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PHAGOCYTES
From Servicio de Reumatología e
Inmunología, Hospital de la Princesa, Universidad
Autónoma de Madrid, Servicio de Reumatología, Hospital
Universitario de Canarias, Universidad de La Laguna, Santa Cruz de
Tenerife, Spain.
L-selectin is an adhesion molecule that plays an essential role in
the early events of the inflammatory response. Our group has recently
described that several nonsteroidal anti-inflammatory drugs
(NSAIDs) are able to induce both in vivo and in vitro the shedding of L-selectin in neutrophils through an unknown mechanism. In
this work, we have studied potential mechanisms involved in the
shedding of L-selectin induced by NSAIDs. This effect of NSAIDs did
not involve any detectable intracellular calcium flux. Pretreatment of
neutrophils either with Ro 31-8220 and H7, 2 specific inhibitors of
protein kinase C (PKC), or with inhibitors of protein tyrosine kinases
such as tyrphostin A25 or herbimycin A did not prevent the
NSAID-mediated L-selectin shedding. However, the KD-IX-73-4, an
inhibitor of L-selectin proteolysis was able to block the effect of
NSAIDs on L-selectin expression. Remarkably, NSAIDs caused a variable
reduction in the neutrophil intracellular ATP concentration that highly
correlated with the differential ability of NSAIDs to trigger
L-selectin shedding (r = 0.8, P < .01). In
agreement with this finding, azide plus 2-deoxy-D-glucose, 2 metabolic
blockers, also induced a rapid L-selectin shedding (65% ± 8%)
without affecting the neutrophil viability, activation, or expression
level of other surface molecules with soluble isoforms such as CD16 and
CD59. These data indicate that the maintenance of L-selectin on the neutrophil surface requires energy consumption, which suggests that
L-selectin is shed in neutrophils by default. Interestingly, NSAIDs
seem to cause the shedding of L-selectin, at least in part, through the
reduction of the intracellular ATP concentration.
(Blood. 2000;96:3592-3600) Nonsteroidal anti-inflammatory drugs (NSAIDs) are a
group of therapeutic agents widely used for symptomatic treatment of
nonsevere human inflammatory disorders. Since the early 1970s, it is
accepted that the mechanism of action of these agents is the inhibition of prostaglandin synthesis1 through the blockade of
cyclooxygenase (COX). However, several clinical2-4 and
experimental5,6 evidences suggest that COX inhibition
might not adequately explain the fully anti-inflammatory action of this
group of compounds. In the last decade, different studies have
described several prostaglandin-independent actions of NSAIDs, such as
the inhibition of oxidative phosphorylation in
mitochondria7-9 or the blockade of transcription factor
NF-kB,10 among many others.2,11-14 In this
regard, our group has reported that leukocyte adhesion molecules
essential for the inflammatory response may be potential targets for
NSAIDs.15-18
The accumulation of leukocytes into tissues is a cardinal event in the
inflammatory response that requires a complex coordination of adhesive
interactions between flowing leukocytes and endothelial cells. The
selectin family is a group of 3 adhesion receptors: L-, E-, and
P-selectin. Selectins recognize carbohydrate moieties expressed by both
leukocytes and activated endothelial cells, causing the slow down of
flowing leukocytes.19 This selectin-mediated reduction in
the leukocyte speed plays an important role in the inflammatory
response because it facilitates the action of other important adhesion
receptors such as integrins and members of the immunoglobulin
superfamily.20 Unlike E- and P-selectins, the cellular
mechanisms involved in the regulation of L-selectin (CD62L) expression
are not fully defined. L-selectin is constitutively expressed on the
cell surface of most leukocytes and is enzymatically cleaved and
released after cell activation both in vitro21,22 and in
vivo.23,24 Although the protease responsible for the cleavage of L-selectin has not been yet characterized, recent observations suggest that it may be a member of the ADAM (a desintegrin and metalloprotease) family of surface metalloproteases.25
Several evidences support the prominent role of L-selectin in the
inflammatory response. A human congenital disorder termed
leukocyte adhesion deficiency (LAD)
type II characterized by an aberrant inflammatory response is due to
the lack of a class of cell-surface carbohydrates, which includes
ligands of selectins.26 In addition, many groups have
confirmed in gene-deficient animals the relevant role of L-selectin in
the recruitment of leukocytes into inflamed tissues.27-29 On this basis, many efforts are currently concentrated in the development of new anti-inflammatory agents that selectively promote the shedding of L-selectin.30 This antiselectin therapy
has been already proved beneficial in several animal models of
inflammation.31-33
Our group has recently described that commonly prescribed NSAIDs such
as indomethacin, diclofenac, ketoprofen, sodium salicylate, aspirin,
and aceclofenac, but not others such as piroxicam, are able to induce
the shedding of L-selectin from the neutrophil surface.15,16 This effect of NSAIDs on L-selectin
expression has been also confirmed in vivo; standard anti-inflammatory
doses of indomethacin cause a significant reduction of L-selectin
expression in flowing neutrophils of healthy volunteers.15
However, the mechanism through which NSAIDs cause the L-selectin
shedding in neutrophils is still unknown.
