(Received for publication, June 28, 1995)
From the
Interleukin 8 (IL-8) is a potent chemoattractant and activating
factor for human polymorphonuclear leukocytes (PMN) and hence plays a
critical role in the pathogenesis of acute inflammation. Two unique but
homologous receptors for IL-8 have been cloned (IL-8RA and -B), each of
which binds the IL-8 ligand with high affinity. PMN stimulated by
cytokines or lipopolysaccharide (LPS) exhibit changes in IL-8R mRNA and I-IL-8 binding. Granulocyte-colony stimulating factor
(G-CSF) treatment of PMN enhances, and LPS inhibits, IL-8R mRNA
expression. Similarly,
I-IL-8 ligand binding to PMN is
increased by G-CSF and decreased by LPS treatment. The stimulatory
effect of G-CSF on IL-8R expression is transcriptional as it is
inhibited by actinomycin D and is evident in nuclear run-on analyses.
In contrast, LPS down-regulates IL-8R by both transcriptional and
post-transcriptional mechanisms. The alterations in IL-8R expression
are associated with similar changes in the IL-8-induced chemotactic
responses of PMN. In conclusion, the two types of IL-8 receptor differ
in their cellular distribution and are regulated in response to
cytokines and LPS. Regulation of IL-8R expression by endogenous and
exogenous immunomodulators may be important in the in vivo control of PMN effector functions in inflammation.
The recruitment and activation of polymorphonuclear leukocytes
is the hallmark of acute inflammation. Chemotactic factors produced at
an inflammatory site regulate vascular adhesion, transendothelial
migration, and the movement of leukocytes through the extracellular
matrix. Interleukin 8 is the prototype of a family of chemoattractant
cytokines known as chemokines(1) , which regulate these
migratory processes and determine the cellular composition of the
inflammatory response by their target cell specificity (reviewed in
Refs. 1 to 3). In particular, several members of the chemokine
subfamily: IL-8, (
)GRO/melanoma growth-stimulating activity,
neutrophil-activating peptide 2 (NAP-2), and ENA-78 are neutrophil
chemoattractants(4, 5, 6, 7) . These
chemokines are produced by a wide range of cell types, notably
macrophages, in response to a diverse array of stimuli including
proinflammatory cytokines, such as IL-1 and TNF, as well as
LPS(1, 2) .
Two receptors exhibiting high affinity
binding (K2 nM) for IL-8
have been cloned, and hence have been designated as type A and B IL-8
receptors. The type A IL-8 receptor was cloned by direct expression
from a PMN library (8) and binds IL-8(9, 10) .
In contrast, the type B receptor was identified from HL60
myelomonocytic cells (11) and has subsequently been
demonstrated to also bind GRO-
/melanoma growth-stimulating
activity with high affinity (K
2
nM), and NAP-2 and ENA-78 with lower
affinity(9, 10) . The IL-8R proteins are members of
the rhodopsin superfamily of seven-transmembrane domain,
G-protein-coupled receptors. They share 77% overall amino acid
identity, including two matching regions of 105 and 64 amino acids, and
their genes co-localize to chromosome 2q35 (12) . The chemokine
receptors, including the IL-8R and the recently cloned
chemokine
receptors(13, 14) , form a new subfamily of the
rhodopsin receptor superfamily. IL-8RA is strongly expressed only in
PMN, although the mRNA is detectable in differentiated HL60 cells (15) . In contrast, IL-8RB is prominently expressed in PMN, and
the mRNA is also widely distributed in myelomonocytic cell lines (U937,
THP-1, and HL60), the Jurkat T cell line, as well as melanoma and
fibroblast lines(15) . In addition, we have recently obtained
evidence that the IL-8R mRNAs are expressed in freshly prepared
peripheral blood T cells, but are progressively diminished by
incubation with or without anti-CD3 stimulation(16) .
In order to better understand the physiological control of IL-8-mediated events in acute inflammation we have investigated the in vitro regulation of the IL-8 receptors A and B in human PMN.
Figure 1:
Regulation of IL-8R mRNA expression in
PMN. RNA samples (5 µg per lane) were prepared from purified
peripheral blood PMN incubated for 6 h in the presence of various
stimuli. Lanes from left to right, medium alone (RPMI/10%
fetal calf serum), IL-8 (50 ng/ml), TNF- (10 ng/ml), fMLP (100
nM), LPS (10 ng/ml), GM-CSF (50 ng/ml), and G-CSF (50 ng/ml).
