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Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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40% of cytosolic protein). Previous work from our
laboratory has demonstrated the extracellular association of these
proteins with vascular endothelium adjacent to transmigrating
leukocytes. We report here a function for MRP-14 as a stimulator of
neutrophil adhesion mediated by the ß2 integrin,
Mac-1. MRP-14 is an affinity regulator of Mac-1 because it promotes
binding of soluble ligand and expression of an "activation
reporter" epitope of high affinity ß2 integrins
recognized by mAb24. The activity of MRP-14 is confined to regulating
integrin function because, unlike other inflammatory agonists, there
was no release of L-selectin, up-regulation of cytosolic Mac-1, or
induction of neutrophil respiratory burst or calcium flux. Furthermore,
MRP-14 does not act as a chemoattractant or cause alterations in cell
shape or cytoskeleton. MRP-8 has a regulatory role in MRP-14 activity,
inhibiting the adhesion induced by MRP-14 through the formation of the
heterodimer. In terms of mechanism of action, MRP-14 does not increase
Mac-1 function by direct binding to this integrin but recognizes a
distinct receptor on neutrophils. This receptor interaction is
pertussis toxin sensitive, indicating that MRP-14-generated signals
leading to a Mac-1 affinity increase are heterotrimeric G protein
dependent. We postulate that MRP-14 and MRP-8 are important in vivo
candidates for the regulated adhesion of neutrophils through control of
Mac-1 activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In human myeloid cells, myeloid-related protein (MRP)3-8 (S100A8) and MRP-14 (S100A9) are coexpressed as a heterodimer, and, in neutrophils, this heterodimer represents 40% of the cytosolic protein (7). Specific mAbs detect these MRPs deposited on endothelia at positions where leukocytes migrate into tissues, suggesting a role in leukocyte trafficking (8). Recently several members of the S100 protein family have been defined as chemoattractants. S100L (S100A2), isolated from bovine lung, stimulated guinea pig eosinophil chemotaxis but exhibited no activity toward neutrophils or monocytes (9). Similarly, psoriasin (S100A7) is highly up-regulated in psoriatic skin and has chemotactic activity for T cells and neutrophils but not for monocytes (10). Most information is available for CP-10, a murine homologue of the human MRP-8 protein (S100A8), which is chemotactic for neutrophils and macrophages and the most potent chemoattractant to date, with optimal activity at 10-13 M (11). In this study we have examined the function of human MRP-14 and found it to be a selective stimulator of Mac-1-mediated adhesion through affinity regulation. This is the first demonstration on neutrophils of the direct activation of Mac-1 by a physiologic protein. MRP-14 activity is distinct from the other S100 family members and classic chemoattractants in promoting Mac-1 adhesion without stimulating chemotaxis or further neutrophil activation. In addition, MRP-8 is identified as a natural and specific inhibitor of MRP-14 function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human MRP-14 and MRP-8 proteins (12) were prepared from pET3a expression vectors containing the cDNAs for these proteins constructed by Dr Paul Hessian in the Leukocyte Adhesion Laboratory (London, U.K.). The proteins were purified to homogeneity by a combination of chromatofocusing on a Mono-P column for MRP-14 (Pharmacia Biotech, St Albans, U.K.) and using the Rotofor system for MRP-8 (Bio-Rad, Hemel Hempstead, U.K.), followed by hydroxyapatite chromatography for both proteins (Bio-Rad) (A. Coffer, unpublished observations). All purification steps were performed in the Imperial Cancer Research Fund Protein Isolation and Cloning Laboratory (London, U.K.). The purified proteins were stored at 2 mg/ml in HBSS containing 10 mM HEPES (H-HBSS), pH 7.5, at -70°C or over the short term at 4°C. MRP-8 was expressed in dimer form and was reduced and alkylated before use as a monomer (13). Recombinant S100A and S100B (Sigma, Poole, U.K.) were reconstituted with water to 2 mg/ml.
Purification of MRP-8/14 heterodimer
MRP-8/14 heterodimer was purified from neutrophil cytosol by fast performance liquid chromatography using a Mono Q anion exchange column as previously described (7).
K562 transfectant cell culture
Mac-1-transfected (KC/16) and untransfected K562 erythroleukemic cells were maintained at 37°C and 5% CO2 in DMEM supplemented with 10% FCS (14). Geneticin G418 (0.5 mg/ml; Life Technologies, Paisley, U.K.) was added to Mac-1-transfected cell medium to maintain selection.
Monoclonal Abs
The following IgG1 mAbs were used in this study: LAM 1.3 (5
µg/ml), specific for L-selectin (CD62L), was a gift from Dr. Tom
Tedder (Boston, MA); ICRF44 (10 µg/ml) is specific for the Mac-1 
subunit (CD11b); LPM19c (20 µg/ml of purified mAb or 1/50 ascitic
fluid), also specific for the Mac-1 subunit (CD11b), was a gift
from Dr. Karen Pulford (Oxford, U.K.); mAb24 (10 µg/ml) is a reporter
of ß2 integrin activation; 3.9 (10 µg/ml) is
specific for the p150,95 subunit (CD11c); 27E10 (1/50), purchased
from BMA Biomedicals AG, Switzerland, is specific for the MRP-8/14
heterodimer; and 52U (10 µg/ml) is an IgG1 isotype control.
FITC labeling of proteins
FITC labeling of MRP-14 and fibrinogen was conducted following the method of Goding (15). Unbound FITC was removed from the FITC-conjugated protein by gel filtration in either H-HBSS (MRP-14) or PBS (fibrinogen) using a pre-equilibrated PD-10 column (Pharmacia Biotech).
