether SAA could induce cell migration via
FPRL1. FPRL1/293 cells showed a potent migratory response to SAA with an EC50 of 200 nM (Fig. 3 A), but
these cells failed to migrate in response to a wide range of
concentrations of fMLP (Fig. 3 B). In contrast, fMLP induced migration of ETFR cells at nanomolar range concentrations, whereas the same cells did not migrate in response to SAA (Fig. 3 C). The chemotaxis experiments indicate that fMLP is only a partial agonist for FPRL1 since
it did not induce cell migration through FPRL1. On the
other hand, SAA showed full agonist activity on FPRL1.
Both SAA-induced Ca2+ mobilization and chemotaxis in
FPRL1/293 cells were inhibited by pretreatment of the
cells with pertussis toxin but not cholera toxin (data not
shown) in correlation with the observation in native cells
(5), suggesting activation of G protein of the Gi type is
required for SAA signaling through FPRL1. In addition, since SAA can form complexes with HDL, which acts as a
natural inhibitor of SAA (5, 6), we examined the effect of
HDL on the chemotactic activity of SAA for FPRL1/293
cells. Fig. 3 D shows that HDL, whether preincubated with
SAA or simultaneously added to SAA, completely abolished SAA-induced FPRL1/293 cell migration. In contrast,
the same concentration of HDL did not affect migration of
FPR-expressing ETFR cells induced by fMLP (data not
shown). These results confirmed that HDL specifically inhibited the agonist activity of SAA on FPRL1.
View larger version (120K):
[in this window]
[in a new window]
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Chemotactic activity of SAA for human monocytes and cells transfected to express chemoattractant receptors. Different concentrations of SAA
were placed in the lower wells of the chemotaxis chamber. Monocytes, FPRL1/293 cells, or FPR-expressing ETFR cells were placed in the upper wells. After incubation, the cells migrated across the polycarbonate filter were counted and photographed (A: SAA 0.8 µM, fMLP 100 nM). The cell migration was
expressed as CI representing the fold increase of the cells migrating in response to stimulants over control medium. (B) Migration of FPRL1/293 cells in
response to SAA and fMLP. (C) Migration of FPR-expressing ETFR cells in response to SAA and fMLP. (D) Effect of HDL on SAA induced FPRL1/293
cell migration. HDL at 1,000 µg/ml mixed with 0.8 µM SAA was preincubated at 37°C for 4 h. The mixture was then tested for chemotactic activity on
FPRL1/293 cells. The HDL/SAA mixture without preincubation was also tested for chemotactic activity and yielded similar results.
|
|
To further verify the usage of FPRL1 by SAA, we performed ligand binding experiments. Fig. 4 shows that radio-iodinated SAA specifically bound to FPRL1/293 cells
with an estimated Kd at 64 nM and 42,000 binding sites per
cell (Fig. 4 A). 125I-labeled SAA also specifically bound to
monocytes (Fig. 4 B) and neutrophils (Kd = 45 nM, R = 6,700/cell) with Kd values comparable to those achieved
with FPRL1/293 cells. In the displacement assay, unlabeled SAA in a dose-dependent manner inhibited its own
binding to monocytes (Fig. 4 C), neutrophils (data not
shown) and FPRL1/293 (Fig. 4 D) with an IC50 at ~50
nM. In contrast, unlabeled fMLP at high concentrations
(10 µM) only partially competed with 125I-SAA for binding. These results confirm SAA to be a far more efficient
agonist for FPRL1 than fMLP.
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 4.
Binding of
125I-labeled SAA to human
monocytes and FPRL1/293 cells.
rhSAA was radio-iodinated with
chloramine T method and the
binding of 125I-labeled SAA to
FPRL1/293 cells (A) or monocytes (B) was measured by adding a constant concentration of
125I-labeled SAA to the cells in
the presence of increasing concentrations of unlabeled SAA.
