(Received for publication, November 11, 1996, and in revised form, December 11, 1996)
From the Center for Experimental Therapeutics and
Reperfusion Injury, Department of Anesthesia, Brigham and Women's
Hospital and Harvard Medical School, Boston, Massachusetts 02115 and
the ¶ Department of Chemistry, Loker Hydrocarbon Institute
219, University of Southern California, Los Angeles, California
90089-1661
Lipoxins (LX) are bioactive eicosanoids that
activate human monocytes and inhibit neutrophils. LXA4 is
rapidly converted by monocytes to inactive products, and to resist
metabolism, synthetic analogs of LXA4 were designed. Here,
we examined the bioactivity of several LXA4 analogs in
monocytes and found, for chemotaxis, 15(R/S)-methyl-LXA4 and 15-epi-LXA4
were equal in activity, and 16-phenoxy-LXA4 was more potent
than native LXA4. Both
15(R/S)-methyl-LXA4 and
16-phenoxy-LXA4 were ~1 log molar more potent than
LXA4 in stimulating THP-1 cell adherence (EC50 1 × 10
10 M). Dimethylamide
derivatives of the LXA4 analogs also possessed agonist
rather than antagonist properties for monocytes. Neither LXA4 nor 16-phenoxy-LXA4 affected
monocyte-mediated cytotoxicity. We cloned an LXA4 receptor
from THP-1 cells identical to that found in PMN. Evidence of
receptor-mediated function of LXA4 and the stable analogs
in monocytes included desensitization of intracellular calcium
mobilization to a second challenge by equimolar concentrations of these
analogs, but not to LTB4. Increases in
[Ca2+]i by LXA4 and the analogs were
specifically inhibited by an antipeptide antibody to the
LXA4 receptor; and both LXA4- and
analog-induced adherence and increments in Ca2+ were
sensitive to pertussis toxin. Together, these results indicate that the
LXA4 stable analogs are potent monocyte chemoattractants and are more potent than native LXA4 in stimulating THP-1
cell adherence, at subnanomolar concentrations. Moreover, they provide additional evidence that the LXA4 stable analogs retain
selective bioactivity in monocytes and are valuable instruments for
examining the functions and modes of action of LXA4.
Lipoxins (LX)1 are members of the eicosanoid family of bioactive lipid mediators with trihydroxytetraene structures (1). They are generated during cell-cell interactions and transcellular transfer of intermediates (2, 3) and lipoxin A4 (LXA4) was recently identified in vivo from human patients with rheumatoid arthritis (4) or asthma (5). The original pathways of LX formation identified were via lipoxygenase-lipoxygenase interactions and an additional route of novel LX formation was recently demonstrated (6). This pathway, via aspirin-triggered acetylation of cyclooxygenase-2 in human endothelial cell/neutrophil (PMN) coincubations, results in generation of 15R-epimers of LX (e.g. 15-epi-LXA4). Thus, LX are the first products identified from both lipoxygenase-lipoxygenase and cyclooxygenase-lipoxygenase interactions.
Lipoxins display selective activities on human leukocytes that are either stimulatory or inhibitory, depending on the target cell type involved. In human PMN, LXA4 induces chemokinesis but inhibits chemotaxis toward leukotriene B4 (LTB4) and N-formylmethionylleucylphenylalanine (FMLP) (7). LXA4 also inhibits FMLP-induced PMN transmigration across intestinal epithelium (8). In addition, LTB4- and LTC4-induced PMN adherence to human umbilical vein endothelial cells (HUVEC) is reduced by ~70% by LXA4 and LXB4 (9) and aspirin-triggered 15-epi-LXA4 also inhibits LTB4-stimulated PMN adherence to HUVEC at nanomolar concentrations and is ~two times more potent than LXA4 in this setting (6). LXA4 and LXB4 also down-regulate peptidoleukotriene-induced P-selectin expression on HUVEC (9). LXA4 displays in vivo activity, with inhibition of PMN migration into the kidney in rat glomerulonephritis models (3) and inhibition of PMN diapedesis from postcapillary venules (10). In contrast to the down-regulation of PMN, LX exhibit selective stimulatory activities in the monocyte, as we recently described potent activation of human monocyte migration and adherence to laminin by both LXA4 and LXB4 (11).