Neutrophil adhesion assay
Human neutrophils were isolated from EDTA-anticoagulated whole blood from healthy volunteers by dextran sedimentation and density gradient centrifugation (16). Purified neutrophils (>95%) were washed and resuspended at 5 x 106 cells/ml in H-HBSS. Cells were labeled with 1 µM of the intracellular fluorescent dye 2', 7'-bis(carboxyethyl)-5(6')-carboxyfluorescein pentaacetoxymethyl ester (BCECF/AM; Calbiochem, Nottingham, U.K.) for 30 min at room temperature. After washing, labeled cells were resuspended in H-HBSS at 2 x 106/ml, and 50 µl were added to Nunc-Immuno Maxisorp plate wells (Life Technologies) that had been coated overnight with 2 mg/ml fibrinogen (50 µl/well; Sigma). These wells contained an equal volume of H-HBSS with 2x cations (2 mM CaCl2, 2 mM MgSO4, 20 µM ZnSO4) together with stimulating or blocking reagents. The plate was allowed to incubate at room temperature for 30 min, and the resultant adherent cells (after washing) were quantitated using a 96-well plate fluorescence reader (Fluoroskan II; Labsystems, Basingstoke, U.K.). Percentages of adherent cells were determined using fluorescence values of 50 µl of total cells added per well. For the pertussis toxin inhibition experiments, cells were preincubated with various concentrations of Bordetella pertussis toxin (Sigma) for 30 min at room temperature.
Flow cytometry
Purified neutrophils (5 x 105/sample) were incubated at room temperature in H-HBSS containing 1 mM Ca2+, 1 mM Mg2+ and 10 µM Zn2+ in the presence or absence of MRP-14 or FMLP together with primary mAbs at concentrations stated. After 30 min the cells were washed with ice cold PBS/0.2% BSA/0.1% azide (FACSwash) and resuspended in FITC-conjugated goat anti-mouse IgG (1/200; Sigma). After 30 min on ice, the cells were washed and resuspended in FACSwash. The fluorescence intensity was then determined using a FACScan flow cytometer (Becton Dickinson). In experiments measuring soluble FITC-fibrinogen binding, the FITC-fibrinogen replaced the primary Ab, and, after 30 min at room temperature, the cells were washed and maintained in ice until analysis. To test for cation dependence, cells were incubated with FITC-fibrinogen and MRP-14 in H-HBSS without Ca2+, Mg2+, and Zn2+. In experiments measuring FITC-MRP-14 binding, neutrophils or K562 cells (1 x 105/sample) were incubated on ice in H-HBSS containing 1 mM Ca2+, 1 mM Mg2+, 10 µM Zn2+, and 0.1% BSA (incubation buffer) with varying amounts of FITC-MRP-14. After 30 min, the cells were washed and resuspended in ice cold incubation buffer. To test for specific binding, increasing amounts of unlabeled MRP-14 and a control S100 protein (S100A) were incubated, as above, with 2 µM FITC-MRP-14. FITC-phalloidin was used to measure filamentous actin (F-actin) according to the method of Cornish et al. (17), with the exception that cells were incubated in H-HBSS containing 1 mM Ca2+, 1 mM Mg2+, and 10 µM Zn2+.
Confocal microscopy
For immunofluorescence analysis of F-actin staining, 13 mm glass coverslips were precoated with 2 mg/ml fibrinogen overnight at 4°C and then washed in H-HBSS. Buffer conditions were as for the 96-well plate adhesion assay. Unlabeled neutrophils (4 x 105) were added to each coverslip in the presence or absence of stimulants in a total volume of 400 µl. Coverslips were spun at 40g and then incubated for 30 min at room temperature. Nonadherent cells were removed by gentle washing in H-HBSS. Cells were fixed, permeabilized, and stained for 30 min on ice by the addition of H-HBSS/1% formaldehyde/0.2% Triton X-100/0.25 µg/ml FITC-phalloidin. Coverslips were then washed and mounted on slides. Confocal microscopy was performed using a Leica TCS NT microscope equipped with a 60x oil immersion objective (Leica, Wetzlar, Germany).
ELISA
Nunc-Immuno Maxisorp 96-well plates (Life Technologies) were incubated overnight at 4°C with 2 µM of MRP-14 or MRP-8 alone, or together in H-HBSS/2 mM Ca2+/2 mM Mg2+/20 µM Zn2+ (50 µl/well). Following incubation, the liquid was aspirated and the plate was blocked with 150 µl/well of PBS/0.1% Tween-20 for 1 h at room temperature. The plate was washed and incubated sequentially (including washes) with 50 µl/well of the MRP-8/14 complex-specific mAb 27E10 (18) (1/50 in PBS/0.1% Tween-20) for 1 h at room temperature, followed by 30 min at room temperature with peroxidase-conjugated goat anti-mouse Ig (1/2000; DAKO, High Wycombe, U.K.) in PBS/Tween. Bound Ig was detected using O-phenylenediamine dihydrochloride (OPD; Sigma) according to the manufacturer’s instructions.
Superoxide production
The production of superoxide was measured according to the method of Smith and Weidemann (19).
Measurement of intracellular calcium ([Ca2+]i)
[Ca2+]i was measured using fura
2-AM according to the method of Tsien et al. (20). Stimulants were
added at time points indicated on Figure 5
. Fluorescence was monitored
using a Perkin-Elmer LS-5 luminescence spectrophotometer (Perkin-Elmer,
Beaconsfield, U.K.).