The data was analyzed and plotted with the Macintosh computer
aided program LIGAND. C
shows the displacement of
125I-labeled SAA binding on
monocytes by unlabeled SAA and
fMLP. The same results were obtained with neutrophils (data not
shown) and FPLR1/293 cells (D).
|
|
| |
Discussion |
In this study, we demonstrate that SAA uses FPRL1, a
seven-transmembrane, G protein-coupled receptor expressed on phagocytes as a chemotactic receptor, suggesting
a molecular basis for our previous observations that SAA is
a potent chemoattractant and activator for human peripheral blood monocytes and neutrophils (5). In addition to
SAA, FPRL1 has previously been shown to be a low affinity receptor for fMLP (11, 13) and a high affinity receptor
for lipid metabolite LXA4 and its analogues (15). Our data suggest that fMLP is a partial agonist incapable of inducing chemotaxis via FPRL1 in this model system. Analysis of LXA4 induction of chemotaxis via FPRL1 has not
been reported. Thus, SAA is the first chemotactic agonist
identified for FPRL1.
FPRL1 was identified and molecularly cloned from human phagocytic cells by low stringency hybridization of the
cDNA library with the FPR sequence and initially was defined as an orphan receptor (10, 11, 13, 14). The cloning of
the same receptor termed FPRH2 from a genomic library
was described by Bao et al. (12). FPRL1 possesses 69%
identity at the amino acid level to FPR, the prototype receptor for synthetic and bacterium-derived formylated peptides (8, 9). Both FPR and FPRL1 are expressed by
monocytes and neutrophils and are clustered on human
chromosome 19q13 (12, 36). Although fMLP is a high affinity agonist for FPR, it interacts with FPRL1 and transduces signals in response to fMLP only at high concentrations (Fig. 2 and references 11, 13, 36). SAA, on the other
hand, selectively bound and activated only FPRL1 at physiologically relevant concentrations, which under inflammatory stimulation could reach 80 µM in the serum (1).
FPRL1 is mainly expressed in monocytes and neutrophils.
However, cells other than phagocytes, such as hepatocytes,
have also been shown to express FPRL1 (8). Recently, the
expression of this receptor (also termed LXA4R) has been
reported to be highly inducible in epithelial cells by specific
cytokines (20). Our previous study showed that CD3+ human peripheral blood T lymphocytes were induced by
SAA to migrate and adhere to endothelial cell monolayers
(6), suggesting that T lymphocytes may also express a receptor(s) for SAA. In fact, we detected specific binding sites
for 125I-labeled SAA on human peripheral blood CD3+ T
lymphocytes (Kd = 300 nM, R = 2,200 sites/cell). However, whether these binding sites on T lymphocytes represent FPRL1 or an additional receptor(s) for SAA is not yet known.
Despite the fact that the chemotactic formyl peptide
fMLP has been shown to be a low efficiency agonist for
FPRL1, a lipid metabolite LXA4 has been reported to be a
high affinity ligand and potent agonist for this receptor
(15). LXA4 is an eicosanoid generated during a number of
host reactions such as inflammation, thrombosis, and atherosclerosis (22), and was initially discovered as an inhibitor
of immune response (for review see reference 37). LXA4
was subsequently reported to inhibit neutrophil chemotaxis (38) and transepithelial migration induced by chemotactic
agents (23). A seven-transmembrane, G protein-coupled
receptor identical to FPRL1 was recently identified for
LXA4 (15, 16, 22). LXA4 bound to CHO cells transfected
with this receptor with high affinity and increased GTPase
activity and the release of esterified arachidonate (15).
Thus, LXA4 has been proposed to be an endogenously produced ligand for FPRL1 (15, 16). Although LXA4 has
not been documented to induce Ca2+ mobilization in neutrophils or FPRL1-transfected cells (15), it was reported to
induce Ca2+ flux and chemotaxis in monocytes, presumably through FPRL1 (17, 22). Thus, differential activation
of second messengers in monocytes versus neutrophils by
LXA4 was postulated. In our study, we did not detect significant induction of Ca2+ flux or chemotaxis in FPRL1/
293 cells by a commercially available LXA4 (Biomol, Plymouth Meeting, PA), nor did we observe inhibition of
SAA signaling or binding by this LXA4 in either phagocytes or FPRL1/293 cells. Further study, beyond the scope
of this report, will be needed to compare the interaction of
FPRL1 with its peptide ligands, SAA and fMLP, versus its
lipid ligand, LXA4, to clarify these results.
Our previous studies showed that both SAA-induced
leukocyte chemotaxis and activation were inhibited by pertussis toxin (6, 7). This study also showed that the signaling
of SAA through FPRL1 was sensitive to pertussis toxin.