Human monocytes were also found to rapidly convert more than 80% of added LXA4 to novel metabolites via dehydrogenation and reduction of double bonds and the products were identified as 15-oxo-LXA4; 13,14-dihydro-15-oxo-LXA4 and 13,14-dihydro-LXA4 (12). The oxo- and dihydro-LX produced by monocytes were essentially inactive in stimulating monocyte adherence, in contrast to the native compounds (11). The dehydrogenation of LX (i.e. production of oxo-LX) by human monocytes appears to be carried out by 15-hydroxyprostaglandin dehydrogenase, which is present in these cells as determined by reverse transcriptase-polymerase chain reaction and Western blotting (11) and was confirmed using recombinant enzyme (13).
We identified an orphan seven-transmembrane receptor cDNA as a high-affinity receptor for LXA4 (14). LXA4 binding to this receptor activates both phospholipase A2 and phospholipase D (14, 15), responses that are inhibited by pretreatment of cells with pertussis toxin (PTX). The magnitude of Ca2+ mobilization via this receptor appears to be cell type-specific. For example, Ca2+ mobilization by LXA4 in PMN is less than 10% of that induced by an equal concentration of FMLP (16), and in monocytic cells, LXA4, but not LXB4, induces increases in intracellular Ca2+ greater than 50% of that induced by equimolar FMLP (17). These findings imply different stimulation of second messengers with cell type specificity and are in concordance with the different responses of monocytes versus PMN to LX. They also suggest that LX could contribute to resolution of injury at sites of inflammation by suppressing PMN influx and stimulating a self-limiting (by monocyte metabolism) monocyte migration to promote healing.
In view of the rapid transformation and inactivation of the LX by monocytes and, potentially, other cells in vivo, it was highly desirable to design LX analogs that would resist this metabolism and maintain their structural integrity and potential beneficial biologic actions to use as tools in disease research. To this end, LXA4 analogs have been synthesized, and they were evaluated for their structural stability in incubations with monocytic cells and with 15-hydroxyprostaglandin dehydrogenase and were resistant to conversion in both cell and enzyme incubations (13). Here, we examined the biologic activity of the synthetic LXA4 analogs with monocytes and THP-1 cells and determined the relative potency of these compounds versus native LXA4 in stimulating migration and adherence as well as Ca2+ mobilization in these cells. In addition, we provide the first evidence that the analogs act via the same G-protein-linked receptor as LXA4.
LXA4 was purchased from Cascade Biochem Ltd. (Berkshire, United Kingdom) and synthetic analogs were prepared and characterized, including NMR spectroscopy, as in Ref. 13. The integrity and concentration of each synthetic LX analog was assessed before each series of experiments. Concentrations of analogs were determined using an extinction coefficient of 50,000.
Cell Culture and IsolationThe human acute monocytic
leukemia cell line, THP-1 (ATCC, Rockville, MD), was maintained in RPMI
(Biowhittaker, Inc., Walkersville, MD) supplemented with 10% fetal
bovine serum (Biowhittaker) and antibiotics in a 37 °C incubator
with 5% CO2 atmosphere. Human monocytes were isolated
using a modification of the method of Denholm and Wolber (18). Briefly,
whole blood collected in acid citrate dextrose from healthy volunteers
was centrifuged (200 × g) at 25 °C for 15 min for
removal of platelet-rich plasma. The plasma was aspirated and the cells
were resuspended to the original blood volume with Dulbecco's
phosphate-buffered saline (DPBS2, Biowhittaker), layered
over Ficoll-Hypaque (Organon Teknika Corp., Durham, NC), and
centrifuged (500 × g) at 25 °C for 35 min. The
mononuclear cell layer was collected, washed once, and resuspended in
DPBS2
plus 0.1% bovine serum albumin. A Percoll:10 × Hanks' balanced salt solution (10:1.65) mixture was prepared and 8 ml mixed with 4 ml of mononuclear cells in a 10 × 1.5-cm
round-bottom silanized polypropylene tube and centrifuged (370 × g) at 25 °C for 30 min. Monocytes were collected from the
upper 5 mm of the gradient and washed with 50 ml of PBS2
before counting and viability assessment.