The aim of this article was to study the mechanism involved in the
NSAID-mediated L-selectin shedding in neutrophils. We found that the
intracellular ATP levels play an important role in the maintenance of
L-selectin on the neutrophil surface. A group of NSAIDs seems to cause
the shedding of L-selectin, at least in part, through its
ability to reduce the intracellular ATP concentration.
Antibodies and reagents
Aceclofenac was provided by Almirall-Prodesfarma (Barcelona, Spain) and
meloxicam from Boëhringer Ingelheim GmbH (Ingelheim, Germany).
Diclofenac, mefenamic acid, flufenamic acid, aspirin (acetylsalicylic
acid), sodium salicylate, piroxicam, phenylbutazone, and indomethacin
were purchased from Sigma Chemical Co (St Louis, MO). The
2-deoxy-D-glucose (2-DG), azide, ionophore A23187, N-formyl-met-leu-phe
(f-MLP), phorbol myristate acetate (PMA), and prostaglandin E2 (PGE2)
were purchased from Sigma Chemical Co.
The protein tyrosine kinase inhibitors tyrphostin A25 and herbimycin A
and the protein kinase C (PKC) inhibitors H-7 and Ro 31-8220 were
purchased from Calbiochem-Novabiochem (San Diego, CA). The protein
kinase A inhibitor H-89 and the tyrosine phosphatase inhibitor sodium
orthovanadate were from Calbiochem-Novabiochem. NS-398, a selective
inhibitor of COX-2 was also obtained from Calbiochem-Novabiochem.
Fluo-3 AM was from Molecular Probes Inc (Eugene, OR).
The hydroxamic acid-based L-selectin shedding inhibitor,
KD-IX-73-4,36 was a gift from Dr Takashi Kei Kishimoto
(Boëhringer Ingelheim Pharmaceuticals, Inc, Ridgefield,
CT). Hanks' balanced salt solution (HBSS) was obtained from
BioWhittaker (Walkersville, MD).