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Dilutions of MRP-14 from 0 to 0.5 µM in H-HBSS containing 1 mM Ca2+, 1 mM Mg2+, 10 µM Zn2+, and 0.1% BSA (assay medium) were added to Costar 24-well tissue culture plates (Life Technologies) in a final volume of 600 µl in quadruplicate. FMLP, at 0.1 µM in the lower wells, was used as a positive control. Costar Transwells (6.5 mm diameter; 3 µM pore size; Life Technologies) were precoated on both sides with 50 µg/ml fibrinogen (Sigma) overnight at 4°C. Either 100 µl of BCECF/AM-labeled neutrophils (5 x 106/ml) in assay medium alone or containing concentrations of MRP-14 from 0 to 0.5 µM were added to the top chamber. Cells were allowed to transmigrate for 1 h at 37°C. After this time, the migrated cells were detached using 5 mM EDTA and counted using a flow cytometer (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we have identified the S100 protein MRP-14 as a new
candidate for activation of ß2 integrin-mediated
adhesion. Our results show that MRP-14 is directly able to stimulate
neutrophil adhesion to fibrinogen (Fig. 1). Adhesion was maximal between 0.5 to 1
µM and could be completely inhibited by CD11b mAb LPM19c, confirming
that adhesion was mediated by the ß2 integrin Mac-1
(CD11b/CD18) (Fig. 1, inset). This stimulation of
adhesion is unique to MRP-14 because the other S100 proteins tested,
including MRP-8 (the heterodimeric partner of MRP-14), S100A (Fig. 1),
and S100B (data not shown) failed to induce any adhesion. Furthermore,
the native MRP-8/14 complex was unable to induce any adhesion (Fig. 1).
The response to MRP-14 was equivalent to that of FMLP at saturating
levels of each mediator (2 µM MRP-14 and 0.1 µM FMLP) (Fig. 1, inset, and Fig. 2). Several
S100 proteins, including MRP-14, have been shown to bind
Zn2+ and, in so doing, increase their affinity for
Ca2+, presumably by a conformational change (21, 22). Our
results can be similarly interpreted because the addition of 10 µM
Zn2+ increases the potency of MRP-14 as an inducer of
neutrophil adhesion by approximately 10-fold (data not shown).
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). Titration of MRP-8 from 0.125 to
10 µM caused a dose-dependent inhibition of 2 µM MRP-14-induced
adhesion (Fig. 2), with the 50% point occurring at an average ratio of
1:1 MRP-14 to MRP-8 (n = 6). MRP-8, in the same
concentration range, had no effect on 0.1 µM FMLP-induced adhesion
(Fig. 2). To eliminate the possibility that MRP-8 inhibition occurred
by Ca2+ depletion, another S100 protein, S100A, was
similarly titrated and had no inhibitory effect on MRP-14-induced
adhesion (Fig. 2). The specificity of MRP-8 inhibition was confirmed by
the expression of the MRP-8/14 complex-specific epitope, recognized by
mAb 27E10 (18) upon coincubation of MRP-8 with MRP-14 (Fig. 2, inset). The ability of MRP-8 to inhibit
MRP-14-induced adhesion and the fact that heterodimer formation occurs
suggests that the mechanism of MRP-8 inhibition is by direct binding to
MRP-14 with a 1:1 stoichiometry between the two proteins. This
identifies MRP-8 as a physiologic antagonist of MRP-14 and confirms the
specificity of MRP-14 function. MRP-14 is a modulator of Mac-1 affinity
Increased integrin affinity is measured by the ability to bind
ligand in solution. MRP-14 induces neutrophil binding of soluble
FITC-fibrinogen with saturation at 10 nM (Fig. 3a). In contrast,
MRP-8/14 complex did not stimulate any soluble FITC-fibrinogen binding,
as predicted from the immobilized fibrinogen assays (data not shown).
The specificity of binding is shown by the ability of 10-fold excess
unlabeled fibrinogen to reduce FITC-fibrinogen binding to background
levels (Fig. 3b). Removal of divalent cations (Fig. 3a) and blocking with CD11b mAb showed the binding to
be Mac-1-dependent with no involvement of other ß2
integrins, such as p150,95, which also recognizes fibrinogen (Fig. 3c) (23). The low level of background binding of
FITC-fibrinogen to untreated neutrophils (Fig. 3a)
was also attributed to Mac-1 (data not shown).
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a). Similarly, MRP-14
was able to induce mAb24 epitope expression on the surface of
neutrophils (Fig. 4b) in a concentration-dependent
manner that mirrored the induction of adhesion (data not shown). The
expression of this epitope further confirms that ß2
integrins are in a high affinity state as a result of exposure to
MRP-14 (and FMLP).
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The stimulated neutrophil undergoes a number of cell surface and
intracellular changes. For example, engagement of certain cell surface
receptors can invoke quantitative changes in expression of at least two
adhesion molecules (26). Characteristically, for the classical
chemoattractants, there is shedding of L-selectin and an up-regulation
of Mac-1 on the neutrophil surface. As expected, FMLP used at 0.1 µM
gave the characteristic loss of L-selectin (Fig. 4c);
however, MRP-14 at 1 µM, or at concentrations up to 8 µM (data not
shown), failed to alter the levels of neutrophil L-selectin (Fig. 4d). We then investigated whether MRP-14, like FMLP
(Fig. 4e), might cause up-regulation of Mac-1 from
cytoplasmic granules. However, MRP-14 at 1 µM, or at concentrations
up to 8 µM (data not shown), failed to alter the levels of neutrophil
Mac-1 (Fig. 4f). Neutrophil stimulants also cause
exocytosis of granules additional to those containing Mac-1 with
ß-glucuronidase release from azurophilic granules frequently taken as
a measure of this activity. MRP-14 failed to induce release of
ß-glucuronidase, further supporting the inability of MRP-14 to induce
neutrophil granule exocytosis (data not shown).
