1 Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612; and 2 Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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Interleukin (IL)-8 is a C-X-C chemokine that plays an important
role in acute inflammation through its G protein-coupled receptors
CXCR1 and CXCR2. In this study, we investigated the role of IL-8 as an
autocrine regulator of IL-8 production and the signaling mechanisms
involved in human peripheral blood mononuclear cells (MNCs).
Sepharose-immobilized IL-8 stimulated a sevenfold increase in IL-8
production within 2 h. IL-8 induced the expression of its own
message, and IL-8 biosynthesis was inhibited by cycloheximide and
actinomycin D, indicating de novo RNA and protein synthesis. In
contrast to MNCs, polymorphonuclear neutrophils did not respond to the
immobilized IL-8 with IL-8 production despite cell surface expression
of CXCR1 and CXCR2. Melanoma
growth-stimulatory activity/growth-related protein-
(MGSA/GRO
), which binds CXCR2 but not CXCR1, was unable to
either stimulate IL-8 secretion in MNCs or desensitize these cells to
respond to immobilized IL-8. The involvement of mitogen-activated protein kinase (MAPK) in IL-8-induced IL-8 biosynthesis was suggested by the ability of PD-98059, an inhibitor of MAPK kinase, to block this
function. Furthermore, IL-8 induced a significant increase in
extracellular signal-regulated kinase 2 phosphorylation, whereas MGSA/GRO
was much less effective. These findings support the role of
IL-8 as an autocrine regulator of IL-8 production and suggest that this
function is mediated by CXCR1 through activation of MAPK.
cytokines; chemokines; inflammation; monocytes
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INTRODUCTION |
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CHEMOATTRACTANTS PRODUCED at sites of inflammation are critical for the attraction and activation of leukocytes. Two major classes of leukocyte chemoattractants have been identified. The classic chemoattractants, including C5a, C3a, formyl-Met-Leu-Phe (fMLP), platelet-activating factor, and leukotriene B4, are potent activators of many phagocyte functions, although their chemical structures vary considerably. In contrast, the chemotactic cytokines (chemokines) are a group of peptides with a molecular mass of 8-10 kDa that share a structural homology including the positioning of cysteine residues (e.g., C-X-C vs. C-C) (1, 25). Chemokines bind and activate lymphocytes, monocytes, macrophages, and polymorphonuclear neutrophils (PMNs). In addition to their primary function of attracting leukocytes to the sites of inflammation, chemokines may affect biological processes such as the development of stem cells, angiogenesis, and virus entry into host cells (reviewed in Ref. 1).
Interleukin (IL)-8 is one of the best-characterized C-X-C chemokines,
the potent chemotactic activity of which has been associated with
numerous acute and chronic inflammatory disorders (1). Two
receptors for IL-8 have been characterized and named CXCR1 (IL-8
receptor type A) and CXCR2 (IL-8 receptor type B) (10, 19,
23). CXCR1 is specific for IL-8, whereas CXCR2 exhibits typical
chemokine promiscuity in agonist recognition, being able to bind not
only IL-8 but also neutrophil-activating peptide-2, melanoma
growth-stimulatory activity/growth-related protein- (MGSA/GRO
),
and 78-amino acid endothelial cell-derived neutrophil activator
(26). IL-8 receptors are widely expressed in PMNs, peripheral blood mononuclear cells (MNCs), T lymphocytes, and natural
killer cells. In addition, IL-8 receptors are also found in endothelial
cells, neuronal cells, fibroblasts, and keratinocytes. Accordingly,
IL-8 has been shown to possess functions such as the regulation of
growth (4, 24) and angiogenesis (15). In
leukocytes, IL-8 has been shown to stimulate the activation of G
proteins and several downstream serine/threonine kinases (14,
16) that are responsible for chemotaxis, degranulation, and
production of superoxide anions by phagocytes.
