Differential Mechanisms of Recognition and Activation of
Interleukin-8 Receptor Subtypes*
Katsutoshi
Suetomi,
Zhijian
Lu
,
Tonia
Heck,
Thomas G.
Wood,
Deborah J.
Prusak,
Karen J.
Dunn, and
Javier
Navarro§
From the Department of Physiology and Biophysics and Sealy Center
for Molecular Science, University of Texas Medical Branch,
Galveston, Texas 77555-0641 and the
Genetics Institute,
Cambridge, Massachusetts 02140
 |
ABSTRACT |
We have probed an epitope sequence
(His18-Pro19-Lys20-Phe21)
in interleukin-8 (IL-8) by site-directed mutagenesis. This work shows that single and double Ala substitutions of His18 and
Phe21 in IL-8 reduced up to 77-fold the binding affinity to
IL-8 receptor subtypes A (CXCR1) and B (CXCR2) and to the Duffy
antigen. These Ala mutants triggered neutrophil degranulation and
induced calcium responses mediated by CXCR1 and CXCR2. Single Asp or
Ser substitutions, H18D, F21D, F21S, and double substitutions,
H18A/F21D, H18A/F21S, and H18D/F21D, reduced up to 431-fold the binding
affinity to CXCR1, CXCR2, and the Duffy antigen. Interestingly, double
mutants with charged residue substitutions failed to trigger
degranulation or to induce wild-type calcium responses mediated by
CXCR1. Except for the H18A and F21A mutants, all other IL-8 mutants
failed to induce superoxide production in neutrophils. This study
demonstrates that IL-8 recognizes and activates CXCR1, CXCR2, and the
Duffy antigen by distinct mechanisms.
 |
INTRODUCTION |
Interleukin-8 (IL-8)1 is
a chemokine secreted in response to injury and infection that
selectively attracts and activates neutrophils. IL-8 is a 72-79-amino
acid peptide that belongs to the CXC chemokine subfamily because its
first two Cys are separated by a single residue. The NMR and crystal
structures of IL-8 reveal that the polypeptide chain is folded into
three antiparallel
-sheets and a C terminus
-helix (1, 2). IL-8
receptors belong to the superfamily of seven transmembrane G
protein-coupled receptors. Several IL-8 receptor subtypes have been
identified. Two structurally homologous receptors, CXCR1 and CXCR2, are
expressed in neutrophils (3-5). CXCR1 selectively binds IL-8, whereas
CXCR2 binds IL-8 and the structurally related CXC chemokines Neutrophil
Activating Protein-2 (NAP-2) and MGSA (6-8). The Duffy antigen of red
blood cells is a promiscuous chemokine receptor that binds several
chemokines including IL-8 (9, 10). Moreover, G protein-coupled
receptors encoded by the Kaposi's sarcoma-associated herpesvirus bind
IL-8 plus other chemokines (11). The mechanisms of recognition and activation of IL-8 receptor subtypes are not well defined.
Studies with IL-8 mutants created by site-directed mutagenesis and
synthetic peptides have demonstrated that the N-terminal triad, ELR, is
a major determinant for binding affinity and activation of IL-8
receptors in neutrophils (12, 13). Further studies with chemokine
chimeras have shown that the region in between Cys7-Cys50 is also important for binding to the
IL-8 receptors (14, 15). In particular, this region contains a
surface-exposed hydrophobic pocket formed by Phe17,
Phe21, Ile22, and Leu43 which is
separated by over 20 Å from the N-terminal ELR triad, and it has been
suggested that residues in or adjacent to the hydrophobic pocket are
major determinants for binding selectivity to both CXCR1 and CXCR2 (16,
17). This hydrophobic pocket may be essential for recognition of CXCR1.
