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CHEMOKINES
From the Department of Immunology and Cell Biology and
the Division of Biophysics, Forschungszentrum Borstel, Borstel,
Germany.
The platelet-derived neutrophil-activating peptide 2 (NAP-2,
70 amino acids) belongs to the ELR+ CXC subfamily of
chemokines. Similar to other members of this group, such as IL-8, NAP-2
activates chemotaxis and degranulation in neutrophils
(polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and
CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine
but as the C-terminal part of several precursors that lack
PMN-stimulating capacity. As we have previously shown, PMN themselves
may liberate NAP-2 from the precursor connective tissue-activating
peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of
inducing cell activation, continuous accumulation of the chemokine in
the surroundings of the processing cells results in the down-regulation
of specific surface-expressed NAP-2 binding sites and in the
desensitization of chemokine-induced PMN degranulation. Thus, NAP-2
precursors may be regarded as indirect mediators of functional
desensitization in neutrophils. In the current study we investigated
the biologic impact of another major NAP-2 precursor, the platelet
basic protein (PBP, 94 amino acids). We show that PBP is considerably
more potent than CTAP-III to desensitize degranulation and chemotaxis
in neutrophils. We present data suggesting that the high desensitizing
capacity of PBP is based on its enhanced proteolytic cleavage into
NAP-2 by neutrophil-expressed cathepsin G and that it involves
efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is
hardly affected. Correspondingly, we found PBP and, less potently,
CTAP-III to inhibit CXCR-2- but not CXCR-1- dependent chemotaxis of
neutrophils toward NAP-2. Altogether our findings demonstrate that the
anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.
(Blood. 2000;96:2965-2972) ELR+ CXC chemokines, such as IL-8 and
neutrophil-activating peptide 2 (NAP-2), are well-known neutrophil
(polymorphonuclear [PMN]) agonists that are considered important for
PMN recruitment during many inflammatory processes. Bearing the
functionally important amino acid motif consisting of glutamic acid
(E), leucine (L), and arginine (R) directly in front of the first of
their 4 conserved cysteines (C), ELR+ CXC chemokines
specifically bind to receptors CXCR-1 and CXCR-2 on their target cells.
This interaction can lead to subsequent activation of PMN functions
such as chemotaxis and degranulation. To attenuate PMN-mediated
deleterious effects during inflammation, considerable efforts have been
focused on elucidating how ELR+ CXC chemokine activity
is regulated.
In most cases, the generation of ELR+ CXC chemokines is
controlled at the level of gene and subsequent protein expression after induction by inflammatory stimuli.1 The only
ELR+ CXC chemokine regulated in a completely different
manner is the 70 amino acids (aa) containing NAP-2 that originates
essentially from platelets and megakaryocytes. The
neutrophil-activating capacity of NAP-2 is strictly controlled by
proteolytic processing of N-terminally extended NAP-2 precursor
molecules (compare Figure 1), which
themselves lack PMN-activating properties.2-4 These
precursors, which together with NAP-2 are comprised under the term
In the current study we were interested in the role of the next most
common NAP-2 precursor after CTAP-III, the platelet basic protein (PBP,
94 aa), which differs from CTAP-III by having an extended N-terminus
(Figure 1). PBP reportedly not only contributes 11% to 49% of the
total Preparation of PBP, CTAP-III, and NAP-2
Preparation of human neutrophils