Because IL-8 plays a critical role in acute inflammation, it is
important to understand how IL-8 expression is regulated. IL-8
production at the sites of inflammation requires de novo biosynthesis
(1, 22). The IL-8 gene promoter contains sites for nuclear
factor (NF)-B, NF-IL-6, and activator protein-1 (22), transcription factors that regulate the expression of a large number of
proinflammatory cytokines and growth factors. Accordingly, IL-8
production in various cell types can be readily induced by lipopolysaccharide, IL-1
, and tumor necrosis factor (TNF)-
. Chemoattractants also have varying potencies as inducers of IL-8 production (5). Recent findings (30)
suggested that chemoattractant receptors, like a number of other G
protein-coupled receptors, mediate the activation of NF-
B and
activator protein-1 and actively regulate the expression of genes that
are controlled by these transcription factors. We have shown that both
fMLP and C5a are able to stimulate the expression of IL-8 in monocytes
and macrophages (3, 11). Like these chemoattractants, IL-8
activates a signaling pathway that involves G protein activation and
therefore can possibly lead to transcription activation. In this study,
we sought to identify the role of IL-8 as an autocrine regulator of
IL-8 production in peripheral blood leukocytes.
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MATERIALS AND METHODS |
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Reagents.
The chemokines were purchased from PeproTech (Rocky Hill, NJ); and
fMLP, cycloheximide, and actinomycin D were from Sigma (St. Louis, MO).
Indo 1-AM was from Molecular Probes (Eugene, OR). Mouse TNF- was a
gift from Dr. V. Kravchenko (Scripps Research Institute, La Jolla, CA).
The reagents used for nuclear runoff assay, including RNase-free DNase
I, RNase A, yeast tRNA, and salmon sperm DNA, were from Boehringer
Mannheim (Indianapolis, IN). Unless otherwise indicated, all other
reagents were purchased from Sigma.
Preparation of cells.
Human peripheral blood leukocytes were fractionated with Percoll as
previously described (3). Briefly, blood was collected from healthy donors, with acid citrate-dextrose (citrate buffer containing 2% dextrose) as an anticoagulant. Erythrocytes were removed
by sedimentation with HESPAN (6% hetastarch; Baxter, Highland, CA).
MNCs and PMNs in the supernatant were further separated by centrifugation at 450 g for 40 min at 10°C through Percoll
step gradients (70 and 55%). Viability of the cells in a routine
preparation was ~98% as determined by trypan blue exclusion. In some
cases, monocytes and lymphocytes were further separated from
preparations of MNCs. Briefly, MNCs were washed four times in PBS
containing 1 mM EDTA, resuspended in RPMI 1640 medium at a density of
106 cells/ml, and layered on top of isotonic 54% Percoll.
After centrifugation for 30 min at 2,500 rpm, monocyte-enriched
populations found at the interface and lymphocytes found at the bottom
of the tube were collected. This method produced enrichment to 85% of
monocytes (CD14+) and lymphocytes (CD14) in
the respective fraction.
Immobilization of IL-8.
Affi-Gel 10 beads (Bio-Rad, Richmond, CA) were washed three times in
PBS followed by resuspension to ~50% (vol/vol) by adding an equal
volume of PBS containing 1 µM IL-8 (or MGSA/GRO). After rotation
overnight at 4°C, the beads were collected by centrifugation and
washed four times with PBS. The beads were resuspended in PBS to obtain
a 50% slurry and maintained at 4°C until used. The amount of IL-8
bound to the beads was quantified by incorporating a small amount of
radioiodinated IL-8 (specific activity 2,200 Ci/mmol; NEN, Boston, MA)
into the binding reaction. The radioactivity associated with the beads
was determined before and after the washing step. By this method, it
was determined that 86% of the IL-8 was initially associated with the
beads. After an extensive wash, only 14% of the total IL-8 added to
the binding reaction remained attached to the beads. The final
preparation (a 50% slurry) contained IL-8 at a concentration of ~70
nM. Beads conjugated with a trace amount of 125I-labeled
IL-8 were incubated in medium in the presence and absence of cells for
the duration of the experiments, and no release of 125I-IL-8 was detected.
Ca2+ mobilization.
The cells were loaded with indo 1-AM (5 µM) in Hanks' balanced salt
solution. Intracellular Ca2+ mobilization experiments were
conducted with cells in suspension and were monitored by continuous
fluorescent measurements in an SLM 8000 photon-counting
spectrofluorometer (SLM-Aminco, Urbana, IL), with an excitation
wavelength of 340 nm and detection at 400 and 490 nm. Relative
intracellular Ca2+ level is expressed as the ratio of
fluorescence determined at 400 nm to that at 490 nm. Intracellular free
Ca2+ concentration ([Ca2+]i)was
determined with the formula [Ca2+]i = 250(F Fmin)/(Fmax
F), where F
is the ratio of fluorescence obtained after ligand stimulation,
Fmax is the ratio of fluorescence obtained with Triton
X-100 (0.1%) and reflects the total available free Ca2+,
and Fmin is the ratio of fluorescence obtained with EDTA (2 mM) included in the assay buffer to remove free Ca2+
released by Triton X-100 treatment.