Cell isolation and treatments
Experiments on the effect of NSAIDs on the expression of adhesion molecules were carried out in 15 mL disposable polypropylene tubes (Falcon Labware, Oxnard, CA). Neutrophils were resuspended in PBS and incubated either alone or in the presence of 20 µg/mL of piroxicam, meloxicam, phenylbutazone, aceclofenac, diclofenac, indomethacin, mefenamic acid, and flufenamic acid for 30 minutes at 37°C. Aspirin and sodium salicylate were used at 200 µg/mL. Dose-response experiments were performed under the same experimental conditions. Flufenamic acid, diclofenac, aceclofenac, and indomethacin were used at doses ranging from 1 to 50 µg/mL, whereas aspirin and sodium salicylate from 100 to 500 µg/mL. Some experiments with NSAIDs were performed in the presence of 3 g/L of human albumin (Grifols, Barcelona, Spain). PMA was used at 20 ng/mL, azide at 10 nmol/L and 2-DG at 50 nmol/L. In experiments with inhibitors, neutrophils were pretreated with the specific inhibitor for 15 minutes at 37°C before adding the different NSAIDs. All NSAIDs and kinase/phosphatase inhibitors were solubilized in dimethyl sulfoxide (DMSO) (Panreac, Barcelona, Spain) and added to the cell solution in a final concentration below 0.2% DMSO. In experiments with prostaglandins, neutrophils were treated with PGE2 1 µmol/L and incubated for 30 minutes at 37°C. All control data were obtained in the presence of 0.2% DMSO. Flow cytometry analysis Cells were treated as described previously and then incubated with the different MAbs at 4°C for 30 minutes. After washing in PBS, the cells were labeled with fluorescein isothiocyanate (FITC)-labeled goat antimouse immunoglobulin (Ig) (Dako, Salstrup, Denmark). At least 5 × 103 cells of each sample were analyzed in a FACScan flow cytometer (Becton Dickinson) and the data were collected in both linear and logarithmic scales. In experiments with mononuclear cells, the L-selectin expression was analyzed in cells with side and forward scatter characteristics of lymphocytes. Mean fluorescence intensity (MFI) in linear scale was obtained by adjusting the fluorescence gain so that about 5% of the cells of the sample with the greatest fluorescence were positive in the highest fluorescence channel. The fluorescence produced by the myeloma P3X63 supernatant was considered background. Because the fluorescence conditions were different from experiment to experiment, data were normalized to express relative mean fluorescence intensity (rMFI), as follows: rMFI = (MFI NSAID MFI P3X63) / (MFI medium MFI P3X63) × 100.
In all experiments with metabolic inhibitors and in some ATP quantification assays, cell viability was determined in the FACScan through the spontaneous uptake of propidium iodide (10 µg/mL final concentration) (Sigma Chemical Co). Soluble L-selectin enzyme-linked immunosorbent assay Neutrophils (7 × 106 cells/mL) were incubated alone or with the different NSAIDs in PBS at doses and times indicated. After 30 minutes at 37°C, cells were centrifuged and the cell-free supernatants were tested for soluble L-selectin (sL-selectin) by an enzyme-linked immunosorbent assay (ELISA) kit (Bender Medsystem, Vienna, Austria) according to the manufacturer's instructions.ATP quantification assays Intracellular ATP concentration in neutrophils was measured with an ATP Bioluminiscence Assay Kit obtained from Boëhringer Mannheim (Mannheim, Germany). Neutrophils (7 × 106 cells/mL) were incubated in PBS alone and in the presence of metabolic inhibitors, PGE2, or different NSAIDs at doses indicated. After times indicated at 37°C, cells were pelleted and resuspended in the lysis buffer provided with the kit. ATP was assayed following the manufacturer's instructions and measured in a luminophotometer Lumat LB9501 (Berthold, Wildbad, Germany).Intracellular calcium assays Neutrophils (3 × 106 cells/mL) were loaded with Fluo-3 AM 3 µmol/L in PBS for 15 minutes at 37°C. After washing twice in PBS, cells were resuspended in HBSS at 1 × 106 cells/mL final concentration. Calcium influx was analyzed in a FACScan studying the variations in the basal fluorescence after the addition of the respective NSAID along 6 minutes. Ionophore (0.5 µmol/L) and f-MLP (0.1 µmol/L) were used as positive controls.Statistical analysis Results were expressed as arithmetic mean ± SD or standard error (SE) of the mean. Student t test for paired samples was used to determine significant differences between means.
NSAIDs cause the shedding of L-selectin in neutrophils through a physiologic mechanism To complement previous data from our group,15,16 we decided to test the effect of an ample collection of NSAIDs on the expression of L-selectin. We examined by flow cytometry, the effect of sodium salicylate, aspirin, indomethacin, aceclofenac, diclofenac, piroxicam, meloxicam, flufenamic acid, mefenamic acid, and phenylbutazone on the basal expression of L-selectin in human neutrophils. As shown in Figure 1, flufenamic acid, mefenamic acid, diclofenac, aceclofenac, aspirin, or indomethacin, caused, in this order of potency, a significant decrement in the basal surface expression of L-selectin. In contrast, neutrophils incubated with piroxicam, meloxicam, or phenylbutazone did not show any change in the basal expression of this adhesion molecule. In some experiments sodium salicylate, the circulating aspirin metabolite at 200 µg/mL was effective in triggering L-selectin shedding, even in the presence of human albumin (data not shown).
