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Department of Physiology and Pathology, University of Trieste, Trieste, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains (CD11a,
CD11b, CD11c) of LFA-1, CR3, and gp150/95. Anti-CD18 mAbs triggered a
marked release of Cl- ions, which was accompanied by
spreading and activation of the respiratory burst. Cross-linking of
gp150/95 and LFA-1 generated the most powerful signals for the
activation of Cl- efflux. The results of three independent
experimental approaches, i.e., kinetic studies, use of Cl-
transport inhibitors, and modulation of Cl- efflux with
different amounts of anti-ß2 integrin mAbs, indicated
that Cl- efflux regulates both spreading and respiratory
burst triggered by ß2 integrin cross-linking.
Cl- efflux appears to be independent on either alterations
of [Ca2+]i or changes in the plasma
membrane potential and shows sensitivity to a raise in pHi.
This study uncovers a new signaling ability of ß2
integrins and contributes to highlight the role of Cl-
efflux in the outside-in signal transduction pathway regulating
adherence-dependent PMN responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF), granulocyte-CSF, and granulocyte/macrophage-CSF.
These molecules are unable to elicit a metabolic response from PMN in
suspension, but become powerful stimulatory agents when they interact
with adherent cells (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Surface proadhesive molecules belonging to
the ß2 (CD11/CD18) integrin subfamily, namely LFA-1
(CD11a/CD18), CR3 (CD11b/CD18), and gp150/95 (CD11c/CD18), which are
all expressed on neutrophil plasma membrane (11), play a crucial role
in this respect. This is suggested by the inhibition by
anti-ß2 mAbs of the TNF-induced adherence and
respiratory burst in PMN in contact with surfaces coated with FBS,
fibrinogen, or fibronectin (FN) (3, 7, 8, 9). A definitive proof is
provided by the finding that PMN of patients with the type 1 leukocyte
adhesion deficiency (LAD-1) syndrome, which genetically lack
ß2 integrin expression (12), fail to respond with a burst
of their oxidative metabolism to TNF when in contact with biologic
surfaces (3). A distinctive feature of resting PMN is their unusually high intracellular content of chloride ions, which has been estimated to be 80–90 mM (13). We have recently shown that PMN, exposed to TNF on immobilized FN, display a long-lasting and sustained Cl- efflux accompanied by a decrease in intracellular chloride levels, which is causally related to activation of the respiratory burst (10). The observation that the Cl- efflux was, at least in part, dependent on ß2 integrin-mediated adherence of PMN to FN suggested that these proadhesive molecules may play a direct role in eliciting signals that activate chloride release from PMN.
It has become increasingly clear that the role of ß2
integrins in mediating cytokine-stimulated PMN functions goes beyond
their well recognized role of proadhesive molecules (reviewed in 14 . Studies by Nathan and coworkers first showed that two key events
in TNF-induced activation of PMN respiratory burst, namely decrease in
intracellular cAMP levels (6) and protein tyrosine phosphorylation
(15), require the engagement of ß2 integrins with
surfaces coated with proteins of the extracellular matrix.
Subsequently, experiments performed by cross-linking the common
ß-chain or the distinct -chains of LFA-1, CR3, and gp150/95 of
resting PMN by immobilized mAbs provided the first direct evidence of
the ability of ß2 integrins to deliver signals for
selective neutrophil functions, such as the release of oxygen reduction
products, spreading, and protein tyrosine phosphorylation
(16, 17, 18). Furthermore, ß2 integrins have been
shown to modulate other PMN responses, such as the elevation in
cytosolic free calcium (19, 20, 21, 22), the activation of phospholipase D (23, 24), actin polymerization (22, 25), leukotriene B4 production
(26), the degranulation and modulation of L-selectin (22), changes in
intracellular pH (27), and p21ras activation
(28).
In this paper, we have tested the ability of PMN ß2
integrins to elicit Cl- efflux, independently of other
stimuli, by inducing their cross-linkage with surface-bound mAbs
directed against either the common ß-chain (CD18) or the individual
-chains (CD11a, CD11b, CD11c) of ß2 integrins. The
results show that: 1) cross-linking of PMN ß2 integrins
by specific mAbs is sufficient to trigger Cl- efflux; 2)
the order of potency of the three ß2 integrin
heterodimers is gp150/95 > LFA-1 > CR3; 3) Cl-
efflux regulates both the spreading and the respiratory burst triggered
by ß2 integrin cross-linking, as shown by the results of
three independent experimental approaches, i.e., kinetic studies, use
of Cl- transport inhibitors, and modulation of
Cl- efflux with different amounts of
anti-ß2 integrin mAbs; 4) Cl- efflux is
independent on either alterations of [Ca2+]i
or changes in the plasma membrane potential; and 5) the activation of
Cl- efflux appears to be regulated by a raise in
pHi and is independent on protein tyrosine phosphorylation
and decrease of cAMP levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-aminomethyl-4-(1,1-dimethylethyl)-6-iodophenol
hydrochloride (MK-447),
2-aminomethyl-4-(1-methyl-1-phenylethyl)-6-chlorophenol
hydrochloride (MK-447/A), and
2-aminomethyl-4-(1,1-dimethylethyl)-6-methylketone hydrochloride
(MK-447/B) were generously provided by Merck Sharp & Dohme Research
Laboratories (Rahway, NJ); BSA, -cyano-4-hydroxy-cinnamic acid
(CHC), cytochrome c (type VI, from horse heart), dibutyryl
cAMP, [2,3-dichloro-4-(2-methylene-butyryl)phenoxy]acetic acid
(ethacrynic acid, EA),
5-[aminomethylsulfonyl]-4-chloro-2-[(furanylmethyl)amino]benzoic
acid (furosemide), genistein, isobutylmethylxantine, protein G, and
5-N,N-hexamethylene amiloride (NHA) were obtained
from Sigma (St. Louis, MO).