Measurement of IL-8 transcripts.
Freshly isolated cells were stimulated with the appropriate ligands
(100 nM each IL-8 and MGSA/GRO or 40 ng/ml of TNF-
) at 5 × 105 cells/ml in a total volume of 10 ml. After 2 h,
the cells were harvested, and total RNA was extracted with the
guanidinium thiocyanate method. cDNA was prepared with Superscript
reverse transcriptase (GIBCO BRL, Life Technologies, Gaithersburg, MD).
Amplification of IL-8 transcripts was accomplished with primers located
within the coding region of IL-8 with the following sequences: forward primer, 5'-TGACTTCCAAGCTGGCCGTG-3' and reverse primer,
5'-ACAGAGCTCTCTCCATCAG-3'. PCR amplification was conducted for 18 cycles to avoid overamplification. Equal amounts of the PCR products
were analyzed on 1% agarose gels by electrophoresis and ethidium
bromide staining. The relative intensity of the IL-8 message in each
sample was determined with ImageQuant software (Molecular Dynamics,
Mountain View, CA) and standardized against the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplified in
the same tube.
Nuclear runoff.
Nuclear transcripts of IL-8 were measured as follows. Briefly, 3 × 107 freshly isolated MNCs were incubated for 2 h
with 100 nM IL-8 or MGSA/GRO or without chemokines (basal) in 4 ml
of RPMI 1640 medium at 37°C. Nuclear suspensions were prepared by
washing the cells with PBS followed by resuspension in 4 ml of lysis
buffer (10 mM Tris · HCl, pH 7.4, 3 mM MgCl2, 10 mM
NaCl, and 0.5% Nonidet P-40) added dropwise with continuous vortexing
followed by centrifugation at 500 g for 5 min. Nuclear
pellets were washed one more time in 4 ml of lysis buffer and then
maintained at
80°C in 230 µl of freezing buffer (50 mM
Tris · HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, and
0.1 mM EDTA, pH 8.0) until used. The nuclear transcripts were elongated
by incubating the nuclei with 60 µl of 5× reaction buffer (150 mM
Tris · HCl, pH 8.0, 750 mM KCl, 25 mM MgCl2, 1 mM
EDTA, 12.5 mM dithiothreitol, 2.5 mM each ATP, GTP, and CTP, and 100 µCi of [32P]UTP) for 30 min at 30°C. Labeled nuclei
were treated with 5 U of DNase I for 5 min at 30°C and then with 50 µg of proteinase K for 30 min at 37°C. Before isolation of the
labeled transcripts, 200 µg of yeast tRNA were added as a carrier,
and RNA was prepared with the guanidinium isothiocyanate method with
the TRI Reagent (Molecular Research Center, Cincinnati, OH). RNA was
denatured with 0.1 N NaOH on ice for 10 min followed by neutralization
with 1 M HEPES, pH 7.4. RNA was precipitated by ethanol and resuspended in 1 ml of hybridization solution (see below) before use. For preparation of the filters, 1 µg of circular DNA was denatured by
boiling in 0.1 N NaOH for 6 min followed by neutralization with HEPES
buffer (pH 7.4) and dilution with 6× saline-sodium citrate (SSC)
buffer before application to the filter with a slot blot apparatus.
Filters were rinsed briefly in 6× SSC and baked for 2 h at
80°C. The filters were prehybridized [10 mM
N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES), 2% SDS,
10 mM EDTA, and 300 mM NaCl] for 2 h at 60°C followed by
hybridization in the same solution with labeled RNA probe for an
additional 36 h. Filters were washed twice for 15 min each in 2×
SSC at 60°C followed by incubation for 30 min at 37°C in 2× SSC
containing 1 µg/ml of RNase A. After an additional wash at room
temperature in 2× SSC, the filters were air-dried and exposed to a
phosphorimaging screen.