Degranulation assay PMN (1 × 107/mL) suspended in D-PBS/BSA (Dulbecco-PBS/0.1% BSA) were preincubated for 10 minutes with 5 µg/mL cytochalasin B (Sigma, Deisenhofen, Germany) and supplemented with CaCl2 (1.8 mmol/L) and MgCl2 (1 mmol/L). Then 100 µL cells were added to 100 µL of samples preheated at 37°C. Samples acidified to stop cathepsin G activity were reneutralized with NaOH immediately before cells were added. After 30 minutes of incubation, cells were pelleted and supernatants were monitored for elastase enzymatic activity, as described elsewhere.9 Release rates were determined as percentage of total elastase activity in hexadecyl-trimethylammoniumbromide (0.1%)-treated PMN lysates. Backgrounds, as determined in the presence of buffer alone, were subtracted.Desensitization of NAP-2-induced degranulation To assess the effect of fixed concentrations of desensitizing agents on PMN degranulation in response to NAP-2, the degranulation assay was modified by pretreating PMN with the given agent 10 minutes before the stimulus was added. For analysis of various concentrations of desensitizing agent on the degranulation induced by 40 nmol/L NAP-2, PMN were suspended at 2 × 107 cells/mL and preincubated with cytochalasin-B. Then 50 µL of cell suspension was added to the same volume of 2-fold-concentrated desensitizing agent. After a 10-minute incubation, 100 µL of 80 nmol/L NAP-2 in D-PBS/BSA/CaCl2 (1.8 mmol/L/MgCl2(1 mmol/L)) was added. Subsequently, the degranulation assay was performed as described. Desensitization was expressed as percentage of release rates obtained with control PMN receiving no desensitizing agent.Processing of NAP-2 precursors by neutrophils and cathepsin G and analyses of truncation products NAP-2 precursors (1 µmol/L in 100 µL D-PBS/BSA) were mixed with either 100 µL PMN (1 × 107/mL) or cathepsin G (1 µg/mL) (Calbiochem, Frankfurt, Germany) in D-PBS/BSA and incubated for different time periods at 37°C (processing period). To terminate the enzyme reaction, samples were acidified with 0.1% TFA. Samples containing PMN were centrifuged and supernatants were analyzed for the presence of NAP-2 biologic activity. In the PMN degranulation assay, a standard of purified NAP-2 was run in parallel for the determination of NAP-2 activity equivalents. The initial velocity (V) of NAP-2 formation from precursors was determined and is given as an increase in NAP-2 activity equivalents per minute. The same samples were concomitantly monitored for the presence of NAP-2 by SDS-PAGE and Western blot analysis. Alternatively, supernatants were separated on a C2/C18 column (µRPC PC3.2/3; Amersham Pharmacia Biotech, Uppsala, Sweden) using a SMART chromatography unit (Amersham Pharmacia Biotech AB). Five hundred microliter volumes of samples acidified with TFA were loaded, and the column was developed with a linear acetonitrile-gradient (17.5%-37.5%). Peaks detected at 214 nm were collected and analyzed by mass spectroscopy (see "Mass spectrometry").Chemotaxis Neutrophil chemotaxis was assayed in a modified 48-well Boyden chamber (Costar, Bodenheim, Germany), essentially as described.12Flow cytometric analyses of CXCR-1 and CXCR-2 expression on neutrophils Flow cytometric (FACS) analyses were performed as described.12 PMN (1 × 106/mL) suspended in D-PBS/BSA were incubated for 1 hour on ice with monoclonal antibodies RII 115 (2 µg/mL) and SE-2 (5 µg/mL) specific for CXCR-2 and CXCR-1, respectively. RII 115 was generated in our laboratory as described,12 and SE-2 was kindly provided by Dr O. Götze13 (University of Göttingen, Göttingen, Germany). Cell-bound antibodies were detected by fluorescein-conjugated goat -mouse immunoglobulin (H+L) antibody
(Dianova, Hamburg, Germany) (15 µg/mL), and analyses were performed
on a flow cytometer (FACStar PLUS; Becton Dickinson,
Heidelberg, Germany).