Chamber chemotaxis was evaluated using a microchamber technique according to the method of Falk et al. (19). Monocytes were isolated as above and suspended at 5 × 106/ml in DPBS2+. Thirty µl of a chemoattractant solution or vehicle (DPBS2+ plus 0.05% EtOH) were added to the lower wells of a 48-well chemotaxis chamber (Neuroprobe, Cabin John, MD). A polycarbonate membrane (5 µm pore size) was layered on top of the chemoattractant wells and 40 µl of monocytes added to the top wells and the chamber incubated at 37 °C for 90 min. After incubation, the membrane was removed, scraped of cells from the upper surface, and stained with modified Wright-Giemsa stain. Cells that migrated through the membrane in four high power fields were counted.
Laminin AdhesionMonocytes were resuspended in
DPBS2 (4 × 106/ml) and
[2
,7
-bis-(carboxyethyl)-5(6
)-carboxyfluorescein acetoxymethyl
ester] (BCECF-AM, Calbiochem, La Jolla, CA) was added (1.0 µM) and incubated with cells for 20 min at 37 °C.
Cells were washed once with DPBS2
and suspended (3.3 × 106/ml) in DPBS2+ plus 0.1% bovine serum
albumin. Aliquots (90 µl) of cells were added to each well of a
96-well flat-bottom tissue culture plate coated with laminin
(Collaborative Biomedical Products, Bedford, MA) and allowed to settle
for 10 min. Ten µl of agonist or vehicle were added to each well and
plates incubated at 37 °C for 20 min. Following incubation, wells
were aspirated and washed once with DPBS2+ plus 0.1%
bovine serum albumin. Adherent cells were solubilized with 100 µl of
0.025 M NaOH plus 0.1% sodium dodecyl sulfate and the
plate stirred on a rotary shaker for 20 min, followed by fluorescence quantitation. Adherence of THP-1 cells with exposure to vehicle alone
was 6.3-8.3% of total cells added.
Monocytes and/or THP-1 cells (2.5 × 106 cells/ml) were loaded with Indo-1 (2 µM) (Molecular Probes, Inc., Princeton, NJ) for 30 min, washed twice, and suspended in Hanks' balanced salt solution supplemented with Ca2+ (1 mM). Indo-1 excitation was at 358 nm, with detection of fluorescence at 405 nm (Photon Technology International Deltascan 4000, South Brunswick, NJ). The intracellular Ca2+ concentrations were calculated as described in Tsien et al. (20).
Cell-mediated CytotoxicityMonocytes (2.5 × 105 cells/50 µl) were added to 96-well tissue culture plates in RPMI plus 10% fetal bovine serum and incubated overnight at 37 °C with either vehicle (DPBS2+ plus 0.1% EtOH), lipopolysaccharide (100 ng/ml) from Escherichia coli serotype O26:B6 (Sigma), LXA4 (1 µM), 16-phenoxy-LXA4-Me (1 µM), or a combination of lipopolysaccharide (100 ng/ml) and LX (1 µM). Following overnight incubation, target cells (THP-1 cells) were added (5.0 × 104 cells/50 µl) to achieve a ratio of 5 effector cells/1 target cell. Cells were incubated together overnight at 37 °C. Following the second overnight incubation, 50-µl aliquots of medium supernatant were removed for use in a lactate dehydrogenase based cytoxicity assay (Cyto Tox 96®, Promega Corp., Madison, WI).