1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic
acid tetrakis(acetoxymethyl ester) (BAPTA-AM), and
2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein
acetoxymethyl ester (BCECF-AM) were purchased from Molecular
Probes Europe BV (Leiden, The Netherlands). Glutardialdehyde (electron
microscopic grade) and Tween 20 were obtained from Merck (Darmstadt,
Germany). 3,3',5,5'-tetramethylbenzidine was purchased from Serva
Feinbiochemica (Heidelberg, Germany). Percoll was obtained from
Pharmacia (Uppsala, Svezia). FN was purified from human plasma by
affinity chromatography on gelatin as previously described (29). Human
rTNF, produced in the yeast Pichia pastoris, was obtained
from Bissendorf Biochemicals (Hannover, Germany).
Na36Cl- (sp. act. 14–15 µCi/g
Cl-) was purchased from Amersham International (Amersham,
U.K.). All other reagents and chemicals were of the highest purity
grade available.
Antibodies
The following murine mAbs were used in this study: mAb TS1-18 (IgG1) and mAb 60.3 (IgG2a), recognizing the CD18 subunit (common ß-chain) of the CD11/CD18 Ag complex (30, 31); mAb TS1-22, mAb MY-904, and mAb 3.9, all belonging to the IgG1 subclass, which recognize CD11a (31), CD11b (32), and CD11c (33), respectively; and mAb BB7.5 (IgG1), directed against common determinants on the HLA-A,B,C (class I) molecule (34). mAb 60.3 and mAb 3.9 were kindly donated by Dr. J. M. Harlan (Washington University, Seattle, WA) and Dr. N. Hogg (Imperial Cancer Research Fund, London, U.K.), respectively. mAbs TS1-18, TS1-22, MY-904, and BB7.5 were affinity-purified from ascite fluids recovered from mice injected with the corresponding cell lines obtained from the American Type Culture Collection (Manassas, VA). Affinity-purified mAb A3G5 (IgG1) recognizes eosinophil peroxidase.
PMN isolation
PMN were isolated onto a Percoll gradient following the method described by Metcalf et al. (35), with slight modifications. Briefly, fresh blood collected in EDTA was layered onto a discontinuous gradient of 62% and 75% Percoll in PBS and centrifuged at 200 x g for 10 min and then at 400 x g for a further 15 min. The neutrophils were collected at the interface between the 62% and 75% Percoll and washed once in HEPES buffer (145 mM NaCl, 5 mM KCl, 5 mM glucose, 5 mM HEPES buffer, pH 7.4, and 0.2% BSA). Isolated PMN were freed of contaminating erythrocytes by a 10-s hypotonic lysis, washed again in HEPES buffer, and resuspended in the same medium. The resulting cell population contained 95–97% neutrophils, 2–3% eosinophils, and 1–2% mononuclear cells.
Preparation of FN-coated surfaces
The coating of flat-bottom microtiter plate-wells (MaxiSorp Immuno microwell plates, catalogue no. 442404; Nunc, Roskilde, Denmark) with FN was performed as previously described (36). Briefly, 50 µl of 20 µg/ml FN in PBS were deposited into replicate wells and the plate was left at 37°C for 1–2 h in a humidified incubator. Just before use, the wells were washed three times with PBS.
Preparation of mAb-coated surfaces
Immobilization of mAbs onto plate microwells with hydrazide (HZ) surface (HZ-wells) (carbohydrate-binding 8-well Strip Plate, catalogue no. 2508, Costar, Cambridge, MA), which allows site-specific binding of Abs through the carbohydrate moieties of the Fc region (37), was performed following the manufacturer’s protocol, with slight modifications. Briefly, mAbs were diluted to 10 µg/ml in 10 mM sodium acetate buffer, pH 4.0, containing 2.5 mM sodium periodate. Periodate-mediated activation of Fc-associated carbohydrate residues was continued for 30 min. Afterward, 50 µl aliquots of mAbs bearing dialdehydes groups of oxidized carbohydrates were transferred into HZ-wells and left to react for 1 h with the amine groups present on the HZ surface. The wells were then washed with two well-volumes of PBS-BSA 0.1% (w/v) added with 0.1% (v/v) Tween 20 (Merck, Darmstadt, Germany) and further rinsed extensively with PBS. Quenching of the remaining active sites of the wells was obtained by 1 h incubation with a 1% (w/v) solution of BSA in 10 mM Tris-HCl buffer, pH 8.8. Just before adding the cells, the wells were again extensively washed with PBS. Binding of mAbs was detected by ELISA with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Igs (Sigma) diluted 1/5000 in PBS-BSA-Tween 20 and revealed by using 3,3',5,5'-tetramethylbenzidine as substrate. After blocking the peroxidatic reaction with 2 N H2SO4, absorbance was read at 405 nm with a microplate reader (Multiskan MCC/340; Labsystem Oy, Helsinki, Finland). Binding of mAbs to protein G-coated surfaces was performed according to the method described by Schramm and Paek (38), with some modifications. Briefly, protein G (20 µg/ml in PBS) was incubated overnight at 4°C in microtiter plate wells (Nunc). After extensively washing with PBS, the wells were filled with a 1% (w/v) solution of BSA in PBS and incubated for 1 h at room temperature. After another washing, 50 µl of the mAb solution (10 µg/ml in PBS) were placed in the wells and further incubated for 1 h at room temperature. After another quenching step with the BSA solution, the wells were washed again with PBS just before adding the cells. Binding of mAbs was detected by ELISA with HRP-conjugated sheep anti-mouse IgG F(ab')2 (Sigma) diluted 1:10,000 in PBS-BSA-Tween 20 and revealed as described above. Binding of mAbs (250 µg/ml) to Staphylococcus aureus (Pansorbin, Calbiochem, San Diego, CA) was performed exactly as previously described (17). HZ-wells, protein G-coated wells, and S. aureus particles were coated with saturating doses of mAbs.