Electrophoresis and immunoblotting SDS-PAGE was performed according to Schägger and von Jagow.14 Samples were reduced 10 minutes at 95°C, treated 30 minutes with iodoacetamide (2%) and loaded onto a 13% polyacrylamide gel with a 10% spacer gel and a 4% stacking gel on top. Rainbow Protein Markers (Amersham Buchler, Braunschweig, Germany) served as molecular mass markers. Western blotting was carried out as described previously.15 Rabbit antisera used for Western blot analyses were R- -TG raised against a purified preparation of
native -TG Ag, reacting to all known variants of -TG Ag and
containing an antibody subpopulation interacting with residues 1 to 24 in PBP but not with residues 1 to 15 in CTAP-III (data not shown), and
R-70/2 raised against the C-terminal octapeptide of NAP-2
(LAGDESAD). R-70/2 had characteristics identical to previously
described antiserum R-70, requiring the presence of the C-terminal
residue D70 for binding to -TG Ag.15
Mass spectrometry Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed with a Bruker-ReflexIII (Bruker-Franzen Analytik, Bremen, Germany) in reflector time-of-flight configuration at an acceleration voltage of 20 kV and with delayed ion extraction. The compounds dissolved in 0.1% TFA at a concentration of less than 0.3 µg/µL, were diluted 1:2 with a freshly prepared matrix solution consisting of saturated 3,5-dimethoxy-4-hydroxy cinnamic acid (Sinapic; Aldrich, Steinheim, Germany) in a 2:1 mixture of 0.1% trifluoroacetic acid/acetonitrile. Aliquots of 0.5 µL were deposited on a metallic sample holder and analyzed immediately after drying in a stream of air. Mass scale calibration was performed externally.N-terminal amino acid sequencing N-terminal sequence analyses of-TG Ag isoforms dissolved in
0.1% TFA were performed by Dr A. Petersen (Forschungszentrum Borstel,
Borstel, Germany) on a gas-phase sequencer (model 473A; Applied
Biosystems, Foster City, CA). When proteins were analyzed directly from
the blot, the area containing the unstained protein was excised, washed
with bidistilled water for 1 hour, dried, and sequenced.
Statistical analysis Data were statistically analyzed using Microcal Origin 4.10 software (Microcal Software, Northampton, MA).
PBP in comparison with CTAP-III shows enhanced potency to desensitize chemokine-induced neutrophil degranulation We have previously shown that CTAP-III, the quantitatively prevailing NAP-2 precursor released by platelets, functions as a desensitizing agent for chemokine-induced PMN degranulation. In the current study we were interested in whether PBP another major
platelet-secreted NAP-2 precursor bearing an N-terminal extension by 9 residues longer than that in CTAP-IIIwould function in a
corresponding way. For comparison, we used highly purified PBP and
CTAP-III isolated from platelet-release supernatants. As shown in
Figure 2, PBP turned out to be
considerably more effective than CTAP-III in desensitizing PMN
degranulation. On preincubation of PMN with a fixed dosage (50 nmol) of
either PBP or CTAP-III for 10 minutes, degranulation in response to
increasing concentrations of NAP-2 was significantly reduced (Figure
2A). Compared with unexposed control cells, pretreatment with PBP
turned the cells approximately 23-fold less sensitive to NAP-2
stimulation, whereas CTAP-III pretreatment reduced their
sensitivity by only 4-fold. To determine the relative potencies of the
2 precursors, cells were preincubated with increasing concentrations of
PBP or CTAP-III for 10 minutes, and PMN degranulation in response to a
fixed dosage of NAP-2 (40 nmol/L) was assessed (Figure 2B). As seen by
the respective dose-response curves running parallel to each other, half-maximal down-regulation of the degranulation response required approximately 120 nmol/L CTAP-III and 16 nmol/L PBP, respectively, indicating that PBP is approximately 7-fold more potent a desensitizing agent than CTAP-III. Similarly, the threshold concentration for PBP to
induce a measurable degree of desensitization was approximately 6- to
7-fold lower (3 nmol/L) than that for CTAP-III (20 nmol/L).