L-selectin is cleaved and released from the cell surface to plasma where it is detected as a soluble isoform (sL-selectin) at high concentrations.37 An ELISA detected a significant increment in the sL-selectin concentration ranging from 26 to 11 ng/106 cells in the cell-free supernatant of neutrophils incubated with flufenamic acid, aceclofenac, mefenamic acid, diclofenac, sodium salicylate, aspirin, and indomethacin. However, the supernatant of neutrophils treated with meloxicam, piroxicam, and phenylbutazone did not show any variation in the concentration of sL-selectin respect to cells incubated with the medium alone (Figure 1A). The vehicle DMSO had no effect on L-selectin shedding at the concentration tested. Figure 1B shows the concentrations of different NSAIDs that caused the 50% of decrement in the basal expression of L-selectin in neutrophils (IC50). Under physiologic stimuli, L-selectin is proteolytically cleaved
by a still not fully characterized metalloprotease. Several reports
have shown that such protease is a surface molecule that can be blocked
by hydroxamic acid-based inhibitors.36,38 One of such
compound is the KD-IX-73-4, recently described as a protease inhibitor
that blocks L-selectin shedding from stimulated neutrophil surface
without affecting CD11b mobilization or general neutrophil activation.36,39 To rule out the possibility of an
inherent proteolytic activity of NSAIDs on L-selectin, we tested the
effect of KD-IX-73-4 on NSAID-dependent L-selectin down-regulation in neutrophils. As shown in Table 1,
pretreatment of neutrophils with 20 µmol/L of KD-IX-73-4 prevented
the loss of L-selectin expression induced by flufenamic acid,
aceclofenac, mefenamic acid, diclofenac, aspirin, and indomethacin.
These data indicate that a group of NSAIDs differentially induces the shedding of L-selectin through a mechanism that requires a functionally active KD-IX-73-4 sensitive-protease. NSAIDs induce L-selectin shedding in neutrophils through a PKC and tyrosine kinase-independent mechanism L-selectin is constitutively expressed on essentially all leukocytes and is rapidly released from the cell surface in response to a variety of stimuli.19 However, neither the physiologic significance of L-selectin shedding nor the signal transduction pathways involved in its regulation have been fully elucidated. Several recent reports suggest that, in a cell type-specific fashion, the PKC system and tyrosine phosphorylation and dephosphorylation cascades are involved in the regulation of L-selectin surface expression.40-43 Therefore, we decided to ascertain the role of both signaling pathways in the L-selectin down-regulation induced by NSAIDs in neutrophils.Ro 31-8220, a new and selective PKC inhibitor (Figure
2), and H7, a broad-based
serine-threonine kinase inhibitor, reverted the rapid L-selectin
shedding induced by the activation of PKC by PMA in neutrophils (data
not shown). However, none of these inhibitors was able to prevent the
down-regulation of L-selectin in neutrophils induced by flufenamic acid
and aspirin (Figure 2, and data not shown). In a similar manner to PKC
inhibitors, the incubation of resting neutrophils with tyrphostin A25
and herbimycin A, 2 tyrosine kinase inhibitors, did not prevent the effect of NSAIDs on L-selectin expression (data not shown). It has been
described that some MAbs against the tyrosine phosphatase CD45 elicit
L-selectin down-regulation in lymphocytes,42 suggesting that the phosphorylation of tyrosine might play a role in the maintenance of L-selectin on the cell membrane. Thus, if NSAIDs induce
the L-selectin shedding in neutrophils through a tyrosine dephosphorylation mechanism, an inhibitor of tyrosine phosphatase should rescue the down-regulation of L-selectin induced by NSAIDs. However, as shown in Table 2,
pretreatment of neutrophils with sodium orthovanadate, an inhibitor of
tyrosine phosphatases, did not prevent the loss of L-selectin
expression induced by flufenamic acid and aspirin.