The autoradiographic signals were measured by densitometry and
normalized to those of
-actin. The figure represents one of three
replicate experiments with the quantitative data (upper panel)
and the Northern analysis (lower
panel).
Figure 2:
A, kinetics of IL-8R regulation in
peripheral blood PMN induced by G-CSF and LPS. RNA samples (5
µg/lane) were prepared from purified peripheral blood PMN incubated
for 1-18 h in the presence or absence of G-CSF (50 ng/ml) or LPS
(10 ng/ml). The autoradiographic signals were measured by densitometry
and normalized to -actin. The figure represents quantitative data
from one of three replicate experiments. A Northern blot containing
data representative of the LPS effect is included with Fig. 4. B, effect of actinomycin D on the regulation of IL-8R mRNA
expression in PMN by G-CSF. RNA samples (5 µg/lane) were prepared
from purified peripheral blood PMN incubated for 1-18 h with
G-CSF (50 ng/ml) or preincubated for 30 min with actinomycin D (10
µM) followed by the addition of G-CSF (50 ng/ml). The
effect of actinomycin D on IL-8R mRNA regulation by LPS is shown in Fig. 4.
Figure 3:
Nuclear run-on analysis of the IL-8R mRNA
in PMN treated with G-CSF or LPS. Nuclei were prepared from 2-5
10
purified peripheral blood PMN immediately after
incubation for 3 or 6 h in medium alone (RPMI 1640, 10% fetal calf
serum), or with G-CSF (50 ng/ml) or LPS (10 ng/ml), or a combination of
both stimuli. The labeled nuclear RNA was hybridized with denatured
plasmid cDNAs of IL-8RA and -B,
-actin, and pGEM-1. The
autoradiographic signals were measured by densitometry. For
quantitative analyses, the signals from nonspecific hybridization (with
pGEM-1) were subtracted from those of IL-8RA and -B and then normalized
to the signals of
-actin. The figure represents one of three
replicate experiments.
Figure 4:
Half-life of the IL-8R mRNA in PMN treated
with LPS. RNA samples (5 µg/lane) were prepared from purified
peripheral blood PMN incubated for 1-18 h: with medium alone,
with actinomycin D (10 µM), with LPS (10 ng/ml), or
preincubated with actinomycin D (10 µM) for 30 min
followed by the addition of LPS (10 ng/ml). The autoradiographic
signals were measured by densitometry and normalized to those of
-actin. The upper panel is quantitative data from three
replicate experiments (mean ± S.E.). A set of Northern blots
from a representative experiment are included in the lower
panel.
Figure 5:
Regulation of IL-8 binding and
IL-8-induced chemotaxis on peripheral blood PMN by G-CSF or LPS.
Purified peripheral blood PMN were incubated for 1-20 h in the
presence or absence of G-CSF (50 ng/ml) or LPS (10 ng/ml) before being
included in standard I-IL-8 binding assays performed at 4
°C in the presence or absence of excess unlabeled ligand (A). Specific counts were determined by subtracting the counts
from binding in the presence of excess unlabeled ligand from the total
bound radioactivity by chemotaxis assays, utilizing a modified Boyden
chamber technique, and various doses of IL-8 or medium alone (B). The data represent the mean number of cells per high
power field (±S.D.) detected in 5 fields for the optimal
chemotactic dose of IL-8 (50 ng/ml).
The IL-8RA and -B are abundantly expressed on human PMN. The IL-8R mRNAs are reciprocally regulated in response to stimuli such as G-CSF and LPS, which are likely to be present in sites of acute inflammation.