Neutrophils generate NAP-2 more rapidly from PBP than from CTAP-III To address the mechanism underlying the enhanced desensitizing effect of PBP on PMN degranulation, we first examined whether this could be caused by the direct interaction of PBP with specific cellular binding sites. In competition binding assays performed at 4°C with up to 10 nmol/L radioiodinated PBP in the absence or presence of a 100-fold molar excess of cold PBP, no specific binding to neutrophils could be detected (data not shown). These results corresponded to those previously obtained with radiolabeled CTAP-III3 and excluded the possibility that PBP-induced desensitization was a directly receptor-mediated event. More likely, the proteolytic generation of an NAP-2-like molecule was involved, similar to the generation of NAP-2 from CTAP-III. To examine this we looked for the presence of NAP-2-like proteolytic truncation products in the supernatants of PMN that had been incubated with 500 nmol/L PBP or, for comparison, with 500 nmol/L CTAP-III. Initially, a relatively long incubation period (240 minutes) was chosen to obtain sufficient amounts of the potential truncation products for biochemical analyses. Separation by SDS-PAGE and subsequent visualization of the blotted proteins by immunostaining with an antiserum to-TG Ag (Figure
3A) revealed that PBP (lane P/0) was
almost completely converted to a molecule similar in size to purified
NAP-2 (compare lanes P/240 to lane N). In fact, sequencing of this
truncation product revealed an N-terminus (A-E-L-R) identical to that
of NAP-2. We suspected that this molecule could represent a
C-terminally truncated NAP-2 variant. The formation of such a variant
from PBP could explain the precursor's enhanced desensitizing effect;
we previously found that C-terminally truncated NAP-2 binds to
receptors with higher affinity and thereby desensitizes PMN
degranulation more potently than full-length NAP-2.16
However, as seen on probing with a -TG Ag antiserum (R-70/2) that
required the outermost C-terminal amino acid (D70) for
binding to NAP-2, the PBP-derived molecule was fairly immunoreactive
(Figure 3A, lane P/240, right). This observation indicated that the
protein indeed represented full-length NAP-2 and thus excluded that
C-terminal truncation was responsible for the enhanced desensitizing
capacity of PBP.
More detailed analysis of the time course of NAP-2 formation by
neutrophils exposed to either PBP or CTAP-III pointed to a different
mechanism that might be effective and that appeared to be based on a
higher susceptibility of PBP to cleavage by PMN. Although the
240-minute coincubation of PMN with PBP led to practically complete
conversion of the precursor to NAP-2, this was not the case with
CTAP-III, which became only partially processed, as indicated by a more
than 50% proportion of the precursor remaining detectable on
immunoblotting (Figure 3A; compare left panels of lanes C/240 and P/240
and right panels of lanes C/240 and P/240). Further comparison of
supernatants obtained after various shorter incubation periods indeed
showed that with PBP-exposed cells, a detectable NAP-2 band appeared
approximately 4- to 6-fold earlier than with cells exposed to CTAP-III
(Figure 3A; compare lanes P/10 and C/60). This observation correlated
well with the data shown in Figure 3B, in which, using purified NAP-2
as a standard, the same supernatants were analyzed for the presence of
NAP-2-like biologic activity in the PMN degranulation assay.