Receptor CloningTotal RNA was isolated from THP-1 cells
using TRIzolTM reagent (Life Technologies, Inc.). One µg of total RNA
was reverse-transcribed and used as the template for a PCR. PCR was
performed using the sense primer 5-CACCAGGTGCTGCTGGCAAG-3
(corresponding to the immediate 5
side of the starting codon ATG of
the LXA4 receptor cloned from HL-60 cells (21)) and
antisense primer 5
-AATATCCCTGACCCCATCCTCA-3
(corresponding to the
immediate 3
side of the stop codon TGA) for 30 cycles (94 °C for
30 s, 64 °C for 45 s, and 72 °C for 80 s) with
TaqPlusTM DNA polymerase (Stratagene, La Jolla, CA). A single band of
approximately 1.1 kilobase pairs was obtained and subcloned into the
EcoRV site of pBluescript II KS(+) (Stratagene). Three
independent clones were subjected to sequencing by an automated sequencer (ABI PRISM model 373A, version 1.20).
LXA4 is rapidly transformed by monocytes by initial
dehydrogenation at carbon 15 to 15-oxo-LXA4, which is
biologically inactive (11, 13). Therefore, a series of analogs was
designed with bulky substitutions on carbon 15, or at the carbon 20 end
of the molecule (Fig. 1). The aspirin-triggered
compound, 15-epi-LXA4, and free acid, methyl ester (Me),
and dimethylamide forms of 15(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 (Fig. 1) were synthesized and used in
the present experiments.
Several of these LXA4 analogs proved to be potent
chemotaxins for monocytes with 15-epi-LXA4,
15(R/S)-methyl-LXA4, and
16-phenoxy-LXA4 stimulating migration of monocytes at 100 nM concentrations (Fig. 2).
15-epi-LXA4 and 15(R/S)-methyl-LXA4
were similar in potency to LXA4.
16-Phenoxy-LXA4 stimulated greater numbers of monocytes to
migrate than LXA4 and was ~23% fewer than the number of
cells migrating with FMLP at this concentration (Fig. 2).
LXA4 analogs were also potent stimuli of THP-1 cell
adherence to laminin (Fig. 3).
15(R/S)-methyl-LXA4 and
16-phenoxy-LXA4 were more effective than LXA4
in stimulating THP-1 cell adherence, especially at concentrations less
than 1 nM (EC50 analogs 8 × 10
11 M, EC50 LXA4 = 8.3 × 10
10 M).
A seven-transmembrane-spanning receptor cloned from neutrophilic HL-60
cells was recently characterized by our laboratory as a high-affinity
LXA4 receptor (14). LXA4 activity with PMN appears to be mediated via binding to this receptor, confirmed by
specifically inhibiting LXA4 actions via treatment of cells with LXA4-receptor antisense oligonucleotides or an
antireceptor antibody (15). To determine if the same receptor is
present in monocytic cells, we used primers from the PMN receptor
sequence and reverse transcribed total RNA from THP-1 cells. One band
of approximately 1.1 kilobase pairs in size was produced from this reverse transcriptase-polymerase chain reaction and, when sequenced (GenBankTM accession number U81501[GenBank]), proved to be identical to the
LXA4 receptor cloned from the differentiated
(neutrophil-like) HL-60 cells. A band of identical size was also seen
in reverse transcriptase-polymerase chain reaction using human
peripheral blood monocyte RNA (data not shown). The deduced amino acid
sequence in Fig. 4 shows the seven-transmembrane regions
as well as the peptide sequence in the third extracellular domain to
which specific antisera were raised (15). This domain was chosen for
production of antiserum, because it is a region of high antigenicity
and is an area predicted to interact with ligand.
LXA4 increases intracellular calcium in monocytes (17);
therefore we examined the impact and potency of LXA4
analogs in this system. Both
15(R/S)-methyl-LXA4-Me and
16-phenoxy-LXA4-Me stimulated
concentration-dependent increases in intracellular Ca2+ in monocytes (Fig. 5). Similar results
were obtained for both the methyl ester and free acid forms of
LXA4 and the analogs (data not shown).
16-Phenoxy-LXA4-Me stimulated greater increments in Ca2+ than LXA4, while
15(R/S)-methyl-LXA4-Me mobilized less
Ca2+ than LXA4 within this concentration range.