Recently, it has been suggested that calcium influx might be involved in the L-selectin shedding through the induction of conformational changes in the calmodulin.44 Consequently, we performed experiments to investigate whether NSAIDs were able to induce directly variations in the intracellular calcium concentration in resting neutrophils, as described in "Materials and methods." Flufenamic acid, aceclofenac, diclofenac, and indomethacin at 20 µg/mL did not significantly modify the intracellular calcium levels in neutrophils preloaded with the calcium indicator fluo-3 (data not shown). These results suggest that NSAIDs-induced L-selectin shedding occurs in neutrophils through a likely PKC and tyrosine kinase/phosphatase-independent pathway. In addition, the effect of NSAIDs on L-selectin expression is not associated with an increment in intracellular calcium concentration, an event that used to follow neutrophil activation.45,46 Correlation between L-selectin expression and intracellular ATP concentration in neutrophils treated with NSAIDs Studies with several NSAIDs indicate that these agents are able to reduce the intracellular ATP concentration through a mechanism that affects the mitochondrial respiration.7-9 We therefore decided to test the potential relationship between the NSAIDs-mediated reduction of intracellular ATP concentration and L-selectin shedding in neutrophils. Figure 3A shows the effects of different NSAIDs in the intracellular concentration of ATP in neutrophils. Although NSAIDs such as piroxicam, meloxicam, or phenylbutazone did not modify the concentration of ATP, the flufenamic acid was able to reduce 4 times the basal ATP concentration in neutrophils. When data of ATP concentration and L-selectin expression in NSAID-treated neutrophils were plotted together, a highly significant direct correlation between reduction of ATP intracellular and surface expression of L-selectin was observed (Figure 3B) (correlation coefficient r = 0. 8, P < .01). Moreover, kinetics studies showed that intracellular ATP concentration decreased in correlation with L-selectin expression in neutrophils incubated with flufenamic acid (Figure 3C). In some kinetics experiments, the neutrophil viability was assessed as described in "Materials and methods" without showing any significant changes (data not shown).
To rule out the potential role of cyclooxygenase-derived products in the regulation of ATP by NSAIDs, we tested the effect of PGE2 in the intracellular ATP levels in neutrophils. The incubation of neutrophils with PGE2 1 µmol/L neither modified the basal levels of ATP concentration nor the L-selectin surface expression. In addition, pretreatment with PGE2 showed no effect in the reduction of ATP and L-selectin induced by flufenamic acid in neutrophils. Moreover, the neutrophil incubation with NS-398, a selective COX-2 inhibitor, did not either significantly modify the basal expression of L-selectin or prevent the shedding of L-selectin induced by flufenamic acid (data not shown). These data suggest that the ability of NSAIDs to induce both the shedding of L-selectin and the reduction of the intracellular ATP concentration may be linked effects. Inhibition of cell metabolism causes the shedding of L-selectin in neutrophils The foregoing experiments suggested a potential relationship between intracellular ATP reduction and L-selectin shedding in neutrophils. To further characterize this phenomenon, we tested the effect of standard metabolic inhibitors in the expression of L-selectin in neutrophils (Figure 4). The incubation of resting neutrophils with a combination of inhibitors of oxidative phosphorylation (azide) and anaerobic glycolysis (2-deoxy-D-glucose [2-DG]) induced a reduction in the basal intracellular ATP concentration of about 2 orders of magnitude (data not shown). Under those conditions, neutrophils displayed a reduction of 65% ± 8% (n = 8) in the basal surface expression of L-selectin (see Figure 5). Pretreatment of neutrophils with KD-IX-73-4 fully prevented the loss of L-selectin expression induced by the metabolic inhibitors (data not shown). To rule out the possibility that a decrement in cell viability might be responsible of such effects, the percentage of dead cells was assayed in all experiments in which metabolic inhibitors were used, as described in "Materials and methods." The inset of Figure 4 shows that under our experimental conditions, the neutrophil viability, assessed by the uptake of propidium iodide, was not modified by azide and 2-DG.