Consistently, NAP-2-like biologic activity was generated by
neutrophils more quickly from PBP than from CTAP-III, and this
difference appeared most drastic at early time points. Thus, the
initial velocity of NAP-2 generation from 500 nmol/L PBP ( Conversion of PBP to NAP-2 is mediated by a single, cathepsin G-like enzyme To obtain further insight into the mechanism(s) responsible for enhanced NAP-2 generation from PBP, we next focused on the enzyme(s) involved in precursor processing by PMN. Concerning CTAP-III, we showed15 that its conversion to NAP-2 is catalyzed by a single PMN-associated cathepsin G-like enzyme and could be mimicked by purified cathepsin G. Under either condition, cleavage occurred in a single step behind the only tyrosine within CTAP-III, giving rise to biologically active NAP-2 (compare Figure 1). Because PBP bears the same cleavage site, cathepsin G was likely to be involved in the processing of this NAP-2 precursor as well. However, as evident in the Western blot shown in Figure 3, panel A, PMN-mediated processing of PBP, in contrast to that of CTAP-III, not only yielded the one truncation product we identified as NAP-2, it also gave rise to another molecule that became visible as an immunoreactive band (intermediate size, approximately 9 kd) and that comigrated with CTAP-III. Different from NAP-2, the latter molecule exhibited only a transient appearance; it was detectable after 10 minutes of processing and disappeared after 240 minutes. Furthermore, with longer developing times of the blot (leading to overstaining of the major bands), a faint band became detectable that migrated at approximately 2 kd (data not shown). These observations led us to assume that PBP was targeted by several enzymes that cleaved the precursor at different sites and possibly cooperated in its conversion to NAP-2. However, control experiments performed by coincubating PBP with purified cathepsin G (Figure 4) surprisingly led to results essentially identical to those obtained with neutrophils. Cathepsin G alone was capable of generating the same 3 fragments from PBP (Figure 4A), ie, a protein comigrating with NAP-2 and accumulating over time, a transiently appearing protein of intermediate size similar to CTAP-III, and a faintly visible 2-kd fragment. Moreover, the enzyme similarly catalyzed the generation of NAP-2-like biologic activity approximately 6 times more rapidly from PBP than from CTAP-III, as seen by a comparison of the initial velocities for NAP-2 formation, which were approximately 41 nmol/L per minute and 7 nmol/L per minute, respectively (Figure 4B). Indeed, N-terminal sequencing and detection by the C-terminus-specific antiserum R-70/2 identified this
NAP-2-like molecule as NAP-2. These data, demonstrating that cathepsin
G alone catalyzes the enhanced formation of NAP-2 from PBP in a way
comparable to that seen with neutrophils, strongly suggest that
cell-mediated PBP conversion depends on a single enzyme, most likely
identical to neutrophil cathepsin G.
Molecular identity and functional impact of the PBP fragments generated by neutrophils Further analyses revealed the identity and the functional involvement of the other fragments generated from PBP by neutrophils. N-terminal sequence analyses of the 2 kd band directly from the blot
yielded a sequence reading S-S-T-K, indicating that the fragment represented an N-terminal stretch of PBP. Because of an insufficient amount of material, no result was obtained for the 9-kd truncation product. We therefore chose a different approach by using reverse-phase HPLC on a C2/C18 column for the separation of supernatants obtained after 10 minutes of coincubation of neutrophils with 2 µmol/L PBP
(Figure 5). Mass spectrometric analyses
of the single-protein peaks yielded molecular masses for the major
components contained in peaks 3 and 4 of 10 281 and 10 265,
respectively, corresponding to the calculated masses of PBP in its
oxidized (10 278) and nonoxidized (10 262) forms. Additionally, peak
4 contained low amounts of 2 components of intermediate size. These had
molecular masses of 9447 and 8220, which corresponded most closely to
PBP cleavage products of 86 and 75 residues in length, representing
CTAP-III extended by one N-terminal residue (R-CTAP-III) and NAP-2
extended by 5 N-terminal residues (DSDLY-NAP-2) (calculated masses,
9446 and 8219, respectively). Peaks 1 and 2 contained only one
component each, with masses of 2651 and 7624, respectively. Although
the former mass was closest to that of the N-terminal stretch of PBP, encompassing residues  24 through 1 (calculated mass, 2655), the
latter was identical to that calculated for NAP-2 (7624). These results
indicate that PMN cleave PBP at 3 different sites, first directly
behind the Tyr at position 1, giving rise to NAP-2 and the 24-aa
residue N-terminal peptide, second at a dibasic cleavage site between
Lys-Arg at positions 16 and 17, leading to the formation of the
fragment one residue longer than CTAP-III, and third behind the Leu at
position 6, giving rise to the molecule 5 residues longer than NAP-2.