Responses to chemoattractant cytokines, including IL-8, may be desensitized by continued stimulation(22) , confirming that the chemokine receptors are regulated via agonist-dependent mechanisms as has been shown for other rhodopsin family members (reviewed in (23) ). In the present work, we have demonstrated that the two IL-8R also exhibit regulation in response to exogenous stimuli. Treatment of PMN with G-CSF resulted in increased IL-8R mRNA expression, as well as IL-8 binding and chemotactic responsiveness. G-CSF has been shown to enhance PMN survival in vitro by inhibition of apoptosis(24) . However, the up-regulation of IL-8R expression we have demonstrated is not explicable simply on the basis of improved PMN survival, as the IL-8R mRNA and chemotactic response to IL-8 are significantly increased above baseline levels in G-CSF-treated PMN ( Fig. 2and Fig. 5). G-CSF enhances IL-8R expression by a transcriptional mechanism, as actinomycin D treatment blocked its effect, and nuclear run-on studies showed increased IL-8R signals after G-CSF treatment of PMN. This regulatory pathway, if direct, may indicate a novel G-CSF-responsive transcriptional regulatory element in the IL-8R genes. Indeed, we have recently isolated, sequenced, and characterized the genomic structure of the IL-8RB gene(25) . IL-8RB promoter region-CAT constructs demonstrated enhanced expression in HL60 cells after treatment with G-CSF, thus providing evidence for a direct transcriptional effect of G-CSF on the IL-8RB gene. Comparison of the promoter region of this gene with that of another PMN chemoattractant receptor, the fMLP receptor, has demonstrated the presence of several novel, but highly conserved, sequence motifs, which may represent tissue-specific transcriptional regulatory elements(25) . Interestingly, these motifs were not detected in the promoter region of the IL-8RA gene(26) .
Down-regulation of IL-8R expression
and IL-8 responsiveness was demonstrated when PMN were incubated with
LPS. This treatment induced a significant decrease in the half-life of
the IL-8R mRNA, consistent with an effect on the stability of the
message. In addition, IL-8R transcriptional activity appeared to be
inhibited in nuclear run-on studies. LPS also has been shown to enhance
the survival of PMN in vitro(24) , and to induce the
expression of several genes including IL-1 (27) and
TNF-
(28) . Hence, the inhibitory effect of LPS on IL-8R
expression contrasts with these findings and is not related to reduced
PMN survival. Hormone-induced reduction in mRNA stability has been
documented for several G-protein-coupled receptors including the
-adrenergic receptor and thyrotropin-releasing hormone
receptor(23) . Although the mechanisms of this altered RNA
stability are not yet clear, the 3`-untranslated region of the mRNA for
the
-adrenergic receptor, as well as several other
rhodopsin family members, contain AU-rich elements correlated with
highly regulated, short-lived mRNAs(23, 29) . We have
identified multiple regions of AU-rich sequence in the 3`-untranslated
region of the IL-8RB gene(25) .
The inhibitory effect of LPS
on the chemotactic response of PMN to IL-8 was detected earlier than
expected from the alteration in the mRNA. This may have been
attributable in part to clumping and adhesion of PMN to the upper side
of the polycarbonate filter. We have observed similar clumping effects
with fMLP, yet LPS treatment enhances fMLP receptor mRNA expression in
PMN and augments the PMN chemotactic response to fMLP. ()Similarly, the stimulatory effect of G-CSF on chemotactic
responses to IL-8 was more rapid than would be expected from the
transcriptional regulation. These apparent discrepancies may relate to
differences in assay sensitivity or to independent effects of these
stimuli on receptor turnover. Nevertheless, our data suggest that
regulation of the IL-8R after some hours of PMN exposure to G-CSF or
LPS is correlated with the transcriptional regulatory mechanisms
defined here.
Our findings suggest that stimuli occurring in
vivo in acute inflammation may alter the expression of IL-8R on
PMN. We have provided preliminary evidence for the functional
significance of this regulation in PMN chemotaxis. The capacity of
G-CSF to stimulate PMN functions, including phagocytosis and superoxide
generation, may be mediated in part by enhanced responsiveness to IL-8.
Interestingly, exposure of PMN to LPS may result not only in the
synthesis and release of a secondary cascade of proinflammatory
cytokines including IL-1, TNF-
, and IL-8(30) , but
also in anti-inflammatory effects leading to decreased PMN
responsiveness such as IL-1RA production (30) and
down-regulation of IL-8R as we have documented here. Perhaps these
immunomodulatory effects of LPS may preferentially direct PMN toward
bacterially derived chemoattractants, such as fMLP. The kinetics and
magnitude of these respective PMN responses may be important
determinants of the outcome of acute neutrophilic inflammation.