The effect of azide and 2-DG on L-selectin expression on neutrophils was time-dependent. Figure 5A shows that metabolic blockers induced a maximum down-regulation of L-selectin expression in neutrophils after 10 to 30 minutes of treatment. On activation, L-selectin is shed from neutrophils both in vivo23,24 and in vitro.22,47 Variations in the expression of CD11b and CD45, 2 glycoproteins contained in the membrane of secretory granules of neutrophils, can be considered as an indirect measurement of neutrophils degranulation (activation).34,48 As shown in Figure 5A, the inhibition of cell metabolism did not cause any significant effect on CD11b basal expression on neutrophils. Similar results were obtained when the CD45 expression was studied (data not shown) that indicate the L-selectin shedding induced by metabolic inhibitors is not due to nonspecific cell activation. Azide and 2-DG caused the loss of L-selectin expression in neutrophils by inducing the shedding of the molecule from the cell surface. An ELISA detected a significant increment in the concentration of sL-selectin in the cell-free supernatant of neutrophils treated with azide and 2-DG (14 ± 2 ng/mL) in respect to the medium (6 ± 2 ng/mL) (Figure 5B). On peripheral blood lymphocytes, these metabolic inhibitors did not cause any effect on the basal expression of L-selectin, even as long as 90 minutes after treatment (data not shown). In addition to L-selectin, numerous other integral plasma membrane
proteins are released from the cell membrane by
proteolysis.49 CD16 and CD59 are 2 glycophosphoinositol
lipid-anchored surface molecules that can be shed from cell surface
during cell activation.50,51 The basal expression of these
2 surface molecules in neutrophils was not altered on exposure to the
metabolic inhibitors. A representative experiment of the effect of
azide plus 2-DG on the basal expression of L-selectin, CD16, and CD59
on neutrophils is shown in Figure 6.
These data indicate that the maintenance of L-selectin in the surface of neutrophils is an energy-dependent process.
The major findings of this study are as follows: (1) Some NSAIDs cause the down-regulation of L-selectin expression through the action of the physiologic membrane metalloprotease responsible of the L-selectin cleavage in neutrophils; (2) this effect seems to be intracellular calcium concentration and protein kinase and phosphatase-independent; (3) our data also indicate that the maintenance of L-selectin on the surface of neutrophils is an energy-dependent process, suggesting that L-selectin is shed in neutrophils by default; and (4) interestingly, that the capability of NSAIDs to reduce the intracellular ATP concentration could account for their ability to induce the shedding of L-selectin in neutrophils. It has been widely accepted that the mechanism of action of NSAIDs is the inhibition of prostaglandin synthesis. However, a significant body of evidence suggests that prostaglandins, as the sole target of anti-inflammatory action of NSAIDs is no longer tenable. The fact that certain prostaglandins have anti-inflammatory activity in animal models of arthritis52 supports this contention, as does the failure of some NSAIDs to act as COX inhibitors, despite their anti-inflammatory activity.4 Furthermore, there is no correlation between the small doses of aspirin that inhibit prostaglandin synthesis and the higher doses required to exert an anti-inflammatory effect in vivo.2 All these data, in addition to the high variations in clinical response of patients to NSAIDs,3 support the idea of many groups, including ours that these agents must have more than just one anti-inflammatory mechanism of action. In this regard, it has been reported that some NSAIDs are able to inhibit membrane-associated processes, to promote synthesis of anti-inflammatory eicosanoids as well as to block a number of intracellular events, including transmembrane anion transport, oxidative phosphorylation in mitochondria, and activation of transcription factor NF-kB.2,9,11,13,53 However, none of these prostaglandin-independent effects fully explains the in vivo anti-inflammatory action of NSAIDs. Our group has recently described that leukocyte adhesion molecules may be potential targets for the anti-inflammatory action of NSAIDs.54 Several, but not all of these therapeutic agents