Most likely, the amounts of the latter truncation product were too low
to become detected by Western blotting. Nevertheless, these data
demonstrate that formation of the intermediate-size fragments is not a
prerequisite for the generation of NAP-2. Although it appears most
unlikely that R-CTAP-III and DSDLY-NAP-2 contribute to neutrophil
functional desensitization before becoming converted into NAP-2, a
potential desensitizing function for the N-terminal fragment PBP (24
to 1) could not be excluded. We produced a corresponding synthetic
peptide and evaluated its impact on NAP-2-induced degranulation.
However, in assays using up to 2 µmol/L of the peptide for
preincubation of PMN, no change of NAP-2-promoted degranulation was
observed (data not shown). Thus our data demonstrate that NAP-2 is the only PBP-derived truncation product responsible for neutrophil desensitization.
Preincubation of PMN with PBP leads to enhanced down-regulation of CXCR-1 and CXCR-2 compared to preincubation with CTAP-III The evident correlation existing between the extremely rapid conversion of PBP to NAP-2 by neutrophils and its similarly enhanced capacity to desensitize the degranulation response of the processing cells led us to analyze how this precursor affected the surface expression of NAP-2 binding sites on PMN. As we have shown,3 preincubation of PMN with CTAP-III leads to compromised binding of radiolabeled NAP-2 to PMN, and, in fact, the same was the case for PBP. Hence, preincubation of PMN for 5 minutes with 400 nmol/L PBP reduced binding of 0.5 nmol/L radioiodinated NAP-2 to background level (data not shown). However, we wanted to know to what degree the expression of the different receptors known for NAP-2, namely CXCR-1 and CXCR-2, was affected. Therefore, by using monoclonal antibodies SE-2 and RII115 specific for CXCR-1 and CXCR-2, respectively, we assessed by FACS analyses the presence of these receptors on the surface of PMN on 10 minutes pretreatment with different concentrations of PBP or CTAP-III. As shown in Figure 6, preincubation of PMN with PBP reduced the expression of both receptors approximately 4-fold more potently than did pretreatment with CTAP-III. These data correspond relatively well with the6- to 7-fold enhanced desensitizing capacity of PBP
and its similarly more rapid conversion to NAP-2, suggesting that
enhanced receptor down-regulation by PBP could indeed be caused by the
higher quantity of NAP-2 accumulating around the processing cells.
Further support for NAP-2 as the active mediator may be derived from
the observation that approximately 250-fold lower precursor
concentrations are required to down-regulate the NAP-2 high-affinity
receptor CXCR-2 (Kd 1
nmol/L9) compared with the low-affinity receptor CXCR-1
(Kd 200 nmol/L9) (minimal effective doses of PBP, 10 nmol/L and 2.5 µmol/L, respectively). In fact, a comparable (400-fold) difference in potency to
down-regulate CXCR-2 versus CXCR-1 was observed when NAP-2 itself was
tested, which down-regulated 50% of CXCR-2 at 15 nmol/L, whereas it
took 6 µmol/L to see the same effect with CXCR-1 (compare Figure
6). Thus, our data suggest that PBP (and CTAP-III) mediates its
desensitizing effect predominantly through the preferential interaction
of NAP-2 with CXCR-2.