In this work, we have created IL-8 mutants in which residues
corresponding to the epitope of blocking anti-IL-8 mAbs were
substituted by Ala, polar, or charged residues. We determined that this
epitope plays a major role in the differential recognition mechanisms
of CXCR1, CXCR2, and the Duffy antigen. But most importantly, this
epitope also plays a role in the activation mechanism for CXCR1.
 |
EXPERIMENTAL PROCEDURES |
Preparation of Human Neutrophils, Red Blood Cells, and Rabbit
Neutrophil Membranes--
Human blood was drawn from healthy human
donors and layered in a Mono-Poly Resolving Medium (ICN Biochemical,
Inc., Aurora, OH). Neutrophils and red blood cells were isolated in
accordance with the instructions of the manufacturer. Neutrophils were
suspended in physiological buffer containing 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM
Na2HPO4, 5 mM glucose, 20 mM HEPES (pH 7.4), and 1 mg/ml bovine serum albumin. Red
blood cells were stored in Alsevers solution consisting of 114 mM dextrose, 27 mM sodium citrate, 71 mM NaCl (pH 6.1) at 4 °C. Rabbit neutrophil membranes were prepared as described (18).
Expression of CXCR1 and CXCR2--
CXCR1 was subcloned into the
retrovirus vector MSX, and virus stocks were produced from amphotropic
packaging cell lines (19). HL-60 cells were infected with these virus
stocks and selected by limiting dilution and binding to
125I-IL-8. Cell line HL-60 was selected for this study
because it expresses a high density of CXCR1 (19) but undetectable
levels of CXCR2. HL-60 cells expressing recombinant CXCR2 were kindly provided by Dr. Richard Ye (University of Illinois, Chicago).
Protein Expression and Purification--
IL-8 mutants were
created by site-directed mutagenesis as described previously (20). The
mutant constructs were subcloned into the thioredoxin-based vector
GM-TRX for expression of thioredoxin-IL-8 mutant fusion proteins.
Escherichia. coli GI724 transformed with mutant constructs
was induced with 100 µg/ml tryptophan for 4 h at 30 °C. The
cells were lysed by French press, and the fusion protein was purified
by fractionation on a QAE anion-exchange column followed by a G-75 gel
filtration column. Fractions enriched with thioredoxin fusion proteins
were digested with enterokinase to release the IL-8 mutant protein. The
digestion products were further purified by using an SP-650
cation-exchange column.
125I-IL-8 Binding Assays--
IL-8 was iodinated by
the chloramine-T procedure as described (3). Rabbit neutrophil
membranes were suspended at a concentration of 200 µg/ml in binding
buffer (phosphate-buffered saline containing 0.1% (w/v) bovine serum
albumin and 20 mM HEPES (pH 7.4)) and incubated for 30 min
at room temperature in the presence of 2 nM
125I-labeled IL-8 and several concentrations of unlabeled
IL-8 or IL-8 mutants. The binding reaction was terminated as described (3).
Red blood cells, or HL-60 cells expressing CXCR1 or CXCR2 were
suspended at a concentration of 2.5 × 107 cells/ml or
1 × 107 cells/ml in binding buffer containing 2 nM 125I-labeled IL-8 and several concentrations
of unlabeled IL-8 or IL-8 mutants and then incubated for 2 h on
ice. The reaction was terminated by overlaying the incubation mixture
on top of a 10% (w/v) sucrose solution followed by centrifugation.
Radioactivity in the pellet was measured in a
-counter. The
Ki for each IL-8 mutant was calculated according to
the equation derived by Cheng and Prusoff (21): Ki = IC50/(1 + [IL-8]/Kd), where
IC50 is the concentration of mutant that produces 50%
inhibition on IL-8 binding to the receptor subtype, [IL-8] is the
concentration of radiolabeled IL-8, and Kd is the
dissociation constant of IL-8 binding to the receptor subtype.
Intracellular [Ca2+] Measurements--
Neutrophils
were suspended in physiological buffer at a density of 1 × 107 cells/ml and loaded with 5 µM Indo-1/AM
(Molecular Probes, OR) for 30 min at room temperature in the dark.
Neutrophils were subsequently washed once with ice-cold
phosphate-buffered saline and then resuspended in physiological buffer
at 1 × 107 cells/ml. HL-60 cells expressing CXCR1 or
CXCR2 were suspended in physiological buffer at a density of 1 × 107 cells/ml and loaded with Indo-1/AM for 30 min at
37 °C in the dark. HL-60 cells were subsequently washed once with
ice-cold physiological buffer and resuspended in physiological buffer
containing 1 mM probenecid at 1 × 107
cells/ml. Neutrophils and HL-60 cells were placed in a continuously stirred cuvette maintained at 37 °C in a 650-10S spectrofluorometer (Perkin Elmer) and stimulated with IL-8 or IL-8 mutants. Fluorescence intensity was measured using an excitation wavelength of 330 nm and an
emission wavelength of 405 nm (22).