induce a rapid loss of L-selectin expression15,16 through an unknown prostaglandin-independent mechanism.54 Other compounds with anti-inflammatory properties such as colchicine and leumedins have also shown able to reduce the expression of L-selectin in neutrophils.55,56 A group of NSAIDs composed by flufenamic acid, mefenamic acid, aceclofenac, diclofenac, sodium salicylate, aspirin, and indomethacin induced the shedding of L-selectin in neutrophils. This effect was detected at reported therapeutic concentrations.57 The NSAIDs that display a high L-selectin shedding activity seem to share the chemical structure diphenylamine. Indeed, the diphenylamine is able to induce the shedding of L-selectin in neutrophils (González-Alvaro et al, unpublished observations). However, other NSAIDs such as piroxicam, meloxicam and phenylbutazone, clinically as effective as members from the first group, did not show any effect on the expression level of this adhesion molecule; instead, this group of compounds affects the activation-dependent integrin-mediated adhesion of leukocytes to endothelial cell.17 Preliminary data from our group suggest that only those NSAIDs based in the diphenylamine display a high L-selectin shedding activity in neutrophils. The importance of L-selectin shedding as a mechanism of action of NSAIDs in vivo has not been established. However, the relevant role that L-selectin plays in inflammation,27-29 in addition to the fact that indomethacin decreases in vivo the L-selectin expression in human neutrophils,15 make this effect of NSAIDs, at least in potency, biologically relevant. L-selectin is a member of the selectin family of surface adhesion receptors constitutively expressed by most leukocytes. The interaction between L-selectin and its ligands, carbohydrates expressed by activated endothelial cells, plays an important role in the inflammatory response mediating the initial rolling and arrest of flowing leukocytes to the endothelium surface.20,58 Membrane-anchored adhesion molecules can modify its activity through several mechanisms.59 One of those mechanisms is the proteolytic processing of the extracellular domain of the adhesion molecules. L-selectin is proteolytically cleaved from the cell surface by the action of a not-yet-well-characterized cell membrane metalloprotease that can be blocked by hydroxamic acid-based protease inhibitors.38,39 The incubation of neutrophils with the L-selectin shedding inhibitor KD-IX-73-4 prevented the down-regulation of L-selectin expression induced by all NSAIDs tested. These data rule out the possibility of an intrinsic proteolytic activity of NSAIDs on L-selectin and demonstrate that the shedding of L-selectin induced by these agents is dependent on the activity of the physiologic metalloprotease responsible of the L-selectin cleavage in neutrophils. Recently, it has been proposed that calcium-induced conformational changes in the calmodulin, an intracellular calcium regulatory protein, might be involved in the control of L-selectin shedding.44 Therefore, we analyzed the possibility that NSAIDs were able to trigger cytosolic calcium oscillations in resting neutrophils as a potential explanation for the L-selectin down-regulation induced by these agents. However, neither the flufenamic acid,60 the most powerful inductor of L-selectin shedding, nor any of the other NSAIDs tested were able to cause a detectable increment in the intracellular calcium concentration in neutrophils. These data suggest that the NSAID-mediated down-regulation of L-selectin cannot be consistently explained by calcium signals in the neutrophils. The signaling mechanisms involved in the regulation of L-selectin expression in leukocytes are largely unknown. Several studies using protein kinases and phosphatases inhibitors suggest that, in a cell type-specific fashion, the PKC and tyrosine phosphorylation and dephosphorylation cascade may be involved in the regulation of L-selectin surface expression.40-43 Interestingly, it has been reported that some NSAIDs activate the PKC signaling pathway in leukocytes.61,62 However, neither Ro 31-8220, a member of a highly specific new family of PKC inhibitors, nor H7, a broad-based serine-threonine kinase inhibitor, was able to prevent the NSAID-induced down-regulation of L-selectin in neutrophils. Likewise, protein tyrosine