Measurements of 36Cl- efflux from PMN
PMN (10–15 x 106 cells/ml in HEPES buffer)
were loaded with 36Cl- as described elsewhere
(13). Briefly, PMN were incubated with 36Cl-
(3.0–4.0 µCi/ml) for 2 h at 37°C in a shaking water bath to
equilibrate 36Cl- between the intracellular
and extracellular compartment. The cells were then washed twice with
prewarmed unlabeled HEPES buffer to remove the tracer and suspended in
the same buffer at 2 x 106 cells/ml. To measure
36Cl- release from PMN exposed to TNF on
FN-coated surfaces, PMN were preincubated in suspension for 10 min at
37°C with or without 10 µg/ml of either mAb 60.3 or mAb TS1-18. On
completion of the preincubation, the cell suspensions were
supplemented, unless otherwise stated, with 1 mM CaCl2 and
1 mM MgCl2 (Ca2+/Mg2+-HEPES
buffer). Then, 50 µl of the cell suspensions were added to replicate
wells (8 wells per assay condition) containing, in a 100-µl volume of
prewarmed Ca2+/Mg2+-HEPES buffer, 15 ng/ml TNF
(final concentration 10 ng/ml). To measure
36Cl- release from unstimulated PMN plated
onto mAbs immobilized to either HZ-wells or protein G-coated wells, the
cells (2 x 106/ml in HEPES buffer) were first
prewarmed in suspension for 10 min at 37°C. Then, 50 µl of the cell
suspension were transferred to replicate wells coated with mAbs as
described above and containing 100 µl of prewarmed HEPES buffer.
After 1 h incubation at 37°C in a humidified incubator, 130-µl
aliquots of the assay medium were collected from the replicate wells
and pulled into microfuge tubes. After a 15-s centrifugation step at
12,000 x g, 750-µl aliquots of the supernatants were
withdrawn and the 36Cl--associated
radioactivity was counted by liquid scintillation counting in a Beckman
LS6000TA ß counter (Beckman Instruments, Fullerton, CA).
36Cl- efflux from PMN in suspension was
measured in polypropylene test tubes with 1) cells (2 x
106 in 1 ml of Ca2+/Mg2+-HEPES
buffer) stimulated by 10 ng/ml TNF (Fig. 1
, S-PMN); 2) cells (2 x
106 in 1 ml of HEPES buffer) exposed to mAb-coated S.
aureus particles; and 3) cells (5 x 106 in 1 ml
of HEPES buffer) preincubated for 30 min at room temperature with
anti-ß2 mAbs (final concentration 5 µg/ml) and then
exposed, after washing, to a goat anti-mouse F(ab')2
(final concentration 2.5 µg/ml). After 1 h incubation at 37°C
in a shaking water bath, 800-µl aliquots of the assay medium were
collected from duplicate tubes and centrifuged for 15 s at
12,000 x g. Afterward, 750-µl aliquots were
withdrawn and counted as described above. To evaluate the effect of the
Cl- transport inhibitors, PMN were preincubated in
suspension with the compounds for 10 min at 37°C. The percentage
efflux was calculated as follows: [(cpm in the supernatant of
tx sample) - (cpm in the supernatant of
t0 sample)]/[(total cpm of cell suspension) -
(cpm of t0 supernatant)] x 100.
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To determine the net movement of 36Cl- in PMN exposed to anti-ß2 integrin mAbs, the changes in 36Cl-i were measured. PMN (3–5 x 106 cells/ml) were loaded with 36Cl- and used without washing them free of the tracer. The assay was performed with either cells exposed to anti-ß2 integrin mAbs bound to HZ-plate wells or in the model of cells incubated in suspension with anti-ß2 integrin mAbs and then exposed to a secondary goat anti-mouse F(ab')2 (for details, see previous paragraph). In the HZ-plate method, on completion of the incubation the plate was spun at 250 x g for 5 min and the supernatant was discarded. After exhaustively washing the wells with PBS prewarmed at 37°C, the wells themselves were introduced in the vials and counted for radioactivity. In the assay performed with cells in suspension, the incubation was stopped by diluting the assay mixture with prewarmed PBS. After exhaustively washing the cells in the same medium, the bottom of the tubes were cut and the cell pellet-associated radioactivity was counted. The 36Cl- that remained associated to PMN at the selected incubation time was expressed as a percentage of 36Cl- associated to PMN at t = 0.
Immunofluorescence flow cytometry
Aliquots of 36Cl--loaded PMN were cooled to 4°C and incubated for 1 h with the indicated mAbs (2 µg/ml). After two washes with ice-cold PBS, the cells were incubated for a further 45 min with a FITC-labeled affinity-purified rabbit anti-mouse IgG F(ab')2. After two additional washings, the PMN were suspended in PBS containing 2% formaldehyde and analyzed by a flow cytometer (EPICS-C; Coulter, Hialeah, FL).
Assay of O2- production
Production of O2- was measured by means of the superoxide dismutase-inhibitable cytochrome c reduction, as detailed elsewhere (36). Briefly, 50 µl of PMN suspended at 2 x 106 cells/ml in HEPES buffer were added to mAb-coated wells containing, in a 100-µl volume of the same medium, 0.18 mM cytochrome c. Both the cell suspension and the plate were prewarmed for 5 min at 37°C. At the desired times, the plate was read at 550 nm and 540 nm. The amount of reduced cytochrome c was calculated from the absorbance difference between 550 nm and 540 nm using as a standard an absorbance of 0.037 OD units for 1 nmol of reduced cytochrome c.
Assay of adherence
Adherence assay was performed exactly as previously described (10). The quantitation of adherent PMN was performed by an enzymatic assay based on the measurement of myeloperoxidase activity.
Assessment of cell spreading
Phase contrast photomicrographs of PMN in contact with surface-bound anti-ß2 integrin Abs were taken from the wells where 36Cl- release was assayed by using a Leitz IMDL inverted microscope (Leica Mikroscopie & Systeme, Wetzlar, Germany) equipped with a Pentax K1000 reflex camera (Pentax, Tokyo, Japan).