Thus, although the signal transduction pathways triggered
by SAA in FPRL1 requires further investigation, the high
level homology of FPRL1 to FPR, its sensitivity to pertussis toxin, and its mediation of potent phagocyte activation by SAA suggest that FPRL1 may share major biochemical
events with FPR. It is well known that binding of FPR by
bacterium-derived or synthetic peptide agonists results in a
G protein-mediated signaling cascade leading to phagocytic
cell adhesion, chemotaxis, release of oxygen intermediates,
enhanced phagocytosis, and bacterial killing, as well as gene
transcription (8, 9). Activation of FPR by its agonists can also
result in heterologous desensitization of the subsequent cell
response to other G protein receptor ligands (39, 40), including chemokines. This "desensitizing" effect of FPR activation may also be seen with FPRL1, although more studies are needed to elucidate the mechanism(s) involved. For instance, SAA was initially reported as an inhibitor of neutrophil response to fMLP (41). In these experiments, neutrophils preincubated with SAA showed reduced superoxide
release in response to fMLP (41). Our previous study also
showed that preincubation of monocytes and neutrophils
with SAA reduced cell response to a number of chemoattractants, including fMLP and chemokines (7), suggesting that FPRL1 is capable of transducing intracellular biochemical events leading to desensitization of other G protein-coupled receptors.
The pathophysiological significance of use of FPRL1 by
SAA requires more in-depth investigation. The optimal
concentrations for SAA to induce leukocyte migration, adhesion, and tissue infiltration ranged from 0.8 to 4 µM (5-
7), which are higher than the SAA levels present in normal
serum but well below the concentration seen during a systemic acute phase response (1). Increased serum levels of
SAA have been observed in a number of inflammatory and
infectious diseases as well as after organ transplantation (4).
The SAA concentrations required for activating FPRL1 are well within the range in which native cells are activated, as shown in this study. The overproduction of SAA by hepatocytes can be induced by inflammatory stimuli such as
LPS, IL-1, IL-6, and TNF- (1). Macrophages have also
been reported as an extra-hepatic source of SAA during inflammation (42) and may produce relatively high concentrations in microcompartments. The expression of SAA mRNA in human atherosclerotic lesions and the induction
of SAA by oxidized low density lipoproteins strengthen the
hypothesis that SAA may play an important role in vascular
injury and atherosclerosis (4). Under normal conditions,
most serum SAA will be associated with HDL, which acts
as a natural inhibitor of the chemotactic activity of SAA
(references 5, 6 and Fig. 3 D). However, since SAA binds
to HDL at equimolar ratios (43), a rapid increase in concentration of locally produced SAA could establish a gradient of free active SAA with consequent recruitment of leukocytes into inflammatory sites. Therefore, it is possible
that at local inflammatory sites elevated SAA can attract and
activate leukocytes for the clearance of pathogenic agents.
This process may also cause tissue injury. Furthermore, signals triggered by activated FPRL1, a functional receptor of
SAA, could eventually result in unresponsiveness of leukocytes to additional stimulation, thus immobilizing the cells and limiting the degree of inflammation.
Address correspondence to Ji Ming Wang, LMI, DBS, NCI-FCRDC, Bldg. 560, Rm. 31-40, Frederick,
MD 21702. Phone: 301-846-5454; Fax: 301-846-7042; E-mail: wangji{at}mail.ncifcrf.gov
The authors thank N. Dunlop for technical assistance. The secretarial assistance by C. Fogle is gratefully acknowledged.
| 1.
| Sipe, J.D. 1990. The acute-phase response. In Immunophysiology: The Role of Cells and Cytokines in Immunity and Inflammation. J.J. Oppenheim and E.M. Shevach, editors. Oxford University Press, New York. 259-273.
|
| 2.
|
Kisilevsky, R..
1991.
Serum amyloid A (SAA), a protein
without a function: some suggestions with reference to cholesterol metabolism.
Med. Hypotheses.
35:
337-341
[Medline].
|
| 3.
|
Skinner, M..
1992.
Protein AA/SAA.
J. Intern. Med.
232:
513-514
[Medline].
|
| 4.
|
Malle, E., and
F.C. De Beer.
1996.
Human serum amyloid A
(SAA) protein: a prominent acute-phase reactant for clinical
practice.
Eur. J. Clin. Invest.
26:
427-435
[Medline].
|
| 5.
|
Badolato, R.,
J.M. Wang,
W.J. Murphy,
A.R. Lloyd,
D.F. Michiel,
L.L. Bausserman,
D.J. Kelvin, and
J.J. Oppenheim.