In addition to changes in adhesion molecule expression, activated
neutrophils can mobilize intracellular Ca2+. FMLP gave the
characteristic response of intracellular Ca2+ flux, whereas
1 µM MRP-14 failed to induce such a Ca2+ flux even after
30 min (Fig. 5, a and
b, and data not shown). Indeed, all concentrations of MRP-14
tested, from 0.05 to 4 µM, failed to induce a Ca2+ flux
(data not shown). We then investigated the possibility that MRP-14
might activate the neutrophil respiratory burst (see (27)). FMLP at 0.1
µM (Fig. 5c), PMA, and the chemokine IL-8 (data not
shown) caused neutrophil activation of the reduced nicotinamide-adenine
dinucleotide phosphate (NADPH) oxidase as detected by production of
superoxide, but MRP-14 at 1 µM, or concentrations up to 8 µM (data
not shown), failed to stimulate any production of superoxide (Fig. 5d).
MRP-14 does not act as a chemoattractant or affect neutrophil morphology
The possibility still existed that MRP-14, despite the absence of
exocytosis, Ca2+ flux, or superoxide production, could
act as a neutrophil chemoattractant. For example, the murine MRP-8
homologue, CP-10, is reported to be chemotactic for neutrophils without
inducing a Ca2+ flux (17). The capacity of MRP-14 (up to 2
µM) to act as a potential chemoattractant was investigated using the
Transwell system. When MRP-14 was titrated from 30 nM to 0.5 µM in
either the upper or lower chambers or in both, no migration was
observed above background (Table I).
Concentrations up to 2 µM also failed to induce any migration (data
not shown). In contrast, approximately 50% of the total cells migrated
in response to FMLP (Table I). Therefore, MRP-14 did not act as a
neutrophil chemotaxin or stimulate random migration. Similarly,
experiments with MRP-8, the human equivalent of CP-10, proved negative
for chemotaxis (data not shown).
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a)
with MRP-14-treated neutrophils (Fig. 6b) shows no
difference in general cell shape, whereas FMLP causes substantial
alteration of cell shape and localization of F-actin (Fig. 6c). We next investigated changes in cell shape more
quantitatively by assessing flow cytometric cell scatter profiles (Fig. 7a) and the kinetics of
induction of F-actin (Fig. 7b). MRP-14 failed to
cause distinguishable cell shape change or formation of F-actin,
whereas FMLP, as expected, was able to promote an increase in cell
scatter (Fig. 7a) and a 1.5-fold increase in
neutrophil F-actin after 3 min of stimulation (Fig. 7b). These results confirmed that MRP-14 treatment of
neutrophils had no effect on reorganization of the cytoskeleton and
underlined the conclusion that MRP-14, in promoting Mac-1-mediated
adhesion, is more selective in its activity than chemoattractants such
as FMLP.
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To determine the means by which MRP-14 altered the function of
Mac-1, we investigated the interaction of MRP-14 with the neutrophil
membrane. MRP-14 was found to bind to a specific receptor in a
dose-dependent manner that could be blocked by unlabeled MRP-14 but not
by control protein S100A (Fig. 8a, and
inset). One possibility, given the limited activity
of MRP-14 to modulate Mac-1 affinity, was that the MRP-14 receptor was
Mac-1 itself. To investigate this possibility, we tested the binding
profiles of a Mac-1 K562 transfectant (KC/16 cells) by comparison with
the parent K562 cells (14, 29). Although the KC/16 cells abundantly
express Mac-1 (Fig. 8b, inset), there was
no difference in their ability to bind MRP-14 compared with K562 cells
(Fig. 8b). The binding to both K562 and Mac-1 K562
was prevented by cold MRP-14 but not by S100A protein (data not shown).
In addition, no evidence was obtained for coprecipitation of MRP-14 and
Mac-1 from neutrophil lysates by an anti-MRP-14-specific mAb (data
not shown). The conclusion from these experiments is that MRP-14 binds
to a receptor distinct from Mac-1. Two other S100 proteins have been
demonstrated to bind to pertussis toxin-sensitive receptors (9, 17). To
aid understanding of the function of MRP-14, we investigated the
possibility that MRP-14 also operated through such a receptor.
Neutrophils preincubated with pertussis toxin showed a
concentration-dependent decrease in the ability of MRP-14 to stimulate
adhesion (Fig. 9). The findings suggest
that MRP-14 interacts with neutrophils via a G protein-coupled receptor
and that the subsequent signaling leads to Mac-1 activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is becoming apparent that integrin-mediated adhesion can occur by
two distinct mechanisms. By one means, a combination of integrin
redistribution into clusters on the cell membrane, accompanied by
accessory events such as cell spreading, acts to increase the strength
of adherence to ligand (reviewed in 25, 34). This form of adhesion is
dependent on rearrangement of the actin cytoskeleton and is sensitive
to cytochalasin D. There are fewer examples of a second mechanism that
involves conformational alteration of integrin leading to enhanced
affinity for ligand. This increase in ligand binding affinity can be
brought about by artificial means, such as treatment with agents such
as divalent cations Mn2+ or Mg2+ and activating
mAbs (25). We provide here an example of affinity regulation of Mac-1
integrin by the naturally occurring S100 protein MRP-14, which triggers
Mac-1 on neutrophils to bind soluble fibrinogen with saturation at 10
nM FITC-fibrinogen. High affinity status of Mac-1 is further confirmed
by the ability of MRP-14 to cause expression of the mAb24 activation
reporter epitope, which also detects the Mg2+-treated Mac-1
on neutrophils (24). Therefore, MRP-14 represents one of few examples
of a physiologic protein acting selectively as a modulator of integrin
affinity. Another example of affinity regulation of Mac-1 follows from
the treatment of monocytes with ADP, which induces Mac-1-mediated
binding of soluble ligand fibrinogen (35) and Factor X (36). Also well
described is the activation of platelets with thrombin or ADP, which
causes IIbß3 to bind soluble ligand fibrinogen (reviewed in
37).