-Glucuronidase Assay--
Human neutrophils (1 × 107 cells/ml) were incubated in a Hanks' balanced salt
solution supplemented with 1% (w/v) bovine serum albumin, 2 mg/ml
glucose, 4.2 mM NaHCO3 and 10 mM
HEPES (pH 7.2) in the presence of IL-8 or IL-8 mutants.
-Glucuronidase assay was carried out according to the fluorometric
assay described by Bary et al. (23).
Superoxide Production Assay--
Superoxide release was
monitored by the continuous fluorometric measurement of
2,2'-dihydroxybiphenyl-5,5'-diacetate produced from
p-hydroxyphenylacetate by the enzymatic reduction of
H2O2 by horseradish peroxidase (HRP) (24, 25).
Neutrophils were suspended in physiological buffer containing 130 mM NaCl, 4.6 mM KCl, 1.1 mM
KH2PO4, 1 mM CaCl2, 5 mM glucose, and 20 mM HEPES (pH 7.4) at 1 × 107 cells/ml. Neutrophils were placed in physiological
buffer containing 1 mM p-hydroxyphenylacetate,
20 units of horseradish peroxidase and 100 µM sodium
azide in a continuously stirred cuvette maintained at 37 °C in a
spectrofluorometer. Neutrophils were stimulated with IL-8 or IL-8
mutants and fluorescence intensity was measured using an excitation
wavelength of 334 nm and an emission wavelength of 425 nm.
 |
RESULTS |
Mutations of the IL-8 Epitope Region and Receptor Recognition
Previous studies (20) have shown that the epitope consensus
sequence of the blocking anti-IL-8 mAb is H-X-K-F and corresponds to
residues
His18-Pro19-Lys20-Phe21
in IL-8. These residues are in or adjacent to the surface-exposed hydrophobic pocket in IL-8 which has been previously implicated in the
recognition mechanisms to the IL-8 receptors (16, 17). Moreover,
binding of IL-8 to the N-terminal fragment of CXCR1 causes
perturbations in the NMR chemical shifts of His18 and
Phe21 (26), suggesting that these residues bind to the N
terminus domain of the IL-8 receptors. Single and double IL-8 mutants
were created by substitution of His18 and Phe21
by Ala, Ser, or Asp. All these mutants exhibited reduced binding to the
anti-IL-8 mAb (20). In this study we expressed recombinant IL-8 mutants
as fusion proteins linked to thioredoxin. IL-8 mutants were purified to
near homogeneity. Fig. 1 shows the purity
of the IL-8 mutants, H18D and H18A/F21A, as determined by
SDS-polyacrylamide gel electrophoresis. To examine whether
His18 and Phe21 residues play a role in the
mechanisms of recognition and activation of the IL-8 receptors, we
initially performed displacement of 125I-IL-8 bound to
neutrophil membranes by IL-8 mutants. In Table I we showed that H18A and F21A mutants
exhibited 4- and 3-fold increases in the Ki,
respectively, as compared with that of the wild type IL-8, suggesting a
moderate reduction in binding affinity of the mutants toward IL-8
receptors. The substitution of His18 for Asp, or
Phe21 for Ser or Asp resulted in larger increases in the
Ki (up to 40-fold) (Table I). To determine whether
these two residues interact independently with the receptors or
overlap, we examined the effect of double substitution mutants. As
shown in Table I, mutants with double substitutions exhibited larger
increases in the Ki, up to 636-fold, than that of
the wild type IL-8. Since these binding experiments were carried out
with neutrophil membranes co-expressing both CXCR1 and CXCR2, we
performed similar binding experiments with cells expressing either
CXCR1 or CXCR2 to determine whether the IL-8 mutants exhibit
receptor-subtype selectivity. Interestingly, similar to the binding
with neutrophil membranes, large increases in the
Ki, up to 431-fold, were observed upon binding of
IL-8 mutants to cells expressing CXCR1 (Table I). In contrast, a
clearly distinct binding profile with less dramatic increases in the
Ki, up to 61-fold, were observed upon binding of
IL-8 mutants to cells expressing CXCR2 (Table I). Further studies were
focused on determining the binding profile of these IL-8 mutants to
other IL-8 receptors. We selected to test the binding of IL-8 mutants
to the red blood cells promiscuous chemokine receptor, the Duffy
antigen, that binds IL-8, MGSA, Regulated on Activation, Normal T cell
Expressed and Secreted (RANTES), and Monocyte Chemotactic Protein-1
(MCP-1) (9, 10). Binding of IL-8 to red blood cells showed a
Ki = 13.2 ± 0.4 nM (Table I) that
was similar to the previously reported Kd (10). We
found that single and double substitution IL-8 mutants showed up to
241-fold increases in the Ki (Table I) and a
distinct binding profile from those exhibited against CXCR1 and CXCR2.