PBP inhibits CXCR-2-mediated neutrophil chemotaxis toward NAP-2 more potently than does CTAP-III The observation that PBP potently reduced the expression of CXCR-2 led us to look at its effect on PMN chemotaxis. We recently reported12 that the chemotactic response of PMN toward NAP-2 consists of 2 optima (compare Figure 7B), one at very low NAP-2 concentrations (5 nmol/L) because of the interaction with CXCR-2 and the other at
400-fold higher concentrations because of the interaction with
CXCR-1. Therefore, CXCR-2 especially appeared to be important for PMN
attraction in response to the initially low amounts of NAP-2 arising
from platelet-released precursors immediately after platelet
degranulation. However, during this early phase, the concentration of
precursors would still be high. Hence, we wanted to know whether the
presence of PBP or CTAP-III would have an impact on PMN chemotaxis
induced by NAP-2 concentrations addressing CXCR-2. Thus, we analyzed
the chemotactic migration of PMN toward 5 nmol/L NAP-2 mixed with
increasing dosages of either precursor. In contrast to the conditions
applied in the degranulation assay, PMN were not pretreated with the
respective precursor but were exposed to NAP-2 and precursors
simultaneously. Though without such a prior period of desensitization
PBP or CTAP-III never affected the degranulation response toward NAP-2
(data not shown), this was different for the chemotactic response
(Figure 7A). Most interestingly, both precursors inhibited
NAP-2-induced chemotaxis, with CTAP-III reducing the chemotactic
response by 50% at 1100 nmol/L and PBP exhibiting a corresponding
effect at 160 nmol/L, the latter being approximately 7 times more
potent. Moreover, analyses of the dose-dependent chemotactic response toward NAP-2 in the presence of 1 µmol/L PBP showed that though the
first (CXCR-2-associated) optimum was virtually abrogated, the second
(CXCR-1-associated) optimum remained unchanged (Figure 7B). These data
demonstrate that PBP does not alter the responsiveness of the cells in
general but selectively down-regulates CXCR-2-dependent PMN
chemotaxis.
In summary, the fact that PBP, and to a lesser extent CTAP-III, are not only capable of desensitizing PMN degranulation but also inhibit neutrophil chemotaxis strongly suggests that these molecules play an important role in delimiting neutrophil activation during wound repair.
In the current study we have shown that PBP acts as an
outstandingly potent agent to down-modulate neutrophil activation. Compared with the NAP-2 precursor CTAP-III, PBP proved to be
approximately 6 to 7 times more potent to desensitize NAP-2-induced
neutrophil degranulation. By several lines of evidence we have shown
that the underlying mechanism for enhanced activity of PBP is the more rapid formation of NAP-2 from this precursor. Our data suggest that the
serine protease cathepsin G or a closely related enzyme is responsible
for this conversion. This contention is strongly supported by the
similar kinetics of NAP-2 generation from PBP by the purified enzyme
compared with PMN (compare Figures 2 and 3). Moreover, the pattern of
truncation products observed, composed of 3 different molecules
detectable by antiserum R Based on our data, we suggest that NAP-2 Interestingly, this conclusion is supported by our discovery that NAP-2 precursor molecules inhibit NAP-2-induced PMN chemotaxis (compare Figure 7A). Strikingly, as shown for PBP in Figure 7, panel B, this inhibitory effect was only observed for the first of the 2 optima that characterize the chemotactic response of neutrophils toward NAP-2. Because we had previously shown that this first optimum at approximately 5 nmol/L NAP-2 was mediated by CXCR-2,12 PBP appears to specifically inhibit CXCR-2-mediated chemotaxis. Surprisingly, this effect, in contrast to the desensitization of degranulation, does not require the pretreatment of PMN with NAP-2 precursors but appears to depend on a high precursor-NAP-2 ratio in the lower compartment of the chemotaxis chamber. This phenomenon is most easily explained by the assumption that PMN, before they come upon chemotactically active NAP-2 concentrations, first encounter precursor concentrations that after conversion to NAP-2 are sufficient to counteract the original NAP-2 gradient and to down-modulate CXCR-2. Thus, our data attribute anti-inflammatory properties to PBP and
CTAP-III, which are directed against homologous PMN activation by NAP-2
or other CXCR-2 ligands, such as MGSA, but also affect IL-8. In fact,
we have previously shown3 that CTAP-III pretreatment of PMN
significantly reduces IL-8-induced degranulation. Correspondingly, we
have observed the significantly reduced efficiency of IL-8 to induce
PMN chemotaxis in the presence of PBP (data not shown). In addition,
PMN desensitization may affect heterologous agonists such as fMLP.