The affinity change to Mac-1 in response to MRP-14 occurs in the absence of accessory adhesion events such as neutrophil shape change, actin reorganization, or cell spreading, underlining the restricted scope of the signaling activity of MRP-14. Because intracellular Ca2+ facilitates adhesion through cell spreading for integrins such as LFA-1 (28), the fact that Mac-1 adhesion is evident in the absence of an increase in [Ca2+]i is a further indication that these accessory events are not part of the mechanism of MRP-14 action. Finally, the lack of sensitivity to cytochalasin D of MRP-14-stimulated neutrophil adhesion is additional proof that it is integrin itself that is altered and that the cytoskeleton has no role in MRP-14 function (data not shown).
These very restricted effects of MRP-14 on Mac-1 function raises the question as to its mechanism of action. One possibility is that by direct physical interaction, MRP-14 alters the conformation of Mac-1 leading to an increase in ligand-binding affinity. A precedent for this type of modification is the example of the subversion of Mac-1 function by membrane-associated urokinase-type plasminogen activator receptor (38). In this study however, we could find no evidence for such a mechanism of direct interaction with integrin. In contrast, MRP-14 binds to a distinct pertussis toxin-sensitive receptor on the neutrophil, indicating that it interacts with neutrophils via a G protein-coupled receptor and that subsequent signaling leads to Mac-1 activation. FMLP, PAF (platelet-activating factor), and the chemokines mediate their effects through pertussis toxin-sensitive 7-membrane-spanning receptors that link to heterotrimeric G proteins (reviewed in 39). Two of the chemotactic S100 proteins, CP-10 and S100L, have been shown to signal through pertussis toxin-sensitive receptors (9, 17). The limited scope of MRP-14 signaling suggests two possibilities: first, that the G proteins coupled to their receptors differ from those associated with receptors that signal a larger array of functions, or, second, that the kinetics of receptor interaction or strength of signal might dictate the number of intracellular pathways activated. A final point is that this general class of receptor has reaction kinetics that would theoretically be sufficiently rapid to account for the rate of leukocyte response observed in vivo (39, 40).
The failure of MRP-14 to cause loss of L-selectin, Mac-1 up-regulation, and Ca2+ flux on neutrophils resembles the functional profile of the murine chemotactic S100 protein CP-10 (17). In contrast, the failure of MRP-14 to induce shape change, actin reorganization, or neutrophil migration indicates that MRP-14 does not act as a chemoattractant. Furthermore, human MRP-8 is not chemotactic, unlike the murine homologue (41, and data not shown). The lack of chemotactic activity of MRP-8 and MRP-14 contrasts with CP-10 (11) and the two other chemotactic S100 proteins, S100L (9) and psoriasin (10). None of these proteins have yet been tested for their ability to directly activate integrins, but it can be concluded that functional differences exist among this subset of S100 family members, which operate within the context of an immune response.
The positive effect of MRP-14 on Mac-1 affinity regulation, considered together with its localization on vascular endothelium (8, 30), implicates a function for MRP-14 in stimulating neutrophil adhesion to endothelium either by directly capturing neutrophils from the blood stream or by securing their firm adhesion. The fact that MRP-14 does not recruit new Mac-1 to the membrane is not detrimental to function because it is the constitutively expressed Mac-1 that is responsive to adhesion-activating stimuli (42). When neutrophils are stimulated with classical chemoattractants, such as FMLP, the total expression of Mac-1 on the membrane can increase by up to 10-fold (43), but this newly arrived Mac-1 is inactive until a second round of stimulation (44). If MRP-14 does supply the initial signal for adhesion, then it may be able to influence other events of the adhesion cascade. For example, the failure to activate L-selectin shedding could have a positive effect on neutrophil accumulation at sites of leukocyte trafficking. L-selectin shedding has been considered necessary for effective neutrophil rolling and tethering on the endothelium, but prevention of L-selectin cleavage causes neutrophils to roll more slowly, bringing them into close contact with the adhesive surface (45). Furthermore, under flow conditions, adhering neutrophils will recruit further neutrophils through L-selectin-mediated adhesion between neutrophils (46). This route of enhanced binding is eliminated if L-selectin is shed through neutrophil activation. As well as directly influencing the above adhesion events, other classical chemoattractants, such as FMLP and the chemokines, serve as general neutrophil activators, inducing functions such as Ca2+ flux, the respiratory burst, and degranulation (see 47). Adhering neutrophils with an activated respiratory burst release harmful products, such as hydrogen peroxide and oxygen radicals (48), that damage endothelium and surrounding tissue (49). The restricted action of MRP-14 may be beneficial in minimizing the potential damage that neutrophils could inflict on vascular endothelium.
In summary, the human MRP-14 protein serves as a unique modulator of the affinity of the neutrophil ß2 integrin Mac-1 acting through a pertussis toxin-sensitive receptor. Further investigation is needed to identify whether MRP-14 is able to signal the activation of other integrins on neutrophils and other leukocytes. The restricted activity of MRP-14 also suggests that it might act in conjunction with chemotactic factors such that the recruited neutrophil adherent to the vascular endothelium would then be primed and ready to respond to further signals directing it toward the injured tissue. These findings emphasize the newly recognized importance of this subset of S100 proteins in leukocyte adhesion reactions and suggest that they may function in a manner distinct from those of the classical chemoattractants.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nancy Hogg, Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln’s Inn Fields, London, WC2A 3PX, United Kingdom.
3 Abbreviations used in this paper: MRP, myeloid-related protein; F-actin, filamentous actin; H-HBSS, HBSS containing 10 mM HEPES; [Ca2+]i, intracellular calcium.
Received for publication August 11, 1997. Accepted for publication October 20, 1997.