We recently determined that adherence of monocytes to laminin was not
dependent on an increase in [Ca2+]i, because
addition of an intracellular Ca2+ chelator to monocytes did
not result in inhibition of LX-induced adherence (17). Changes in
intracellular Ca2+ are, nevertheless, a reliable means to
assess receptor-mediated signaling and may be connected with a specific
Ca2+-mediated function, which has not yet been identified;
therefore, it was further employed to determine whether monocyte
activation by LXA4 and analogs is mediated by a common
receptor site. We first examined homologous desensitization by
LXA4 and found that it completely down-regulates
Ca2+ mobilization by a second equimolar addition of
LXA4 (Fig. 6A). The specificity
of this response was determined by addition of LTB4 to the
cells approximately 60 s after LXA4. The
Ca2+ response to LTB4 was unaffected by prior
stimulation with equimolar LXA4 (Fig. 6A). We
next tested for cross-desensitization between LXA4 and the
individual analogs and found that addition of LXA4, followed by either 15(R/S)-methyl-LXA4-Me or
16-phenoxy-LXA4-Me, or vice versa, completely
desensitized the Ca2+ response to the second ligand (Fig.
6B).
To determine if the Ca2+ response to LXA4,
15(R/S)-methyl-LXA4-Me, and
16-phenoxy-LXA4-Me was via the cloned LXA4
receptor, we treated monocytes with the specific antiserum to the third extracellular domain of the receptor. Monocytes were exposed to anti-LXA4 receptor antiserum (anti-LXA4R) or
preimmune rabbit serum (1:500) for 10-30 min prior to exposure to
agonists. Pretreatment with the anti-LXA4R specifically
inhibited intracellular mobilization of Ca2+ by
LXA4 and both analogs, but did not affect the response to LTB4 (Fig. 7).
PMN responses to LXA4 are inhibitable by PTX, indicating
linkage of the receptor to G proteins in this cell type (16, 22). To
investigate this property of the receptor in monocytic cells, we
treated both monocytes and THP-1 cells with PTX. Pretreatment of
monocytes with PTX completely abrogated the Ca2+ response
by LXA4 and both stable analogs (Fig.
8A). Additionally, THP-1 cell exposure to PTX
inhibited LXA4- and analog-induced THP-1 cell adherence
>90%, with no effect on response to phorbol 12-myristate 13-acetate,
a stimulus which bypasses cell surface receptors (Fig. 8B).
This result reinforces that both adherence and intracellular
Ca2+ mobilization elicited by LXA4 and the
stable analogs are via binding to the G-protein-linked LXA4
receptor.
PTX inhibits LXA4- and
analog-induced responses. A, intracellular Ca2+
mobilization: monocytes were loaded with Indo-1 as in Fig. 5 and
incubated with PTX (2 µg/ml) or the inactive -oligomer of PTX (2 µg/ml) for 1 h before agonist (100 nM)
exposure. Values are from a representative experiment performed in
duplicate from n = 2 separate donors. B,
THP-1 cell adherence to laminin: cells were exposed to PTX (100 ng/ml)
for 16 h and labeled with BCECF-AM prior to incubation (3 × 105 cells in 100 µl) with LXA4 analogs (10 nM) or phorbol 12-myristate 13-acetate (150 nM)
for 40 min at 37 °C in laminin-coated plates. Values are expressed
as percent adherence above vehicle and are from a representative
experiment performed in quadruplicate from n = 3 separate experiments.
Because monocytes are involved in chronic conditions such as
atherosclerosis and rheumatoid arthritis, it was important to also
examine potential antagonists of LX-induced monocyte stimulation. To
this end, we prepared dimethylamide derivatives of
15(R/S)-methyl-LXA4 and
16-phenoxy-LXA4 that were modeled after the
PGF2 and LTB4 dimethylamide analogs that are
receptor-level antagonists of their respective native compounds (23,
24). We tested these compounds for potential agonist or antagonist
activity in THP-1 cell adherence and found that both
15(R/S)-methyl-LXA4-dimethylamide (Fig.
9) and 16-phenoxy-LXA4 (data not shown)
stimulated adherence of THP-1 cells with no statistically significant
difference in potency from that of their corresponding free acids.