These findings support the idea that IL-8 recognizes CXCR1, CXCR2, and
the Duffy antigen by distinct mechanisms.

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Fig. 1.
Purity of IL-8 mutants. SDS-PAGE and
Coomassie Blue staining of purified IL-8 mutants. Lane 1 corresponds to molecular weight standards. Lane 2 is 5 µg
of mutant H18D. Lane 3 is 10 µg of mutant H18AF21A.
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Table I
Ki values of binding of IL-8 mutants to IL-8 receptors
Ki values were estimated from displacement of
125I-IL-8 bound to neutrophil membranes, red blood cells, and
HL-60 cells transfected with cDNAs encoding CXCR1 or CXCR2 by
unlabeled IL-8 or IL-8 mutants. Ki values are
expressed as nM ± S.E. of triplicate determinations. The
Ki values were determined according to the equation
(Ki = IC50/(1 + [IL-8]/Kd)), using the following
Kd values of binding of IL-8 to neutrophils, HL-60
cells expressing CXCR1 or CXCR2, or red blood cells, 1.4, 0.265, 0.6, or 15 nM, respectively.
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Activation of IL-8 Receptors
Activation of IL-8 receptors were evaluated by measuring the
agonist-dependent rise in intracellular calcium in
neutrophils and HL-60 cells expressing either CXCR1 or CXCR2. All
single and double mutants triggered maximal calcium responses in
neutrophils (data not shown), suggesting that the activation mechanisms
on the IL-8 mutants are preserved. However, neutrophils co-express both
CXCR1 and CXCR2, and it is possible that some mutants selectively activate either CXCR1, CXCR2, or both. Mobilization of intracellular calcium were examined with HL-60 cells expressing either CXCR1 or
CXCR2. We found that mutants with single substitutions, and the double
mutant H18A/F21A, exhibited calcium responses in a dose-dependent fashion (Figs.
2 and 3).
Maximal calcium responses were achieved at concentrations of IL-8
mutants near their Ki, indicating that full receptor
occupancy is not necessary for maximal calcium response as previously
observed with the fMLP chemoattractant receptor (27). Furthermore,
because these mutants elicited maximal calcium responses as those of
the wild type IL-8, it is likely that these mutant sites are major
determinants for the binding affinity to the receptors but not for the
activation mechanisms of the receptors. On the other hand, double
mutants containing either polar and charged or two charged residues
showed a unique activation profile. For example, the mutant H18A/F21D
triggered a calcium response in HL-60 cells expressing CXCR2 in a
dose-dependent fashion and a maximal calcium response as
IL-8 did (Figs. 3 and 4B). In
contrast, this mutant elicited a weak calcium response in HL-60 cells
expressing CXCR1 (Figs. 2 and 4A). This observation indicates that double mutants containing one or two charged residues are full agonists of CXCR2 but are partial agonists of CXCR1. These
data suggest that IL-8 activates CXCR1 and CXCR2 by distinct mechanisms.

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Fig. 2.
Dose-dependent curves for calcium
responses of HL-60 cells expressing CXCR1. HL-60 cells expressing
CXCR1 loaded with Indo-1 were stimulated with several concentrations of
IL-8 or IL-8 mutants. IL-8 ( ), H18A ( ), H18D ( ), F21A ( ),
F21D ( ), H18A/F21A ( ), and H18A/F21D ( ) were added at the
indicated concentrations for activation of CXCR1. The percentages of
intracellular calcium responses stimulated by 20 nM IL-8 is
referred as 100%. Values are means of triplicate determinations, and
the bars of each point represent the standard errors.