Cell staining and flow cytometry. Fluorescence-activated cell analysis was performed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). To identify monocytes in mononuclear fractions of human blood, cells were labeled with FITC-conjugated anti-CD14 monoclonal antibody (MAb; PharMingen, San Diego CA). Identification of the levels of each type of IL-8 receptor was done by incubating MNCs with phycoerythrin-conjugated MAb (1:500; PharMingen) directed against CXCR1 or CXCR2. All incubations were carried out on ice for 60 min. The cells were washed twice with PBS before flow cytometry analysis.
Detection of phosphorylated extracellular signal-regulated
kinase.
Freshly isolated MNCs (5 × 106 cells in 250 µl of
RPMI 1640 medium) were stimulated with either 100 nM IL-8 or 100 nM
MGSA/GRO. Samples were harvested at different time points over a
period of 15 min. Stimulation was stopped by adding 1 ml of ice-cold PBS followed by centrifugation. The cell pellets were resuspended in
SDS-PAGE sample buffer and immediately boiled for 5 min. The extracts
were then separated on 10% polyacrylamide gels and blotted to
nitrocellulose filters. Filters were treated for 20 min with PBS
containing 0.05% Tween 20 and 4% BSA and then incubated with anti-phospho-extracellular signal-regulated kinase (ERK) 1/2 (New England Biolabs, Beverly, MA). The bands were then stained with a
peroxidase-conjugated goat secondary antibody (Ab) followed by
visualization with chemiluminescence (Pierce, Rockford, IL).
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RESULTS |
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IL-8 stimulates IL-8 production through an autocrine loop.
A previous study (30) showing IL-8 induction of B
binding activity suggested that IL-8 may serve as an autocrine
regulator of IL-8 production. This possibility and the underlying
mechanism were investigated in the present study. A technical
difficulty associated with this endeavor is the separation of newly
synthesized IL-8 from the added ligand. Our initial efforts involved
metabolically radiolabeling cells with [35S]methionine or
[3H]leucine followed by immunoprecipitation of newly
synthesized IL-8 from the culture medium. This method produced results
that varied considerably due to interference of the added ligand with immunoprecipitation of the radiolabeled IL-8. To overcome this problem,
IL-8 protein was conjugated to Affi-Gel (Sepharose) beads, and the
immobilized IL-8 was then used as an agonist. The conjugated beads
contain IL-8 at a concentration of ~70 nM in a 50% slurry. Moreover,
incubation of bead-immobilized 125I-IL-8 with blood
leukocytes for 2 h caused no detectable release of radiolabeled
IL-8. Therefore, it is feasible to detect newly synthesized IL-8 in the
culture medium after removal of the agonist (IL-8-conjugated beads) by
centrifugation. Using this approach, we found that the immobilized IL-8
induced a sevenfold increase in IL-8 production from MNCs over a 2-h
period (Fig. 1A). Further incubation resulted in additional increase in IL-8 secretion at 4 h (Fig. 1B). To minimize the possible secondary effects on
IL-8 biosynthesis, incubation time was limited to 2 h in
subsequent experiments.
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Autocrine production of IL-8 is cell-type specific.
The distribution of the two IL-8 receptors in different leukocytes has
been reported by several groups (9, 18, 20) with slightly
different results. Using specific MAbs against CXCR1 and CXCR2, we
observed that PMNs express more CXCR1 and CXCR2 than either monocytes
or lymphocytes (Fig. 2A). Also
in agreement with the previous findings, donor variation was evident,
but in each preparation, monocytes always expressed relatively low
levels of both receptors, and CXCR2 was the more abundant of the two subtypes. The presence of small amounts of both CXCR1 and CXCR2 in a
lymphocyte subpopulation, probably natural killer cells
(27), necessitated investigation of the source of IL-8
secretion in the MNC preparations. By enrichment of monocytes from the
MNC fractions to ~85%, it was determined that the immobilized IL-8 could stimulate almost twice as much IL-8 secretion in monocytes as in
unfractionated MNCs (Fig. 2B). Furthermore, the enriched lymphocyte population (~85%) responded with less than half as much
secreted IL-8 as with MNCs (Fig. 2B). These data indicate that monocytes within the MNC preparations are the principal cells responding to the immobilized ligand with production of IL-8.
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The role of transcription and translation in autocrine IL-8
production.