1994.
Serum amyloid A is a chemoattractant: induction of
migration, adhesion, and tissue infiltration of monocytes and
polymorphonuclear leukocytes.
J. Exp. Med.
180:
203-209
[Abstract/Free Full Text].
|
| 6.
|
Xu, L.,
R. Badolato,
W.J. Murphy,
D.L. Longo,
M. Anver,
S. Hale,
J.J. Oppenheim, and
J.M. Wang.
1995.
A novel biologic function of serum amyloid A. Induction of T lymphocyte migration and adhesion.
J. Immunol.
155:
1184-1190
[Abstract].
|
| 7.
|
Badolato, R.,
J.A. Johnston,
J.M. Wang,
D. McVicar,
L.L. Xu,
J.J. Oppenheim, and
D.J. Kelvin.
1995.
Serum amyloid
A induces calcium mobilization and chemotaxis of human
monocytes by activating a pertussis toxin-sensitive signaling
pathway.
J. Immunol.
155:
4004-4010
[Abstract].
|
| 8.
|
Prossnitz, E.R., and
R.D. Ye.
1997.
The N-formyl peptide
receptor: a model for the study of chemoattractant receptor
structure and function.
Pharmacol. Ther.
74:
73-102
[Medline].
|
| 9.
| Murphy, P.M. 1996. The N-formyl peptide chemotactic receptors. In Chemoattractant Ligands and Their Receptors.
CRC Press, Boca Raton, FL. 269 pp.
|
| 10.
|
Murphy, P.M.,
T. Ozcelik,
R.T. Kenney,
H.L. Tiffany,
D. McDermott, and
U. Francke.
1992.
A structural homologue
of the N-formyl peptide receptor. Characterization and chromosome mapping of a peptide chemoattractant receptor family.
J. Biol. Chem.
267:
7637-7643
[Abstract/Free Full Text].
|
| 11.
|
Ye, R.D.,
S.L. Cavanagh,
O. Quehenberger,
E.R. Prossnitz, and
C.G. Cochrane.
1992.
Isolation of a cDNA that encodes
a novel granulocyte N-formyl peptide receptor.
Biochem. Biophys. Res. Commun.
184:
582-589
[Medline].
|
| 12.
|
Bao, L.,
N.P. Gerard,
R.L. Eddy Jr.,
T.B. Shows, and
C. Gerard.
1992.
Mapping of genes for the human C5a receptor
(C5AR), human FMLP receptor (FPR), and two FMLP receptor homologue orphan receptors (FPRH1, FPRH2) to
chromosome 19.
Genomics.
13:
437-440
[Medline].
|
| 13.
|
Gao, J.L., and
P.M. Murphy.
1993.
Species and subtype variants of the N-formyl peptide chemotactic receptor reveal
multiple important functional domains.
J. Biol. Chem.
268:
25395-25401
[Abstract/Free Full Text].
|
| 14.
|
Nomura, H.,
B.W. Nielsen, and
K. Matsushima.
1993.
Molecular cloning of cDNAs encoding a LD78 receptor and putative leukocyte chemotactic peptide receptors.
Int. Immunol.
5:
1239-1249
[Abstract/Free Full Text].
|
| 15.
|
Fiore, S.,
J.F. Maddox,
H.D. Perez, and
C.N. Serhan.
1994.
Identification of a human cDNA encoding a functional high
affinity lipoxin A4 receptor.
J. Exp. Med.
180:
253-260
[Abstract/Free Full Text].
|
| 16.
|
Takano, T.,
S. Fiore,
J.F. Maddox,
H.R. Brady,
N.A. Petasis, and
C.N. Serhan.
1997.
Aspirin-triggered 15-epi-lipoxin A4
(LXA4) and LXA4 stable analogues are potent inhibitors of
acute inflammation: evidence for anti-inflammatory receptors.
J. Exp. Med.
185:
1693-1704
[Abstract/Free Full Text].
|
| 17.
|
Maddox, J.F.,
M. Hachicha,
T. Takano,
N.A. Petasis,
V.V. Fokin, and
C.N. Serhan.
1997.
Lipoxin A4 stable analogs are
potent mimetics that stimulate human monocytes and THP-1
cells via a G-protein-linked lipoxin A4 receptor.