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References |
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, S100a (ß), and S100b (ßß) protein: Zn2+ regulates Ca2+ binding of S100b protein. J. Biol. Chem. 261:8192. chain of fibrinogen. Proc. Natl. Acad. Sci. USA 88:1044.
RIIA. Eur. J. Immunol. 26:207.[Medline]
; formerly gp 110), a surface glycoprotein associated with neutrophil adhesion. J. Clin. Invest. 74:1280.
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 D Foell, H Wittkowski, and J Roth Monitoring disease activity by stool analyses: from occult blood to molecular markers of intestinal inflammation and damage Gut, June 1, 2009; 58(6): 859 - 868. [Abstract] [Full Text] [PDF]
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 K. Otsuka, F. Terasaki, M. Ikemoto, S. Fujita, B. Tsukada, T. Katashima, Y. Kanzaki, K. Sohmiya, T. Kono, H. Toko, et al. Suppression of inflammation in rat autoimmune myocarditis by S100A8/A9 through modulation of the proinflammatory cytokine network Eur J Heart Fail, March 1, 2009; 11(3): 229 - 237. [Abstract] [Full Text] [PDF]
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 P L E M van Lent, L Grevers, A B Blom, A Sloetjes, J S Mort, T Vogl, W Nacken, W B van den Berg, and J Roth Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis Ann Rheum Dis, December 1, 2008; 67(12): 1750 - 1758. [Abstract] [Full Text] [PDF]
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 G. Bode, A. Luken, C. Kerkhoff, J. Roth, S. Ludwig, and W. Nacken Interaction between S100A8/A9 and Annexin A6 Is Involved in the Calcium-induced Cell Surface Exposition of S100A8/A9 J. Biol. Chem., November 14, 2008; 283(46): 31776 - 31784. [Abstract] [Full Text] [PDF]
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 J. H. Boyd, B. Kan, H. Roberts, Y. Wang, and K. R. Walley S100A8 and S100A9 Mediate Endotoxin-Induced Cardiomyocyte Dysfunction via the Receptor for Advanced Glycation End Products Circ. Res., May 23, 2008; 102(10): 1239 - 1246. [Abstract] [Full Text] [PDF]
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 M.-A. Raquil, N. Anceriz, P. Rouleau, and P. A. Tessier Blockade of Antimicrobial Proteins S100A8 and S100A9 Inhibits Phagocyte Migration to the Alveoli in Streptococcal Pneumonia J. Immunol., March 1, 2008; 180(5): 3366 - 3374. [Abstract] [Full Text] [PDF]
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 M. Frosch and J. Roth New insights in systemic juvenile idiopathic arthritis--from pathophysiology to treatment Rheumatology, February 1, 2008; 47(2): 121 - 125. [Abstract] [Full Text] [PDF]
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 D. Viemann, K. Barczyk, T. Vogl, U. Fischer, C. Sunderkotter, K. Schulze-Osthoff, and J. Roth MRP8/MRP14 impairs endothelial integrity and induces a caspase-dependent and -independent cell death program Blood, March 15, 2007; 109(6): 2453 - 2460. [Abstract] [Full Text] [PDF]
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H. Y. Sroussi, J. Berline, and J. M. Palefsky Oxidation of methionine 63 and 83 regulates the effect of S100A9 on the migration of neutrophils in vitro J. Leukoc. Biol., March 1, 2007; 81(3): 818 - 824. [Abstract] [Full Text] [PDF]
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D. Foell, H. Wittkowski, T. Vogl, and J. Roth S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules J. Leukoc. Biol., January 1, 2007; 81(1): 28 - 37. [Abstract] [Full Text] [PDF]
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Y. Le Priol, D. Puthier, C. Lecureuil, C. Combadiere, P. Debre, C. Nguyen, and B. Combadiere High Cytotoxic and Specific Migratory Potencies of Senescent CD8+CD57+ Cells in HIV-Infected and Uninfected Individuals J. Immunol., October 15, 2006; 177(8): 5145 - 5154. [Abstract] [Full Text] [PDF]
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T. Shimizu, L. Esaki, H. Mizuno, and K. Takeda Granulocyte macrophage colony-stimulating factor enhances retinoic acid-induced gene expression J. Leukoc. Biol., October 1, 2006; 80(4): 889 - 896. [Abstract] [Full Text] [PDF]
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 H.Y. Sroussi, J. Berline, P. Dazin, P. Green, and J.M. Palefsky S100A8 Triggers Oxidation-sensitive Repulsion of Neutrophils Journal of Dental Research, September 1, 2006; 85(9): 829 - 833. [Abstract] [Full Text] [PDF]
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 S. Wang, M.-B. Voisin, K. Y. Larbi, J. Dangerfield, C. Scheiermann, M. Tran, P. H. Maxwell, L. Sorokin, and S. Nourshargh Venular basement membranes contain specific matrix protein low expression regions that act as exit points for emigrating neutrophils J. Exp. Med., June 12, 2006; 203(6): 1519 - 1532. [Abstract] [Full Text] [PDF]
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 R. R. Montgomery, K. Schreck, X. Wang, and S. E. Malawista Human Neutrophil Calprotectin Reduces the Susceptibility of Borrelia burgdorferi to Penicillin Infect. Immun., April 1, 2006; 74(4): 2468 - 2472. [Abstract] [Full Text] [PDF]
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 M. T. Follettie, M. Pinard, J. C. Keith Jr., L. Wang, D. Chelsky, C. Hayward, P. Kearney, P. Thibault, E. Paramithiotis, A. J. Dorner, et al. Organ Messenger Ribonucleic Acid and Plasma Proteome Changes in the Adjuvant-Induced Arthritis Model: Responses to Disease Induction and Therapy with the Estrogen Receptor-{beta} Selective Agonist ERB-041 Endocrinology, February 1, 2006; 147(2): 714 - 723. [Abstract] [Full Text] [PDF]