Measurement of pHi
pHi was assayed by fluorescence spectrophotometry in PMN loaded with the pH-sensitive dye BCECF-AM using a 650-10S fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT). After loading with 36Cl-, the cells were washed free of the tracer, suspended in HEPES buffer containing 5 µM of BCECF-AM, and then incubated for 30 min at 37°C in a shaking water bath. mAb TS1-18 (final concentration 5 µg/ml) was added to the incubation mixture during the last 15 min of the incubation with BCECF-AM. After a washing step with prewarmed PBS, the cells were counted and suspended at 5 x 106/ml in HEPES buffer. Then, 1 ml of cell suspension was transferred to a cuvette thermostated at 37°C under continuous stirring. Changes in pHi were monitored upon addition of 2.5 µg/ml of a goat anti-mouse F(ab')2. To assay the effect of NHA or EA on pHi changes, the secondary Ab was added to cells previously preincubated with the required compound for 10 min at 37°C. The nigericin/K+ method described by Thomas et al. (39) was used to calibrate pHi.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PMN were loaded with 36Cl- and then
exposed to TNF either in suspension (S-PMN) or in contact with FN
immobilized on a solid support (FN-PMN). As shown in Fig. 1
, a
36Cl- efflux occurred from both FN-PMN and
S-PMN, but the 36Cl- efflux from FN-PMN was
>65% higher than that induced in S-PMN (86.6 ± 16.5% vs
51.2 ± 3.2%, mean ± SD, n = 4). The
following experimental evidence suggested that this extra
36Cl- efflux was dependent on ß2
integrin-mediated adherence of PMN to FN. First,36Cl- efflux from TNF-stimulated FN-PMN was
significantly decreased in the presence of two mAbs (60.3 and TS1-18)
directed against the common ß-chain (CD18) of leukocyte
ß2 integrins. These mAbs also inhibited PMN adherence to
FN (inset in Fig. 1). Second, both adherence
(inset) and 36Cl- efflux of FN-PMN
were significantly decreased in Mg2+-free buffer, a
condition known to prevent ß2 integrin activation (40, 41), (12.8 ± 3.6% adherence and 42.9 ± 1.1%
36Cl- efflux in Mg2+-free buffer
vs 60.2 ± 10.5% adherence and 86.6 ± 16.5%
36Cl- efflux of control, mean ± SD,
n = 4). It is worthy of note that the
36Cl- efflux from FN-PMN in
Mg2+-free buffer was similar to that measured in S-PMN. As
expected, the release of 36Cl- in S-PMN was
unaffected by the absence of Mg2+.
Engagement of the ß-chain (CD18) of ß2 integrins by surface bound anti-CD18 mAbs triggers Cl- efflux from PMN
In the light of the results of the previous section, we set out to demonstrate directly the ability of ß2 integrins to trigger Cl- efflux from PMN independently of other agonists. To this end, we exploited an experimental model based on the cross-linking of ß2 integrins by specific mAbs immobilized through their Fc portion onto solid supports. The use of this method has already led to the demonstration that ß2 integrins deliver signals for some PMN functions, such as the reorganization of the cytoskeleton (17), the release of oxygen reduction products (17, 42), the activation of phospholipase D (23, 24), and protein tyrosine phosphorylation (18, 28).
The cross-linking of ß2 integrins of 36Cl--loaded PMN was obtained by exposing the cells to the anti-CD18 mAb TS1-18 immobilized on different supports, such as HZ-wells, protein G-coated polystyrene wells, and S. aureus particles in suspension, or by adding a secondary goat anti-mouse F(ab')2 to PMN suspensions that have been pre-exposed to mAb TS1-18. 36Cl- efflux was then measured in the supernatant after 60 min of incubation at 37°C as detailed in Materials and Methods.
Fig. 2a shows that the
engagement of CD18 by mAb TS1-18 immobilized on HZ-wells induced
36Cl- efflux. Two unrelated isotype-matched
mAbs were also tested: mAb BB7.5, which reacts with a combinatorial
determinant of the HLA-A,B,C molecule (34) expressed in PMN at levels
not significantly different from those of CD18 (Fig. 2b)
(mean peak fluorescence intensity = 17.5 ± 6.0 of mAb BB7.5
vs 31.0 ± 13.3 for mAb TS1-18; mean ± SD,
n = 4, p = 0.080 by t test
on paired data), and mAb A3G5, which recognizes eosinophil peroxidase
(M. Romano, unpublished observations). Both mAbs triggered only a minor
36Cl- efflux (Fig. 2a), despite the
fact they bound to HZ-wells similarly to mAb TS1-18 (inset
in Fig. 2a). These results support the hypothesis of a
selective ability of the PMN surface proadhesive complex CD11/CD18 to
trigger Cl- release and, at the same time, rule out a
possible Fc-dependent stimulation of 36Cl-
efflux.
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c),
although the overall response was quantitatively lower than that
measured on HZ. Such a difference could be accounted for by the lower
binding of the mAbs to protein G-coated wells (see legend to Fig. 2).
Further evidence of the ability of ß2 integrins to
mediate 36Cl- efflux from PMN was obtained by
using a different experimental approach, in which the cross-linking of
ß2 integrin was induced by exposing cells in suspension
to mAb-coated S. aureus particles. As shown in Fig. 2d, the mAb TS1-18-coated particles triggered a considerable
36Cl- efflux from PMN, which was markedly
higher than that induced by mAb BB7.5. This difference could not be
ascribed to a differential engulfment of the particles by PMN because
we found that 1) the phagocytosis of mAb-coated S. aureus
particles was negligible, in agreement with previously reported results
(24), and 2) 36Cl- efflux did not
substantially vary when assayed in the presence of a phagocytosis
blocking drug, such as cytochalasin B (data not shown).
Fig. 2e shows that Cl- efflux could be well
detected also when ß2 integrin cross-linking was induced
in an alternative way, i.e., by adding a goat anti-mouse
F(ab')2 to PMN suspensions that have been previously
incubated with mAb TS1-18. It is worthy of note that ß2
integrin cross-linking led to a net Cl- efflux, as
indicated by the concomitant decrease in
36Cl-i (Fig. 2e,
inset).
ß2 integrin-mediated Cl- efflux: individual signaling ability of LFA-1 (CD11a/CD18), CR3 (CD11b/CD18), and gp150/95 (CD11c/CD18)
The results reported in the previous paragraph prompted us to
investigate the signaling properties of each member of the
ß2 integrin subfamily. To this end, we measured the
36Cl- efflux from PMN plated onto HZ-bound
mAbs directed against the -chains of either LFA-1, CR3, or gp150/95.