Additionally, pretreatment or simultaneous addition of these
dimethylamide analogs to THP-1 cells did not inhibit adherence
stimulated by their respective free acids (data not shown).
Mobilization of intracellular Ca2+ was also examined and
15(R/S)-methyl-LXA4-dimethylamide increased [Ca2+]i in monocytes in the same potency range as
15(R/S)-methyl-LXA4-Me.
Monocytes are known to play important roles in cancer cell and immune surveillance. To further examine the range and specificity of monocyte activities that LXA4 and stable analogs may induce, we examined their impact on cell-mediated cytotoxicity. LXA4 and the analogs had no effect on monocyte cytotoxicity at concentrations of 1 µM and did not change cytotoxicity when added together with lipopolysaccharide (Table I).
|
LX have been shown, in vitro, to stimulate monocyte adherence and migration, while they inhibit the same responses in PMN (6, 8, 9, 11). Synthetic LX analogs that have longer half-lives in vitro and in vivo than native LX were designed to further evaluate the impact of LX on monocytes and aid in discerning signal transduction via the LX in monocytes. The LXA4 stable analogs used in these determinations were 15(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 (Fig. 1), which we previously showed had longer half-lives in incubations with monocytic cells and with recombinant 15-hydroxyprostaglandin dehydrogenase than native LXA4. These LXA4 stable analogs were shown to have similar bioactivity to native LXA4 in inhibiting PMN adhesion and migration (13), and here is the first report of monocyte stimulation by 15-epi-LXA4 (aspirin-triggered LXA4), 15(R/S)-methyl-LXA4, and 16-phenoxy-LXA4. These analogs were potent monocyte chemoattractants at 100 nM concentrations and 15(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 were more potent in stimulating THP-1 cell adherence to laminin than native LXA4. These results suggest that structural changes made to the native molecule have decreased conversion to inactive products by monocytic cells because, as we recently reported, the monocyte products of LXA4, namely, 15-oxo-LXA4, 13,14-dihydro-15-oxo-LXA4, and 13,14-dihydro-LXA4, were virtually inactive in stimulating monocyte adherence (11).
We cloned an LXA4 receptor from the monocytic cells which, when sequenced, proved to be identical to the receptor on neutrophilic cells. These results imply that the different LX responses seen in monocytes versus PMN are not due to actions via a different receptor. Other eicosanoids, for example prostaglandin E2, have alternate receptor forms in different tissues (25) to explain the diversity of their bioactions. Alternate forms of the LXA4 receptor have not yet been identified; however, this does not preclude their existence and is an area we are pursuing in other cell types.
These LX stable analogs also mobilized intracellular Ca2+
in monocytes in a dose-dependent manner, and the
differences in the relative potency of the analogs
(16-phenoxy-LXA4 > LXA4 > 15(R/S)-methyl-LXA4) in this assay
versus adherence (16-phenoxy-LXA4 15(R/S)-methyl-LXA4 > LXA4)
reinforces our recent findings, in that addition of an intracellular
Ca2+ chelator to THP-1 cells had no effect on LX-induced
cell adherence, suggesting that Ca2+ mobilization and
adherence are independent events in these cells (17). The increase in
[Ca2+]i stimulated by LXA4 showed
homologous desensitization and the analogs also proved to desensitize
the cells to an LXA4 response, but not an LTB4
response. This cross-desensitization implicated a common receptor site
on the monocyte for LXA4, 16-phenoxy-LXA4, and
15(R/S)-methyl-LXA4.
Specific antibodies to G-protein-linked seven-transmembrane receptors have been produced before, such as a polyclonal antibody to the platelet-activating factor receptor (26) and a monoclonal antibody to the receptor for the anaphylatoxin C5a (27). The C5a receptor antibody was shown to specifically inhibit Ca2+ transients and functional responses of both neutrophils (27) and eosinophils (28). We showed previously that the anti-LXA4R antiserum was selective, as characterized by immunoprecipitation. It specifically blocked LXA4 binding with PMN and also inhibited LXA4 activity in PMN (15). Here, the antipeptide antibody to the LXA4 receptor specifically inhibited [Ca2+]i increments by LXA4 and the individual LX analogs in monocytes, which suggests that these compounds are acting specifically via the cloned monocyte LXA4 receptor.