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 M. Ikura and J. B. Ames Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: Two ways to promote multifunctionality PNAS, January 31, 2006; 103(5): 1159 - 1164. [Abstract] [Full Text] [PDF]
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G. Srikrishna, O. Turovskaya, R. Shaikh, R. Newlin, D. Foell, S. Murch, M. Kronenberg, and H. H. Freeze Carboxylated Glycans Mediate Colitis through Activation of NF-{kappa}B J. Immunol., October 15, 2005; 175(8): 5412 - 5422. [Abstract] [Full Text] [PDF]
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D. Viemann, A. Strey, A. Janning, K. Jurk, K. Klimmek, T. Vogl, K. Hirono, F. Ichida, D. Foell, B. Kehrel, et al. Myeloid-related proteins 8 and 14 induce a specific inflammatory response in human microvascular endothelial cells Blood, April 1, 2005; 105(7): 2955 - 2962. [Abstract] [Full Text] [PDF]
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 J. C. Havelock, P. Keller, N. Muleba, B. A. Mayhew, B. M. Casey, W. E. Rainey, and R. A. Word Human Myometrial Gene Expression Before and During Parturition Biol Reprod, March 1, 2005; 72(3): 707 - 719. [Abstract] [Full Text] [PDF]
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K. Hsu, R. J. Passey, Y. Endoh, F. Rahimi, P. Youssef, T. Yen, and C. L. Geczy Regulation of S100A8 by Glucocorticoids J. Immunol., February 15, 2005; 174(4): 2318 - 2326. [Abstract] [Full Text] [PDF]
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 R. Yanagisawa, H. Takano, K.-I. Inoue, T. Ichinose, S.-i. Yoshida, K. Sadakane, K. Takeda, S. Yoshino, K. Yamaki, Y. Kumagai, et al. Complementary DNA Microarray Analysis in Acute Lung Injury Induced by Lipopolysaccharide and Diesel Exhaust Particles Experimental Biology and Medicine, November 1, 2004; 229(10): 1081 - 1087. [Abstract] [Full Text] [PDF]
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C. Ryckman, C. Gilbert, R. de Medicis, A. Lussier, K. Vandal, and P. A. Tessier Monosodium urate monohydrate crystals induce the release of the proinflammatory protein S100A8/A9 from neutrophils J. Leukoc. Biol., August 1, 2004; 76(2): 433 - 440. [Abstract] [Full Text] [PDF]
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 G. Bouma, W. K. Lam-Tse, A. F. Wierenga-Wolf, H. A. Drexhage, and M. A. Versnel Increased Serum Levels of MRP-8/14 in Type 1 Diabetes Induce an Increased Expression of CD11b and an Enhanced Adhesion of Circulating Monocytes to Fibronectin Diabetes, August 1, 2004; 53(8): 1979 - 1986. [Abstract] [Full Text] [PDF]
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S. Ghavami, C. Kerkhoff, M. Los, M. Hashemi, C. Sorg, and F. Karami-Tehrani Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions J. Leukoc. Biol., July 1, 2004; 76(1): 169 - 175. [Abstract] [Full Text] [PDF]
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M. Jaramillo, I. Plante, N. Ouellet, K. Vandal, P. A. Tessier, and M. Olivier Hemozoin-Inducible Proinflammatory Events In Vivo: Potential Role in Malaria Infection J. Immunol., March 1, 2004; 172(5): 3101 - 3110. [Abstract] [Full Text] [PDF]
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M. Frosch, T. Vogl, R. Waldherr, C. Sorg, C. Sunderkotter, and J. Roth Expression of MRP8 and MRP14 by macrophages is a marker for severe forms of glomerulonephritis J. Leukoc. Biol., February 1, 2004; 75(2): 198 - 206. [Abstract] [Full Text] [PDF]
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J. Roth, T. Vogl, C. Sunderkotter, and C. Sorg Chemotactic activity of S100A8 and S100A9. J. Immunol., December 1, 2003; 171(11): 5651 - 5651. [Full Text] [PDF]
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T. Chavakis, A. Bierhaus, N. Al-Fakhri, D. Schneider, S. Witte, T. Linn, M. Nagashima, J. Morser, B. Arnold, K. T. Preissner, et al. The Pattern Recognition Receptor (RAGE) Is a Counterreceptor for Leukocyte Integrins: A Novel Pathway for Inflammatory Cell Recruitment J. Exp. Med., November 17, 2003; 198(10): 1507 - 1515. [Abstract] [Full Text] [PDF]
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 A. Ahmad, D. L. Bayley, S. He, and R. A. Stockley Myeloid Related Protein-8/14 Stimulates Interleukin-8 Production in Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., October 1, 2003; 29(4): 523 - 530. [Abstract] [Full Text] [PDF]
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K. Vandal, P. Rouleau, A. Boivin, C. Ryckman, M. Talbot, and P. A. Tessier Blockade of S100A8 and S100A9 Suppresses Neutrophil Migration in Response to Lipopolysaccharide J. Immunol., September 1, 2003; 171(5): 2602 - 2609. [Abstract] [Full Text] [PDF]
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C. Ryckman, K. Vandal, P. Rouleau, M. Talbot, and P. A. Tessier Proinflammatory Activities of S100: Proteins S100A8, S100A9, and S100A8/A9 Induce Neutrophil Chemotaxis and Adhesion J. Immunol., March 15, 2003; 170(6): 3233 - 3242. [Abstract] [Full Text] [PDF]
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N M Wulffraat, P J Haas, M Frosch, I M de Kleer, T Vogl, D M C Brinkman, P Quartier, J Roth, and W Kuis Myeloid related protein 8 and 14 secretion reflects phagocyte activation and correlates with disease activity in juvenile idiopathic arthritis treated with autologous stem cell transplantation Ann Rheum Dis, March 1, 2003; 62(3): 236 - 241. [Abstract] [Full Text] [PDF]