Fig. 3a shows that, upon
cross-linking with specific mAbs, all three ß2 integrin
subfamily members were able to induce 36Cl-
efflux from PMN and that the extent of this efflux varied, depending on
the type of ß2 integrin engaged. Of the three
ß2 heterodimers present on PMN, gp150/95 was the most
powerful in triggering Cl- efflux, followed by LFA-1
(nearly 80% of the response measured with gp150/95) and CR3 (
50%
of the response of gp150/95). The differences in the release of
36Cl- could not be ascribed to a differential
binding of the three anti--chain mAbs to HZ-wells because all
mAbs bound similarly to the solid support (Fig. 3b).
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-chains mAbs triggered a
36Cl- efflux significantly higher than that
induced by the isotype-matched mAb BB7.5 (p <
0.02; t test on paired data, n = 6), despite
the fact that this mAb bound similarly to HZ-wells (Fig. 3b)
and that the expression of the molecule it recognizes on PMN surface
was comparable to that of LFA-1, nearly twice as high as that of
gp150/95 and almost half of that of CR3, as revealed by flow cytometry
studies (Fig. 3c).
The differential response of the three ß2 integrin
subfamily members can be better appreciated when the data of
36Cl- efflux are expressed as a function of
the degree of expression of the three heterodimers (Fig. 3d). Based on this elaboration, gp150/95 appears to be
largely the most effective in inducing 36Cl-
efflux, whereas the effect of CR3 is modest if not negligible, and
LFA-1 is about 60% less active than gp150/95.
Relationships between Cl- efflux, spreading, and O2- generation induced by ß2 integrin cross-linking
The selective cross-linking of either CR3, LFA-1, or gp150/95 by
mAbs directed against the -chain of these molecules has led to the
demonstration that all of these three leukocyte integrins can generate
signals that trigger PMN spreading, whereas the ability to deliver
signals that activate the respiratory burst seems to be restricted to
LFA-1 and gp150/95 (17).
The question then arises whether the Cl- efflux induced by the cross-linking of ß2 integrins is somehow linked to spreading and metabolic activation, and, if so, which one of the three heterodimers plays the most relevant role.
To address this question, we evaluated, in parallel experiments,
36Cl- efflux, cell shape changes, and
O2- generation in PMNtreated or untreated
with EA, a Cl- transport blocker belonging to the
phenoxyacetates family, which has been shown to inhibit
Cl- fluxes in several cell types (13, 42, 43, 44, 45). This drug
was selected on the basis of a previously reported screening test
showing that EA was far more efficient concentration inhibiting 50% of
response (IC50 in the µmol range) than other known
Cl- transport blockers (IC50 in the mmol
range) in inhibiting Cl- efflux, spreading, and
O2- generation of PMN stimulated by TNF on
FN-coated surfaces (10). Fig. 4 shows
that mAbs TS1-22, MY 904, and 3.9, which recognize the -chains of
LFA-1, CR3, and gp150/95, respectively, as well as mAb TS1-18, which is
directed against the common ß-chain of ß2 integrins,
triggered both Cl- efflux (upper panel) and
cell spreading (middle panel). The mAbs TS1-22, TS1-18 and,
to a lesser extent, mAb 3.9 also stimulated release of
O2- from PMN, whereas cross-linking of CR3 by
mAb MY 904 triggered only a minor metabolic response, which was not
significantly different from the metabolic activation elicited by the
control mAb BB7.5 (9.5 ± 1.9 nmol/106 PMN of
O2- with mAb MY 904 vs 7.4 ± 1.7 nmol
O2-/106 PMN with mAb BB7.5;
mean ± SEM, n = 5, p = 0.090 by
t test on paired data). Cells treated with EA, which failed
to release 36Cl- (upper panel, open
columns), maintained a roundish appearance (f in
middle panel) and did not mount a respiratory burst
(lower panel, open columns), suggesting that the
release of Cl- is required for both spreading and
metabolic activation. A possible toxic effect of EA was excluded
because EA-treated cells were >98% viable and their metabolic
response to other agonists (for example, 10-7 M FMLP) was
not significantly affected (data not shown).
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, the inhibition of
Cl- efflux was, whatever its extent, invariably
accompanied by an almost proportional inhibition of both metabolic
burst and cell spreading (see data with 250 µM and 500 µM CHC, 100
µM MK-447/B, 50 µM and 100 µM MK-447 and MK-447/A). Conversely,
drugs that failed to inhibit Cl- efflux neither modified
the metabolic response nor substantially affected PMN spreading (see
data with 250 µM and 500 µM furosemide and 50 µM MK-447/B).
Interestingly, two compounds of the 2-(aminomethyl)phenols family,
namely MK-447 and MK-447/A, appeared to be very active in inhibiting
all three PMN responses (see Table I and Fig. 5), suggesting that membrane
stretch-activated PMN Cl- channels, described so far as
the specific targets of these compounds (47), may be involved in
mediating Cl- efflux triggered by ß2
integrin cross-linking. PMN viability was not affected by any of the
agents used because in all cases >98% of the cells excluded the dye
trypan blue (data not shown).
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reports the results of time course
experiments in which 36Cl- efflux and
O2- generation were simultaneously measured.
36Cl- release, but not the generation of
O2-, was well detectable by 10 min of
incubation. The half-maximal response time was 15 min and 21 min for
36Cl- efflux and O2-
generation, respectively. This demonstrates that the efflux of chloride
ions precedes the activation of the respiratory burst. Cl-
efflux also precedes PMN spreading, as the latter begins to be clearly
detectable in the majority of cells after 20 min of incubation, judging
by phase contrast microscopy observations (not shown). The results
obtained in experiments performed with PMN placed in wells coated with
different amounts of anti-ß2 integrin mAbs further
supported the hypothesis that efflux of chloride ions may regulate both
spreading and metabolic activation of PMN. In fact, Cl-
efflux increased by increasing the concentration of the mAb solution
used to coat the wells (upper panel of Fig. 7, open columns), accompanied by a
parallel enhancement of both O2- generation
(black columns) and cell spreading (lower panel).