J. Biol.
Chem.
272:
6972-6978
[Abstract/Free Full Text].
|
| 18.
|
Fiore, S.,
M. Romano,
E.M. Reardon, and
C.N. Serhan.
1993.
Induction of functional lipoxin A4 receptors in HL-60
cells.
Blood.
81:
3395-3403
[Abstract/Free Full Text].
|
| 19.
|
Fiore, S., and
C.N. Serhan.
1995.
Lipoxin A4 receptor activation is distinct from that of the formyl peptide receptor in
myeloid cells: inhibition of CD11/18 expression by lipoxin
A4-lipoxin A4 receptor interaction.
Biochemistry.
34:
16678-16686
[Medline].
|
| 20.
|
Gronert, K.,
A. Gewirtz,
J.L. Madara, and
C.N. Serhan.
1998.
Identification of a human enterocyte lipoxin A4 receptor that is regulated by interleukin (IL)-13 and interferon  and inhibits tumor necrosis factor -induced IL-8 release.
J.
Exp. Med.
187:
1285-1294
[Abstract/Free Full Text].
|
| 21.
|
Gewirtz, A.T.,
B. McCormick,
A.S. Neish,
N.A. Petasis,
K. Gronert,
C.N. Serhan, and
J.L. Madara.
1998.
Pathogen-
induced chemokine secretion from model intestinal epithelium is inhibited by lipoxin A4 analogs.
J. Clin. Invest.
101:
1860-1869
[Medline].
|
| 22.
|
Romano, M.,
J.F. Maddox, and
C.N. Serhan.
1994.
Activation of human monocytes and the acute monocytic leukemia
cell line (THP-1) by lipoxins involves unique signaling pathways for lipoxin A4 versus lipoxin B4: evidence for differential Ca2+ mobilization.
J. Immunol.
157:
2149-2154
[Abstract].
|
| 23.
|
Colgan, S.P.,
C.N. Serhan,
C.A. Parkos,
C. Delp-Archer, and
J.L. Madara.
1993.
Lipoxin A4 modulates transmigration
of human neutrophils across intestinal epithelial monolayers.
J. Clin. Invest.
92:
75-82
.
|
| 24.
|
Takano, T.,
C.B. Clish,
K. Gronert,
N. Petasis, and
C.N. Serhan.
1998.
Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin-triggered 15-epi-lipoxin A4 and novel lipoxin B4 stable analogues.
J. Clin. Invest.
101:
819-826
[Medline].
|
| 25.
|
Yokomizo, T.,
T. Izumi,
K. Chang,
Y. Takuwa, and
T. Shimizu.
1997.
A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis.
Nature.
387:
620-624
[Medline].
|
| 26.
|
Owman, C.,
A. Garzino-Demo,
F. Cocchi,
M. Popovic,
A. Sabirsh, and
R.C. Gallo.
1998.
The leukotriene B4 receptor
functions as a novel type of coreceptor mediating entry of
primary HIV-1 isolates into CD4-positive cells.
Proc. Natl.
Acad. Sci. USA.
95:
9530-9534
[Abstract/Free Full Text].
|
| 27.
|
Berger, E.A..
1997.
HIV entry and tropism: the chemokine receptor
connection.
AIDS.
11:
S3-S16
.
|
| 28.
|
Steinkasserer, A.,
E.H. Weiss,
W. Schwaeble, and
R.P. Linke.
1990.
Heterogeneity of human serum amyloid A protein. Five different variants from one individual demonstrated
by cDNA sequence analysis.
Biochem. J.
268:
187-193
[Medline].
|
| 29.
|
Murphy, P.M., and
D. McDermott.
1991.
Functional expression of the human formyl peptide receptor in Xenopus oocytes requires a complementary human factor.
J. Biol. Chem.
266:
12560-12567
[Abstract/Free Full Text].
|
| 30.
|
Ali, H.,
S. Sozzani,
I. Fisher,
A.J. Barr,
R.M. Richardson,
B. Haribabu, and
R. Snyderman.
1998.
Differential regulation
of formyl peptide and platelet-activating factor receptors.
Role of phospholipase C beta3 phosphorylation by protein
kinase A.
J. Biol. Chem.
273:
11012-11016
[Abstract/Free Full Text].
|
| 31.
|
Falk, W.,
R.H. Goodwin Jr., and
E.J. Leonard.
1980.