It has recently been shown that several chemoattractants including
leukotriene B4, platelet-activating factor, and fMLP can activate transcription in peripheral blood monocytes (2, 3, 11,
30). To determine whether the secreted IL-8 was the result of de
novo synthesis, the effect of transcription and translation inhibitors
(actinomycin D and cycloheximide, respectively) on the production of
IL-8 was investigated. Preincubation of MNCs for 1 h with
increasing doses of these inhibitors followed by stimulation of the
cells for 2 additional hours with the immobilized IL-8 resulted in a
dose-dependent inhibition of the IL-8 detected in the medium (Fig.
3A). The dose-response curves
for both actinomycin D and cyclohexamide were similar, suggesting the
importance of both transcription and translation for the production of
IL-8 in these experiments. The inhibition of IL-8 production was
significant (~60% by 1 h pretreatment), suggesting de novo
synthesis of IL-8 in response to IL-8 stimulation.
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Involvement of mitogen-activated protein kinase activation in IL-8
secretion by MNCs.
Mitogen-activated protein (MAP) kinase cascades are important mediators
of transcription factor activation leading to gene expression in
response to a variety of cellular stimuli. The most widely studied MAP
kinase pathway is activated upstream by the small GTPase Ras, which
leads to activation of Raf and MAP kinase kinase kinase (MEK) 1/2 and
ultimately to the phosphorylation of ERK. Activation of ERK has been
linked to IL-8 production in a previous study (6) with the
lung epithelial cell line A549. Although both CXCR1 and CXCR2 can
mediate ERK activation (13, 14), the ability of
MGSA/GRO and IL-8 to cause phosphorylation of ERK1/2 in human
monocytes has not been investigated. The importance of this pathway in
the autocrine production of IL-8 in mononuclear cells was demonstrated
by the dose-dependent inhibition of IL-8 production by the MEK1/2
inhibitor PD-98509 (Fig. 5A).
Because IL-8 but not MGSA/GRO
was able to stimulate IL-8 production
from MNCs, it was of interest to compare the abilities of each of these ligands with respect to phosphorylation of ERK1/2 in these cells. As in
PMNs, phosphorylation of ERK in response to IL-8 was rapid and
transient, peaking within 1 min. However, the phosphorylation of ERK1/2
after stimulation with equimolar concentrations of MGSA/GRO
was
barely detectable relative to IL-8 stimulation (Fig. 5B). These data are consistent with the efficacy of each ligand to induce
IL-8 production and lend support to a role for ERK activation in
autocrine IL-8 production.
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DISCUSSION |
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Results obtained from this work establish IL-8 as an autocrine regulator of IL-8 production in monocytes. As such, IL-8 acts on one of its own receptors and stimulates the biosynthesis of IL-8 through a mechanism that involves de novo mRNA and protein synthesis. Monocytes and tissue macrophages are primary sources of inflammatory cytokines and chemokines. It is generally assumed that IL-8 released by cells at inflammatory sites diffuses to more distal regions where, at a subnanomolar concentration, it serves as a principal chemoattractant for inflammatory cells. In the lung, IL-8 is synthesized and released by epithelial cells (17) as well as by residential macrophages (29) and plays an important role in the recruitment of PMNs and MNCs (28). The discovery that MNCs produce subnanomolar concentrations of IL-8 after stimulation indicates the likelihood of autocrine signaling in vivo. Local production of IL-8 by the stimulated MNCs may facilitate the accumulation of this chemokine over time to a higher concentration than is required for activation of several phagocyte functions. In addition, the autocrine production of IL-8 by MNCs may amplify an inflammatory response by creating a possibly transient and self-sustaining source of this potent chemotactic factor, thus attracting additional leukocytes to the site of inflammation. It has been shown that MNCs may amplify their recruitment into inflammatory lesions by inducing self-production of monocyte chemotactic protein (8). Thus autocrine regulation of chemokines provides an additional mechanism for the recruitment of inflammatory cells. The in vivo production of IL-8 may be negatively regulated by cytokines such as IL-4 and IL-10. Future study will be necessary to determine the conditions under which IL-8 autocrine regulation occurs in vivo.