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Fig. 3.
Dose-dependent curves for calcium
responses of HL-60 cells expressing CXCR2. HL-60 cells expressing
CXCR2 loaded with Indo-1 were stimulated with several concentrations of
IL-8 or IL-8 mutants. IL-8 ( ), H18A ( ), H18D ( ), F21A ( ),
F21D ( ), H18A/F21A ( ), and H18A/F21D ( ) were added at the
indicated concentrations for activation of CXCR2. The percentages of
intracellular calcium responses stimulated by 20 nM IL-8 is
referred as 100%. Values are means of triplicate determinations, and
the bars of each point represent the standard errors.
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Fig. 4.
H18A/F21D is a full agonist of CXCR2 but a
partial agonist of CXCR1. HL-60 cells expressing CXCR1
(A) or CXCR2 (B) were loaded with Indo-1. IL-8
(20 nM) was added to HL-60 cells expressing CXCR1 or CXCR2
to induce maximal calcium responses. A, H18A/F21D (2 µM) was added to HL-60 cells expressing CXCR1.
B, H18A/F21D (500 nM) was added to HL-60 cells
expressing CXCR2. These are representative records of three independent
determinations.
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Neutrophil Responses
Release of
-Glucuronidase--
Except for double mutants
H18A/F21D and H18D/F21D, all single and double mutants triggered
release of
-glucuronidase in neutrophils (Fig.
5). This finding suggests that the rise
in intracellular calcium mediated by binding of H18A/F21D or H18D/F21D
to CXCR2 is not sufficient to elicit neutrophil degranulation.

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Fig. 5.
IL-8 mutants released
-glucuronidase. Human neutrophils were treated
with several concentrations of IL-8 or IL-8 mutants, and degranulation
of -glucuronidase was assayed as described under "Experimental
Procedures." IL-8 ( ), H18A ( ), H18D ( ), F21A ( ), F21D
( ), H18A/F21A ( ), H18A/F21D ( ), H18A/F21S ( ), and H18D/F21D
( ) were added at the indicated concentrations. The percentages of
-glucuronidase release in the presence of 1 µM IL-8 is
referred as 100%. Values are means of triplicate determinations, and
the bars of each point represent the standard errors.
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Superoxide Production--
Previous studies have indicated that
MGSA, in contrast to IL-8, is a poor activator of superoxide production
in neutrophils (28). We have tested the effect of single and double
mutants on superoxide production. IL-8 mutants at concentrations
ranging from 100 nM to 2 µM failed to trigger
superoxide production. The H18A and F21A mutants were only partially
active (Fig. 6). These data indicate that
activation of calcium responses mediated by both IL-8 receptor subtypes
in neutrophils is not sufficient to trigger superoxide production.

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Fig. 6.
IL-8 mutants are poor activators of
superoxide production. Time course of IL-8 or IL-8 mutant-induced
superoxide production, assayed by monitoring the fluorescence intensity
of stable fluorescent product 2,2'-dihydroxybiphenyl-5,5'-acetate as
described under "Experimental Procedures." A, IL-8 (1),
H18A (2), F21A (3), and H18A/F21A (4) were added to neutrophils at a
final concentration of 100 nM. B, IL-8 (1), H18D
(2), F21D (3), F21S (4), H18A/F21D (5), H18A/F21S (6), and H18D/F21D
(7) were added to neutrophils at a final concentration of 100 nM. Arrows indicate the addition of IL-8 or the
IL-8 mutant. The horizontal bar is the 1-min interval. The
vertical bar corresponds to the change of fluorescence
elicited by 50 nM H2O2. These are
representative records of three independent determinations.