kinase inhibitors, herbimycin A and tyrphostin A25, did not reverse the down-regulation of L-selectin induced by NSAIDs. Alternatively, some MAbs against the tyrosine phosphatase CD45 are able to promote L-selectin down-regulation in both lymphocytes42 and neutrophils (Gómez-Gaviro et al, unpublished observation), suggesting that NSAIDs may induce the L-selectin shedding through a tyrosine phosphatase-dependent system. However, the sodium orthovanadate, a tyrosine phosphatase inhibitor, did not modify the activity of NSAIDs on L-selectin expression in neutrophils. Although results obtained in living cells with inhibitors neither support nor preclude definitive conclusions, these data indicate that the NSAIDs-mediated L-selectin shedding involves a protein phosphorylation/dephosphorylation-independent mechanism. NSAIDs are able to interfere with the mitochondrial oxidative phosphorylation reducing the intracellular ATP synthesis in vivo.8 This effect has been proposed as an early pathogenic event in the NSAID-mediated enteropathy.9,63 The incubation of neutrophils with the different NSAIDs showed a differential effect on the intracellular ATP concentration regardless of the presence of cyclooxygenase products (PGE2) in the cell suspension. The effect of NSAIDs on the level of intracellular ATP strongly correlated with the ability of these agents to reduce the basal expression of L-selectin in neutrophils, which suggests that both events may be functionally related. Remarkably, the metabolic inhibitors, azide and 2-DG, also caused the shedding of L-selectin without an evident variation in the neutrophil activation state, as assessed by the CD11b and CD45 expression. These observations suggest that the maintenance of L-selectin in the membrane of neutrophils is an energy-dependent process, raising the idea that, in resting neutrophils but not in lymphocytes, the L-selectin tends to be shed by default. The fact that serum from healthy humans presents high levels of sL-selectin supports this latter contention.37 Other molecules expressed by neutrophils that can be shed from cell surface such as CD16 and CD59 did not modify their basal expression in the presence of azide plus 2-DG. This fact excludes that the shedding of L-selectin is an unspecific effect of metabolic inhibitors on neutrophils. All these data strongly suggest that NSAIDs are able to induce, at least in part, the shedding of L-selectin in neutrophils through the reduction of the energy capacity of the cell. Recently, a cell-surface metalloprotease responsible of the cleavage of
the surface form of TNF-
Submitted December 15, 1999; accepted July 19, 2000.
Supported in part by Boëhringer Ingelheim, by grant SAF 96/0332 from Ministerio de Educación y Cultura and by FIS 99/0222 from Fondo de Investigaciones Sanitarias de la Seguridad Social (F.D-G) and by BEFI 99/9012 from Fondo de Investigaciones Sanitarias de la Seguridad Social (M.V.G-G).
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: Federico Díaz-González, Servicio de Reumatología, Hospital Universitario de Canarias, Universidad de La Laguna, Ofra s/n, La Cuesta, 38320 La Laguna, Santa Cruz de Tenerife, Spain; e-mail: fdiaz{at}cnb.uam.es.
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Top
Abstract
Introduction
) and supernatant
concentration of its soluble isoform (sL-selectin, 
) in neutrophils
treated with different NSAIDs. Neutrophils isolated from peripheral
blood were incubated for 30 minutes at 37°C in medium alone, with 20 ng/mL of PMA, or with 20 µg/mL of the different NSAIDs, except
aspirin and sodium salicylate that were used at 200 µg/mL. After
incubation, cell solutions were centrifuged, and supernatant fluids
were tested for sL-selectin by an ELISA. Values were obtained in
duplicate determinations for each sample. Data represent the mean
(ng/106 cells) ± SD (
) and intracellular ATP concentration (
) in
neutrophils incubated with 20 µg/mL of flufenamic acid. Values are
percentage of expression and concentration of L-selectin and ATP in
respect to the basal conditions (medium alone) in each time. Data
represent the mean ± SD of 3 independent experiments.
) in neutrophils. Cells were cultured in
the presence or absence of metabolic inhibitors at concentrations
described in Figure 
, the TACE, has shown a mild proteolytic
activity on L-selectin.