Cross-desensitizing activity of IL-8 on fMLP-induced PMN calcium flux
has already been demonstrated,20 and we are investigating
the impact of NAP-2 precursors on fMLP-induced degranulation. Possibly,
as PBP proves a more potent anti-inflammatory agent than CTAP-III, the
relative proportion of these agents stored within platelets influences
their desensitizing capacity. Because the contribution of PBP to the
total Despite their anti-inflammatory properties, it has to be kept in mind that, after all, PBP and CTAP-III represent the source for NAP-2 that, by itself, can potently activate PMN. Taking both characteristics into account, we suggest that the NAP-2-precursor system may have a dual function and be effective during different phases of wound repair. Thus, during the initial hemostatic phase on wounding, NAP-2 generated directly after platelet aggregation from PBP and CTAP-III most likely desensitizes degranulation, especially of those PMN entrapped within the hemostatic plug. Such functional attenuation is probably important for protecting the plug and regenerating tissue from deleterious neutrophil enzymes. By the same token, our data suggest that during the initial prevalence of NAP-2 precursors over NAP-2, further PMN will be prevented from migrating to the site of tissue damage. However, because this prevalence will decline within the thrombus over time due to successive PMN-mediated conversion of the precursors to active NAP-2, the inhibitory effect will decline as well. Therefore, NAP-2 in a later, less critical phase of tissue regeneration is probably very important for the attraction and activation of PMN to the site of wound repair to ensure antimicrobial defense.21
We thank Dr A. Petersen for N-terminal sequencing of
Submitted April 14, 2000; accepted June 26, 2000.
Supported in part by Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 367, Projekt C4 and Graduiertenkolleg 288, Projekt B4). J.E.E. is a recipient of fellowship EH 188/1-1 from the Deutsche Forschungsgemeinschaft.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ernst Brandt, Department of Immunology and Cell Biology, Forschungszentrum Borstel, Parkallee 22, D-23845 Borstel, Germany; e-mail: ebrandt{at}fz-borstel.de.
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  D. Frommhold, A. Ludwig, M. G. Bixel, A. Zarbock, I. Babushkina, M. Weissinger, S. Cauwenberghs, L. G. Ellies, J. D. Marth, A. G. Beck-Sickinger, et al. Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation J. Exp. Med., June 9, 2008; 205(6): 1435 - 1446. [Abstract] [Full Text] [PDF]
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K. F. Nolan, V. Strong, D. Soler, P. J. Fairchild, S. P. Cobbold, R. Croxton, J.-A. Gonzalo, A. Rubio, M. Wells, and H. Waldmann IL-10-Conditioned Dendritic Cells, Decommissioned for Recruitment of Adaptive Immunity, Elicit Innate Inflammatory Gene Products in Response to Danger Signals J. Immunol., February 15, 2004; 172(4): 2201 - 2209. [Abstract] [Full Text] [PDF]
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B. I. Schenk, F. Petersen, H.-D. Flad, and E. Brandt Platelet-Derived Chemokines CXC Chemokine Ligand (CXCL)7, Connective Tissue-Activating Peptide III, and CXCL4 Differentially Affect and Cross-Regulate Neutrophil Adhesion and Transendothelial Migration J. Immunol., September 1, 2002; 169(5): 2602 - 2610. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
; PBP, 
; no desensitizing agent, 
.
= 214 nm (solid line) was compared to that of SN from PMN not
having received PBP, and only those peaks (1-4, shaded) that newly
emerged in the SN of PBP-supplemented cells, compared with cells not
having received PBP, were collected and further analyzed by mass
spectroscopy. The molecular masses given above peaks 1 to 3 are
representative for the respective major component detected, whereas for
peak 4 the masses of the major (bold) and 2 minor components
are given.