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 |
DISCUSSION |
The results of this work have indicated that His18 and
Phe21 are involved in the mechanisms of recognition to
anti-IL-8 mAb (20), CXCR1 and CXCR2, and the Duffy antigen of red blood
cells. The data show that IL-8 recognizes CXCR1, CXCR2, and the Duffy
antigen by different mechanisms. Furthermore, despite the high degree of sequence homology of CXCR1 and CXCR2, they appear to be
differentially activated by IL-8. This is consistent with our previous
work indicating that IL-8 binding to IL-8 receptor subtypes causes a
higher rate of internalization of CXCR2 than that of CXCR1 (29),
suggesting that agonist binding to IL-8 receptor subtypes trigger
different internalization signals. Finally, this work shows that the
transient rise of intracellular calcium is not sufficient to trigger
neutrophil responses including release of
-glucuronidase and
superoxide production.
His18 and Phe21 are adjacent or are in the
surface-exposed hydrophobic pocket that has been argued to drive the
association of IL-8 to the receptor by hydrophobic interactions (17).
In our present work, substitution of His18 and
Phe21 for Ala caused modest changes in binding affinity,
calcium responses, and release of
-glucuronidase. These observations
indicate that the hydrophobic nature of these residues does not play a
major role in the mechanisms of recognition and activation of the IL-8 receptors. Although, these single Ala mutants are poor activators of
superoxide production, suggesting that these mutants are not full
agonists in terms of production of superoxide. Single or double
substitutions of His18 and Phe21 by polar or
charged residues produce major changes in binding affinity to CXCR1 and
the Duffy antigen. In contrast, minor changes were observed with CXCR2.
These findings support the idea that this epitope plays a role in
determining binding selectivity among IL-8 receptors.
Substitution of His18 and Phe21 by Ala, polar,
or charged residues may lead to localized, long range, or gross
structural changes. In our studies, the mutants do not appear to show
gross structural changes because all the mutants trigger full calcium
responses in neutrophils, and they exhibit different binding affinities toward CXCR1, CXCR2, and the Duffy antigen. In addition, NMR chemical shifts of the mutant F21A are indistinguishable from the wild type,
suggesting that this mutation causes a localized change (17).
His18 and Phe21 are in close proximity to each
other on the protein (Fig. 7). Whether
these two residues overlap or interact independently with the IL-8
receptor subtypes remain to be established.

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Fig. 7.
Ribbon representation of IL-8. Residues
His18 and Phe21 side chains are shown in
yellow. The IL-8 structure is formed by three antiparallel
-sheets located below the C terminus -helix.
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In summary, our binding data generated with this set of IL-8 mutants
strongly argue that distinct mechanisms operate in the recognition of
IL-8 to CXCR1, CXCR2, and the Duffy antigen. Binding of IL-8 to the N
terminus fragment of CXCR1 causes perturbations in the NMR chemical
shifts of His18 and Phe21 (26). This finding
plus our binding data suggest that the selective recognition of IL-8
receptor subtypes by IL-8 is mediated by the interaction of
His18 and Phe21 with the variable N terminus
domains of IL-8 receptors (6).
On the basis of the patterns of calcium responses elicited with this
set of IL-8 mutants, we can distinguish at least two sets of mutants.
One set composed of H18A, F21A, H18D, F21D, F21S, H18A/F21A, and
H18A/F21S exhibit wild-type calcium responses and release of
-glucuronidase. The other set composed of H18A/F21D and H18D/F21D
are full agonists of CXCR2 but are partial agonists of CXCR1. This
finding suggests that the chemical nature of both residues 18 and 21 in
IL-8 is a major determinant for the calcium responses mediated by CXCR1
but not for the calcium responses mediated by CXCR2. It is likely that
IL-8 contains distinct motifs for activation of IL-8 receptor subtypes.
Interestingly, H18A and F21A release
-glucuronidase as the wild type
IL-8 but are poor activators of superoxide production in neutrophils.
This finding supports the idea that the single Ala mutants are partial agonists in terms of superoxide production. This study provides the
framework to further elucidate the activation motifs in IL-8 that
trigger the biological responses in neutrophils including superoxide
production, phagocytosis, and degranulation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. John McCoy for helpful
discussions, Mei-Yu Xu for technical assistance, and Dr. Bruce Luxon
for the construction of the ribbon representation of IL-8.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant AI34031.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.
§
To whom correspondence should be addressed. Tel.: 409-772-5480;
Fax: 409-772-3381; E-mail: jnavarro{at}utmb.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
IL-8, interleukin-8;
MGSA, melanoma growth stimulating activity;
mAb, monoclonal
antibody.
 |
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