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shows that the cross-linking of
ß2 integrins caused, besides Cl- efflux, a
decrease in Cl-i, thus proving that a net
outward transport of Cl- ions occurred. This decrease in
Cl-i was accompanied by
O2- generation, which was markedly reduced if
the drop in Cl-i was prevented by treating the
cells with EA. In contrast, PMN at rest did not show any appreciable
change in Cl-i or activation of the
respiratory burst. These results suggest that a drop in the cytosolic
chloride content is required for the activation of the metabolic
response to take place.
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Role of chloride ion channels.
Besides stretch-activated channels, whose putative involvement in
ß2 integrin-mediated Cl- efflux is suggested
by the results reported in Table I, two other types of Cl-
channels have been well characterized so far in human PMN, i.e.,
calcium-activated, voltage-independent channels (48), and
voltage-dependent, protein kinase C-regulated channels (49).
Fig. 9 shows that Cl- efflux
measured in cells loaded with the intracellular Ca2+
buffering agent BAPTA-AM and resuspended in Ca2+-free
EGTA-containing buffer was almost undistinguishable from
Cl- efflux of control cells. The effectiveness of the
BAPTA-AM/EGTA treatment in preventing [Ca2+]i
alterations was indirectly demonstrated by the inhibition of the
FMLP-induced PMN respiratory burst, a response known to be dependent on
[Ca2+]i elevations (0.2 nmol of
O2-/106 BAPTA-AM/EGTA-treated PMN
vs 6.7 nmol of O2-/106 untreated
PMN).
|
shows also that ß2 integrin-mediated
Cl- efflux is unlikely to occur through a
voltage-dependent channel, because the efflux appeared insensitive to a
marked elevation of extracellular K+ concentration
([K+]o = 140 mM), a condition known to
markedly impair K+ gradients across the PMN plasma membrane
and, hence, to clamp the membrane potential at highly depolarized
values (50). Moreover, Cl- efflux remained unchanged in
cells treated with H-7, a known inhibitor of the protein kinase C, an
intracellular messenger that regulates the opening of voltage-dependent
Cl- channels in PMA- and FMLP-stimulated PMN (49) (45.7%
of 36Cl- efflux of H-7-treated PMN vs 42.8%
of 36Cl- efflux of untreated PMN; means of two
experiments performed with cells of different donors).
Fig. 9 shows also that Cl- efflux of PMN pretreated with
either BaCl2, known to block both the voltage-dependent and
the Ca2+-activated K+ currents of neutrophils
(48) or quinine, a widely used inhibitor of leukocyte K+
channels (reviewed in 51 , did not differ from those of untreated
cells. This result, together with that obtained in the high
K+ buffer, as discussed above, speaks against a possible
role for K+ efflux in mediating ß2
integrin-dependent chloride release.
Role of chloride ion carriers.
The fact that a nominally HCO3--free buffer is
used throughout this study makes unlikely the involvement of the
electroneutral Cl-/HCO3-
exchanger in ß2 integrin-mediated Cl-
efflux, because this carrier can export internal Cl- only
in the presence of exchangeable external HCO3-
(52). The results presented in Table I, showing that Cl-
efflux is not modified by pretreating the cells with furosemide, speak
against a possible role in such an efflux of the
Na+-K+-2Cl- cotransporter
whose distinct feature is the sensitivity to inhibition by
this drug (53).
Signaling pathways involved in Cl- efflux induced by ß2 integrin cross-inking
Role of tyrosine phosphorylation and lowering of intracellular cAMP levels. Studies performed with PMN exposed to either TNF on biologic surfaces or surface-bound anti-CD18 mAbs demonstrated that ß2 integrins are undoubtedly involved in the generation of intracellular signals, such as protein tyrosine phosphorylation (15, 16, 18, 54) and lowering of intracellular cAMP levels (6), which, in turn, have been shown to modulate PMN spreading and activation of respiratory burst (6, 10, 15, 18, 54).
The results reported in the previous paragraph, showing that
Cl- efflux is involved in the regulation of spreading and
O2- generation in PMN exposed to surface-bound
anti-ß2 integrin mAbs, raised the question as to
whether the release of Cl- ions is in some way related to
the activation of neutrophil tyrosine kinases and/or to a decrease of
the intracellular levels of cAMP. Fig. 10 shows that neither genistein, a
widely used inhibitor of protein tyrosine kinases (7, 15, 55), nor the
phosphodiesterase inhibitor isobutylmethylxantine, used in combination
with the cAMP analogue dibutyryl cAMP, affected the
36Cl- efflux induced by ß2
integrin cross-linking. In agreement with previously reported data
(6, 7, 8, 15), the same drugs markedly inhibited both
O2- generation (inset in Fig. 10)
and cell spreading (not shown). These results strongly suggest that
release of Cl- by PMN exposed to anti-CD18 mAbs does
not depend on either activation of tyrosine kinases or lowering of cAMP
levels.