Our data suggest that autocrine IL-8 production is both cell specific and receptor specific. MNCs, which express less CXCR1 and CXCR2, respond better than PMNs in autocrine IL-8 production. The exact signaling mechanism that dictates this difference is not clear, but there are several possibilities. MNCs are a major source of cytokines and have a well-developed machinery for protein synthesis and secretion. In contrast, PMNs are terminally differentiated cells suited for special functions such as degranulation and superoxide generation. However, their protein synthesis capabilities are quite limited. Published studies indicate that PMNs utilize their remnant transcription and translation machinery to express a selected number of genes (12). Although PMN expression of IL-8 has been documented, the level of production is well below that of MNCs (3, 5). Another possibility is that PMNs and MNCs employ different signaling mechanisms downstream of the IL-8 receptors, which then lead to different cellular functions. In PMNs, IL-8 stimulation triggers the generation of superoxide anions and release of granule contents, whereas in MNCs, IL-8 stimulation causes production of IL-8 and possibly other cytokines. Consistent with this notion is the observation that IL-8 induces a more pronounced Ca2+ mobilization in PMNs, which is critical for such functions as degranulation.
Autocrine IL-8 production in MNCs preferentially utilizes CXCR1, which
binds IL-8 but not MGSA/GRO. The latter is a potent agonist for
CXCR2 and is structurally similar to IL-8, but it does not seem to
stimulate autocrine IL-8 production in MNCs. This discrepancy
apparently is not associated with the expression of IL-8 receptors in
leukocytes because both CXCR1 and CXCR2 have been detected in MNCs and
PMNs. Data presented in this paper provide evidence for signal
transduction events that are potentially important for autocrine
production of IL-8. Experimental results with PD-98059 suggested that
activation of the MEK/ERK MAP kinase pathway was necessary for IL-8
production in MNCs. A previously published study (6) also
suggests a link between ERK2 activation and IL-8 production. The role
of ERK activation in this process was further supported by the
inefficiency with which MGSA/GRO
could stimulate ERK phosphorylation
and IL-8 production in these cells. It is likely that the observed
small increase in ERK phosphorylation by MGSA/GRO
is due to the
overlapping activation of CXCR1, which can bind MGSA/GRO
at the
higher concentration used here. In addition to ERK activation, there
are other differences in the signaling pathways triggered by the two
IL-8 receptors. In PMNs, for example, only CXCR1 induces respiratory
burst, although both CXCR1 and CXCR2 can mediate chemotaxis and
Ca2+ mobilization (14). In addition, although
both receptors can activate phosphoinositide turnover by phospholipase
C in PMNs and transfected Jurkat cells, CXCR1 but not CXCR2 activates
phospholipase D (14). A more recent study
(31) in Jurkat cells links CXCR1 to IL-8 expression in
response to the tax gene product of human immunodeficiency virus-1. It
is noted, however, that MGSA/GRO
can serve as an autocrine regulator
under some conditions such as in melanoma cell lines (24).
We believe that preferential usage of CXCR1 in autocrine IL-8
production occurs in certain types of cells such as MNCs.
In summary, this study provides direct evidence that autocrine IL-8
production occurs in monocytes stimulated with IL-8 and that this
cellular response is regulated at the cell and receptor levels. These
new observations warrant additional studies of the signaling mechanisms
activated by IL-8 and MGSA/GRO. The finding may have profound
applications in leukocytes and in other types of cells as IL-8 is more
and more recognized as a regulator of cell proliferation in addition to
its traditional role in chemotaxis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Eric Prossnitz and Robert Hoch for helpful discussions.
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FOOTNOTES |
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This work was supported by National Institute of Allergy and Infectious Diseases Grant AI-40176 and a Biomedical Science Grant from the Arthritis Foundation (to R. D. Ye).
D. D. Browning and M. H. Hsu are recipients of postdoctoral fellowships from the Arthritis Foundation. R. D. Ye is an Established Investigator of the American Heart Association.
Present addresses: W. C. Diehl, Agouron Pharmaceutical, San Diego, CA 92121; M. H. Hsu, Stratagene, San Diego, CA 92121; I. U. Schraufstatter, La Jolla Institute for Experimental Medicine, La Jolla, CA 92037.
Address for reprint requests and other correspondence: R. D. Ye, Dept. of Pharmacology (MC868), Univ. of Illinois College of Medicine, 835 South Wolcott Ave., Chicago, IL 60612 (E-mail: yer{at}uic.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 June 2000; accepted in final form 24 July 2000.
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