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 M.-P. Manitz, B. Horst, S. Seeliger, A. Strey, B. V. Skryabin, M. Gunzer, W. Frings, F. Schonlau, J. Roth, C. Sorg, et al. Loss of S100A9 (MRP14) Results in Reduced Interleukin-8-Induced CD11b Surface Expression, a Polarized Microfilament System, and Diminished Responsiveness to Chemoattractants In Vitro Mol. Cell. Biol., February 1, 2003; 23(3): 1034 - 1043. [Abstract] [Full Text]
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C. Ryckman, G. A. Robichaud, J. Roy, R. Cantin, M. J. Tremblay, and P. A. Tessier HIV-1 Transcription and Virus Production Are Both Accentuated by the Proinflammatory Myeloid-Related Proteins in Human CD4+ T Lymphocytes J. Immunol., September 15, 2002; 169(6): 3307 - 3313. [Abstract] [Full Text] [PDF]
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 I. Eue, S. Konig, J. Pior, and C. Sorg S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells Int. Immunol., March 1, 2002; 14(3): 287 - 297. [Abstract] [Full Text] [PDF]
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M. J. Robinson, P. Tessier, R. Poulsom, and N. Hogg The S100 Family Heterodimer, MRP-8/14, Binds with High Affinity to Heparin and Heparan Sulfate Glycosaminoglycans on Endothelial Cells J. Biol. Chem., January 25, 2002; 277(5): 3658 - 3665. [Abstract] [Full Text] [PDF]
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H. Husson, E. G. Carideo, D. Neuberg, J. Schultze, O. Munoz, P. W. Marks, J. W. Donovan, A. C. Chillemi, P. O'Connell, and A. S. Freedman Gene expression profiling of follicular lymphoma and normal germinal center B cells using cDNA arrays Blood, January 1, 2002; 99(1): 282 - 289. [Abstract] [Full Text] [PDF]
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R. K. Kumar, Z. Yang, S. Bilson, S. Thliveris, B. E. Cooke, and C. L. Geczy Dimeric S100A8 in human neutrophils is diminished after phagocytosis J. Leukoc. Biol., July 1, 2001; 70(1): 59 - 64. [Abstract] [Full Text] [PDF]
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H. J. Strausbaugh and S. D. Rosen A Potential Role for Annexin 1 as a Physiologic Mediator of Glucocorticoid-Induced L-Selectin Shedding from Myeloid Cells J. Immunol., May 15, 2001; 166(10): 6294 - 6300. [Abstract] [Full Text] [PDF]
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K. Xu, T. Yen, and C. L. Geczy IL-10 Up-Regulates Macrophage Expression of the S100 Protein S100A8 J. Immunol., May 15, 2001; 166(10): 6358 - 6366. [Abstract] [Full Text] [PDF]
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G. Srikrishna, K. Panneerselvam, V. Westphal, V. Abraham, A. Varki, and H. H. Freeze Two Proteins Modulating Transendothelial Migration of Leukocytes Recognize Novel Carboxylated Glycans on Endothelial Cells J. Immunol., April 1, 2001; 166(7): 4678 - 4688. [Abstract] [Full Text] [PDF]
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I. Eue, B. Pietz, J. Storck, M. Klempt, and C. Sorg Transendothelial migration of 27E10+ human monocytes Int. Immunol., November 1, 2000; 12(11): 1593 - 1604. [Abstract] [Full Text] [PDF]
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K. Xu and C. L. Geczy IFN-{gamma} and TNF Regulate Macrophage Expression of the Chemotactic S100 Protein S100A8 J. Immunol., May 1, 2000; 164(9): 4916 - 4923. [Abstract] [Full Text] [PDF]
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 B. H. Schønning, M. Bévort, S. Mikkelsen, M. Andresen, P. Thomsen, H. Leffers, and B. Norrild Human papillomavirus type 16 E7-regulated genes: regulation of S100P and ADP/ATP carrier protein genes identified by differential-display technology J. Gen. Virol., April 1, 2000; 81(4): 1009 - 1015. [Abstract] [Full Text]
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C. Kerkhoff, M. Klempt, V. Kaever, and C. Sorg The Two Calcium-binding Proteins, S100A8 and S100A9, Are Involved in the Metabolism of Arachidonic acid in Human Neutrophils J. Biol. Chem., November 12, 1999; 274(46): 32672 - 32679. [Abstract] [Full Text] [PDF]
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 K. S.C. Weber, L. B. Klickstein, and C. Weber Specific Activation of Leukocyte beta 2 Integrins Lymphocyte Function-associated Antigen-1 and Mac-1 by Chemokines Mediated by Distinct Pathways via the alpha Subunit Cytoplasmic Domains Mol. Biol. Cell, April 1, 1999; 10(4): 861 - 873. [Abstract] [Full Text]
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L. H. K. Lim, E. Solito, F. Russo-Marie, R. J. Flower, and M. Perretti Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: Effect of lipocortin 1 PNAS, November 24, 1998; 95(24): 14535 - 14539. [Abstract] [Full Text] [PDF]
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). MRP-8, the heterodimeric
partner of MRP-14, shows no adhesive activity when titrated over the
same dose range (
). Control S100 protein S100A (
) and native
MRP-8/14 complex (
) were similarly negative. The inset
figure demonstrates the ability of CD11b mAb LPM19c at 20 µg/ml to
inhibit either FMLP (0.1 µM)- or MRP-14 (1 µM)-stimulated adhesion
to fibrinogen. The data shown are from one experiment representative of
six.
) but not that induced by 0.1 µM FMLP (