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To explore such a possibility, Cl- efflux and
pHi were measured in suspended PMN preincubated with the
anti-ß2 integrin mAb TS1-18 and then exposed to a
secondary goat anti-mouse F(ab')2. Fig. 11 shows that ß2 integrin
cross-linking led to appreciable pHi changes. These
exhibited the classical pathway of a quick and transient acidification
followed by an alkalinizing response that was nearly half as high as
that induced by the chemotactic peptide FMLP. The raise in
pHi triggered by ß2 integrins seemed to
precede the full activation of the Cl- efflux, because the
cytoplasmic alkalinization plateaued after 3–4 min, when the release
of 36Cl- was only at its onset (data not
shown). The alkalinizing response was absent in PMN incubated with the
isotype-matched mAb BB7.5, ruling out the possibility that the raise in
pHi was accounted for by a Fc-dependent stimulation of the
Na+/H+ antiporter (57). Fig. 11 shows also that
a potent and specific inhibitor of the antiporter, the amiloride
analogue NHA (58), virtually abrogated the cytoplasmic alkalinization,
suggesting that the raise in pHi depends on the activation
of the Na+/H+ exchanger. Interestingly, NHA had
a marked inhibitory effect also on Cl- efflux
(inset), whereas the Cl- transport blocker EA,
which blunted Cl- efflux (inset), had no
effects on pHi changes. Taken altogether, these results
indicate that the activation of the Na+/H+
antiporter occurs earlier than the activation of the Cl-
transport mechanism and suggest that the raise in pHi may
represent a positive regulatory signal for Cl- fluxes
triggered by ß2 integrin cross-linking.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At least one of the following experimental evidence is usually provided
as a demonstration of the involvement of ß2 integrins in
the modulation of PMN functions: 1) the inhibition of a given function
by mAbs directed against either the common ß-chain (CD18) or the
three distinct -chains (CD11a, CD11b, CD11c) of ß2
integrins (3, 6, 8, 9, 10, 16, 19, 26, 59, 60); 2) the absence, or marked
impairment, of that function in PMN isolated from LAD-1 patients that
lack ß2 integrin expression (3, 6, 16, 18, 26, 54, 61);
and/or 3) the triggering of the selected PMN response upon
cross-linking of ß2 integrins with specific mAbs in the
absence of other agonists (7, 17, 18, 19, 22, 23, 24, 27, 28, 42, 61, 62).
The results presented in this paper fulfill at least two of the above
criteria, because we have shown that 1) in a model of TNF-stimulated
PMN adhering via ß2 integrins to FN, mAbs recognizing
CD18 strongly inhibit Cl- efflux, bringing it back to
values similar to those measured from cells in suspension (Fig. 1); and
2) the cross-linking of ß2 integrins by surface-bound
anti-ß or anti--chains mAbs is sufficient, per se, to
elicit Cl- efflux from PMN (Fig. 2, 3, and 4).
An additional evidence strengthening these results is that, in the
absence of Mg2+, a condition which prevents
ß2 integrin activation (40, 41) and thus inhibits
ß2 integrin-mediated adherence of PMN to FN, the
TNF-induced Cl- efflux of PMN residing on FN is equivalent
to that of PMN in suspension (Fig. 1). Experiments with LAD cells could
not be performed because patients with this syndrome are not available
to us at the moment.
All three ß2 integrin subfamily members are expressed,
although at different levels, on PMN, CR3 (CD11b/CD18) being the most
abundant one, followed by LFA-1 (CD11a/CD18) and gp150/95 (CD11c/CD18)
(11). Over the last 10 years, several studies have clearly demonstrated
that all three ß2 heterodimers, besides their well
known involvement in the adhesive response (see Table I in 14 ,
can also act as outside-in signaling molecules. For example, by
selectively cross-linking each of the three ß2 integrin
subfamily members with mAbs directed against their distinct -chains,
it has been shown that CD11b/CD18 can trigger tyrosine phosphorylation
(50), both CD11a/CD18 and CD11c/CD18 generate signals that activate the
PMN respiratory burst (7, 17), and all three molecules can mediate PMN
spreading (17). Furthermore, it has been shown that CD11b/CD18, but not
CD11a/CD18 or CD11c/CD18, plays a major role in TNF- and FMLP-induced
degranulation of adhered neutrophils (19), in the adherence-dependent
oscillations of cytosolic free Ca2+ in unstimulated PMN
(19), and in the phorbol ester-induced paxillin tyrosine
phosphorylation in nonadhered PMN (54).
Cross-linking studies performed with specific anti--chains mAbs
allowed us to demonstrate that all three ß2 integrin
heterodimers molecules are capable of triggering Cl-
efflux from PMN. This response appears integrin specific and
independent of Fc involvement because cells plated onto an
isotype-matched mAb directed against HLA, the expression of which
on PMN surface is comparable or even higher than that of the three
ß2 heterodimers (Fig. 3c), underwent only a
minimal release of chloride ions (Fig. 3a).
Interestingly, gp150/95, the least expressed of the three
ß2 integrin subfamily members (Fig. 3c),
appears to be the most effective one in triggering Cl-
release because it elicits a response higher than that induced by the
engagement of either CR3 or LFA-1 (Fig. 3a). These findings
are in agreement with previously reported data suggesting that CR3,
LFA-1, and gp150/95 are differentially involved in the generation of
signals that activate selective PMN functions (7, 17, 19, 54, 62).
A further relevant finding of this study is that Cl-
efflux is causally related to two other PMN responses triggered by
ß2 integrin cross-linking, i.e., cytoskeleton
reorganization and activation of the respiratory burst. This relies on
the following evidence: 1) treatment of PMN with agents that inhibit
Cl- movements invariably lead to a parallel inhibition of
both spreading and activation of the respiratory burst (see Fig. 4, Table I, and Fig. 5), 2) Cl- efflux precedes the onset of
O2- generation (Fig. 6); and 3) a
dose-dependent enhancement of ß2 integrin-induced
Cl- efflux, obtained by placing the cells in wells coated
with different concentrations of anti-ß2 integrin
mAbs, proportionately enhances both the metabolic burst and the cell
spreading (Fig. 7).
It is worthy of note that signals generated by the cross-linking of CR3 led the PMN to release Cl- and spread, but failed to activate their respiratory burst. At first glance, this finding could be in line with previously reported data, showing that CR3-dependent signals activate selected PMN functions, e.g. cytoskeleton reorganization, but not others, such as assembly and/or activation of NADPH oxidase, which leads to the respiratory burst (17, 61). However, because the metabolic activation seems to be dependent on Cl- efflux, the inability of CR3 to induce a respiratory burst may be explained in at least two ways: 1) the Cl- release triggered by CR3 is quantitatively insufficient to activate the burst (CR3 is indeed the least effective Cl- releaser) although sufficient to induce spreading; and 2) additional signals provided by LFA-1 and gp150/95, but not CR3, are required, together with Cl- release, to activate the respiratory burst.