This receptor appears to be coupled to a PTX-sensitive G-protein in monocytes, because both adherence and Ca2+ mobilization were inhibited by PTX treatment. LXA4 responses in PMN, including phospholipase A2 activation and release of arachidonic acid (16) and activation of phospholipase D (22), are also PTX-sensitive. This indicates that the receptor coupling in monocytes and PMN is similar to this point, although there could be different PTX-sensitive G-protein subtypes that are coupled to the receptor and must diverge downstream in the signal transduction pathway to stimulate monocytes and inhibit PMN.
Leukocyte chemoattractants have been classified in two categories, the
"classical" chemoattractants, including FMLP, LTB4, and
C5a, which activate phospholipases, induce Ca2+ increments,
trigger generation of reactive oxygen products, and release of granule
enzymes, and the "pure" chemoattractants, including substance P and
transforming growth factor 1, which do not mobilize Ca2+, activate phospholipases, stimulate superoxide, or
enzyme release (29). In common, both of these classes of
chemoattractants act via G-protein-coupled receptors that are sensitive
to PTX. Results of the present and recent reports with human monocytes
(11, 17) on the activity of LXA4 and its stable analogs
place these LX in a unique class of chemoattractants due to the
following characteristics, namely: 1) stimulation of monocyte
chemotaxis and adherence, 2) Ca2+ mobilization, 3) no
impact on cell-mediated cytotoxicity, 4) no generation of superoxide
anion, and 5) action via PTX-sensitive receptors. The LX lie between
the classical and pure chemoattractants in their effects, by inducing
Ca2+ increments and activating phospholipases without
initiating production of reactive oxygen species. Interestingly, in
PMN, LX also inhibit some of these same activities of the classical
chemoattractants FMLP and LTB4, including inhibition of
chemotaxis and adherence (reviewed in Ref. 1). Therefore,
LXA4 has distinctive actions compared with known molecules
that interact with leukocytes.
Dimethylamide versions of two of the more potent LXA4
analogs were prepared as potential antagonists, based on the models of
LTB4- and PGF2-dimethylamides that can act
as specific antagonists to their parent compounds (23, 24), but these dimethylamide-LX analogs proved to have agonist properties as potent as
their corresponding free acids and methyl ester analogs. It is,
therefore, apparent that there is little effect of modifying the
LXA4 molecule at the carbon 1 position carboxylic acid and implies that the ligand interaction of LXA4 with its
receptor differs from that of LTB4 and PGF2
.
Nevertheless, these data provide us with a basis of evidence to develop
other analogs that could prove to be either potent agonists or
antagonists to the actions of LXA4 on monocytes. Developing
antagonists to LX actions with monocytes is of interest, because it is
possible that, in certain chronic inflammatory conditions or viral
infections, when monocytes instead of PMN are the prominent leukocytes
involved, inhibition of overt LX chemotactic properties may be
desirable to the outcome of disease.
These data provide new evidence on the activity of novel LXA4 analogs in human monocytes and indicate that they act via the cloned G-protein linked LXA4 receptor. The increased potency (~1 log molar) of these LX analogs indicates that structural modifications made to the carbon 20 end of LXA4 both inhibited dehydrogenation and oxidation of the molecule, and thus blocked inactivation, and did not hinder receptor-ligand interactions. Together, the results of the present report indicate that the LXA4 analogs are potent activators of monocytes that mimic the activity of native LXA4, but are active at even lower concentrations than the parent compound. This increased potency and decreased transformation of the analogs in vitro provides better tools for dissecting the differences in signal transduction between monocytes and PMN that lead to the selective effects of LX in these two cell types and, additionally, has important implications as far as longer half-lives and increased potency in vivo. Moreover, our results suggest that LX analogs will be vital tools in determining the impact of these and related compounds in selective monocyte and PMN trafficking in inflammatory diseases.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U81501[GenBank].