A 48-well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration.
J. Immunol. Methods.
33:
239-247
[Medline].
|
| 32.
|
Gong, X.,
W. Gong,
D.B. Kuhns,
A. Ben-Baruch,
O.M. Howard, and
J.M. Wang.
1997.
Monocyte chemotactic protein-2 (MCP-2) uses CCR1 and CCR2B as its functional receptors.
J. Biol. Chem.
272:
11682-11685
[Abstract/Free Full Text].
|
| 33.
|
Wang, J.M.,
D.W. McVicar,
J.J. Oppenheim, and
D.J. Kelvin.
1993.
Identification of RANTES receptors on human monocytic cells: competition for binding and desensitization by homologous chemotactic cytokines.
J. Exp. Med.
177:
699-705
[Abstract/Free Full Text].
|
| 34.
|
Gong, W.,
O.M. Howard,
J.A. Turpin,
M.C. Grimm,
P. Gray,
C.J. Raport,
J.J. Oppenheim, and
J.M. Wang.
1998.
Monocyte chemotactic protein (MCP)-2 activates CCR5 and
blocks CD4/CCR5-mediated HIV-1 entry/replication.
J.
Biol. Chem.
273:
4289-4292
[Abstract/Free Full Text].
|
| 35.
|
Ben-Baruch, A.,
L. Xu,
P.R. Young,
K. Bengali,
J.J. Oppenheim, and
J.M. Wang.
1995.
Monocyte chemotactic protein-3
(MCP3) interacts with multiple leukocyte receptors. C-C
CKR1, a receptor for macrophage inflammatory protein-1
alpha/Rantes, is also a functional receptor for MCP3.
J. Biol.
Chem.
270:
22123-22128
[Abstract/Free Full Text].
|
| 36.
|
Durstin, M.,
J.L. Gao,
H.L. Tiffany,
D. McDermott, and
P.M. Murphy.
1994.
Differential expression of members of
the N-formylpeptide receptor gene cluster in human phagocytes.
Biochem. Biophys. Res. Commun.
201:
174-179
[Medline].
|
| 37.
|
Samuelsson, B.,
S.E. Dahlen,
J.A. Lindgren,
C.A. Rouzer, and
C.N. Serhan.
1987.
Leukotrienes and lipoxins: structures, biosynthesis, and biological effects.
Science.
237:
1171-1176
[Abstract/Free Full Text].
|
| 38.
|
Lee, T.H.,
P. Lympany,
A.E. Crea, and
B.W. Spur.
1991.
Inhibition of leukotriene B4-induced neutrophil migration
by lipoxin A4: structure-function relationships.
Biochem. Biophys. Res. Commun.
180:
1416-1421
[Medline].
|
| 39.
|
Ali, H.,
R.M. Richardson,
E.D. Tomhave,
J.R. Didsbury, and
R. Snyderman.
1993.
Differences in phosphorylation of
formylpeptide and C5a chemoattractant receptors correlate
with differences in desensitization.
J. Biol. Chem.
268:
24247-24254
[Abstract/Free Full Text].
|
| 40.
|
Ali, H.,
E.D. Tomhave,
R.M. Richardson,
B. Haribabu, and
R. Snyderman.
1996.
Thrombin primes responsiveness of selective chemoattractant receptors at a site distal to G protein
activation.
J. Biol. Chem.
271:
3200-3206
[Abstract/Free Full Text].
|
| 41.
|
Linke, R.P.,
V. Bock,
G. Valet, and
G. Rothe.
1991.
Inhibition of the oxidative burst response of N-formyl peptide-stimulated neutrophils by serum amyloid-A protein.
Biochem.
Biophys. Res. Commun.
176:
1100-1105
[Medline].
|
| 42.
|
Steel, D.M.,
F.C. Donoghue,
R.M. O'Neill,
C.M. Uhlar, and
A.S. Whitehead.
1996.
Expression and regulation of
constitutive and acute phase serum amyloid A mRNAs in hepatic and non-hepatic cell lines.
Scand. J. Immunol.
44:
493-500
[Medline].
|
| 43.
|
Liang, J., and
J.D. Sipe.
1995.
Recombinant human serum
amyloid A (apoSAAp) binds cholesterol and modulates cholesterol flux.
J. Lipid Res.
36:
37-46
[Abstract].
|
Top
Abstract
Introduction
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