Both protein tyrosine phosphorylation and decrease in intracellular
cAMP levels have been shown to be required for ß2
integrin-dependent spreading and metabolic activation of PMN stimulated
by TNF on biologic surfaces (6, 7, 8, 15, 16) or of resting PMN exposed to
surface-bound anti-ß2 integrin mAbs (7, 17, 18). The
relationships between these two signals and Cl- efflux
remain unresolved at present. The finding that drugs which block
tyrosine kinases or prevent the decrease in cAMP levels do not modify
ß2 integrin-induced Cl- release but, as
expected, inhibit spreading and the respiratory burst (Fig. 10)
indicates that Cl- efflux takes place independently of the
two other signals.
At this point, it seems legitimate to ask the question whether tyrosine
kinase activation and/or decrease of cAMP levels are somehow dependent
on Cl- efflux. In this respect, the data available are
very limited. It has been shown that in both resting PMN exposed to
surface-bound anti-CD18 mAbs (18) and TNF-stimulated PMN adhering
to immobilized FBS (15, 16) protein tyrosine phosphorylation is a
relatively late event, being clearly detectable by 15–60 min
incubation. In another study, an earlier (after 10 s) and
transient (60 s duration) increase in tyrosine phosphorylation of
several PMN proteins induced by cross-linking of ß2
integrins was described (62), but its functional relevance was not
assessed in that paper. Concerning the decrease in cAMP levels, the
dependence of such response on ß2 integrin engagement was
formally documented at 45 min of incubation in PMN exposed to TNF on
FBS- or fibrinogen-coated plastic (6). Because Cl- efflux
elicited by the engagement of CD18 was well measurable as early as 10
min of incubation (Fig. 6), it does not seem unreasonable at present to
hypothesize that signals generated by ß2 integrin
cross-linking trigger the release of chloride ions before tyrosine
kinase activation and decrease in cAMP levels take place.
Several possibilities were explored to investigate the mechanisms underlying the ß2 integrin-activated Cl- efflux. The marked inhibitory effect exerted by the compound MK-447 and its analogue A, which have been described as selective inhibitors of membrane stretch-activated Cl- channels in human PMN (47), is of particular interest. Indeed, because the stretching of cell plasma membrane is likely to occur upon ß2 integrin cross-linking by immobilized ligands, it is conceivable to hypothesize a role for these channels in regulating Cl- efflux. However, because the biological activity of the compounds of the MK-447 series is of recent identification, the possibility should be considered that they possess a broader spectrum of targets and thus may affect other Cl- transport mechanisms. This is suggested also by the inhibitory effect exerted by a low, nontoxic concentration of EA, which has not been reported so far to inhibit Cl- fluxes through stretch-activated channels.
Our results tend to exclude a role for other two types of Cl- channels known to operate in the PMN, i.e., the calcium-activated, voltage-independent channels (48) and the voltage-dependent, protein kinase C-regulated channels (49). In fact, both the inhibition of [Ca2+]i changes and the clamping of the plasma membrane potential to highly depolarized values did not appreciably affect Cl- efflux.
Likewise, a known Cl- transporter of leukocytes, i.e., the electroneutral Cl-/HCO3- exchanger, does not seem to play a role in this outward Cl- transport process, because the use of a nominally HCO3--free buffer makes unlikely the involvement of a carrier that can export Cl- only in the presence of exchangeable HCO3- (52). In addition, the finding that furosemide does not inhibit ß2 integrin-mediated Cl- efflux seems to exclude the possibility that such an efflux is mediated by the furosemide-sensitive Na+-K+-2Cl- cotransporter (53).
An interesting finding is that Cl- efflux appears to be
dependent on a ß2 integrin-induced raise in
pHi (see Fig. 11). This relies on the following evidence:
1) the cross-linking of ß2 integrins is sufficient, per
se, to cause a distinct cytoplasmic alkalinization likely due to
activation of the Na+/H+ exchanger; 2) the
alkalinizing response precedes the full activation of Cl-
release; and 3) Cl- efflux is markedly reduced in PMN
treated with NHA, a specific inhibitor of the
Na+/H+ antiporter, whereas the raise in
pHi is unaffected by the Cl- transport blocker
EA. These findings agree with those previously reported by Demaurex et
al. (27), showing the occurrence of pHi changes during PMN
spreading on surfaces coated with anti-ß2 integrin
mAbs. In a recent paper by Fukushima et al. (57), no changes in
pHi were found to occur in suspended PMN by cross-linking
anti-ß2 integrin mAbs with a secondary anti-mouse
Ab, but differences in the mAb used and in several experimental
conditions with respect to our study may explain this discrepancy.
Because it is well established that both cationic and anionic channels
can be modulated by changes in pHi (63, 64, 65, 66), it is
legitimate to hypothesize that an as yet unidentified
pHi-regulated Cl- channel can operate in
PMN after ß2 integrin cross linking. Experiments
addressing this issue are in progress in our laboratory.
In conclusion, this paper uncovers a new signaling ability of ß2 integrins and contributes to highlight the role of Cl- efflux in the outside-in signal transduction pathway that regulates adherence-dependent PMN responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. R. Menegazzi, Department of Physiology and Pathology, University of Trieste, via A. Fleming, 22, 34127 Trieste, Italy. E-mail address:
3 Abbreviations used in this paper: PMN, neutrophilic polymorphonuclear leukocytes; S-PMN, PMN in suspension; FN, fibronectin; FN-PMN, PMN residing on FN-coated surfaces; LAD-1, type 1 leukocyte adhesion deficiency; MK-447, 2-aminomethyl-4-(1,1-dimethylethyl)-6-iodophenol hydrochloride; MK-447/A, 2-aminomethyl-4-(1-methyl-1-phenylethyl)-6-chlorophenol hydrochloride; MK-447/B, 2-aminomethyl-4-(1,1-dimethylethyl)-6-methylketone hydrochloride; NHA, 5-N,N-hexamethylene amiloride; BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester); BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester; EA, [2,3-dichloro-4-(2-methylene-butyryl)phenoxy] acetic acid (ethacrynic acid); CHC, -cyano-4-hydroxy-cinnamic acid; HZ, hydrazide; HZ-wells, plate microwells with HZ surface; HRP, horseradish peroxidase.
Received for publication March 16, 1998. Accepted for publication September 1, 1998.
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