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*
Immunology Research Center, Institut Armand-Frappier, Université du Québec, des-Prairies, Laval, Québec, Canada; and
Service de pneumologie, Département de médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin, a natural
HLE protease inhibitor. The ability of HLE to degrade ICAM-1 was
further confirmed by electrophoretic analysis using a soluble form of
ICAM-1 (D1-D5). Enzymatic removal of N-linked
glycosylation did not significantly modulate ICAM-1 cleavage by HLE,
while removal of sialic acid residues partially reduced the sensitivity
of ICAM-1 to HLE. We further showed that sputum of cystic fibrosis
patients contains high levels of HLE activity capable of cleavage of
cell surface ICAM-1. The cleavage induced by incubation of cells with
the sputum sample was totally inhibited by 1-antitrypsin and the
specific peptidic HLE inhibitor
N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone.
Moreover, the cleavage of ICAM-1 was concomitant to that of CD4 at the
surface of the same cell, at the same amplitude, and at all HLE
concentrations. The capacity of HLE to modulate the expression of
ICAM-1 on the surface of leukocytes by proteolytic cleavage brings
support to the hypothesis that overproduction of HLE can cause severe
immunologic lung disorders by affecting intercellular
adhesion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the cellular proteases that are potentially taking part in the physiologic cleavage of leukocyte Ags are the various neutral serine proteases that are stored in the primary granules of neutrophils (PMN)3 and released following their activation. Human leukocyte elastase (HLE; PMN elastase, E.C. 3.4.21.11) and cathepsin G (E.C. 3.4.21.20) are the major serine proteases secreted by PMN; they were reported to play important roles in PMN-mediated proteolytic events. These two proteases were also found on the plasma membrane of resting and activated PMN as noncovalently membrane-bound forms. Their expression is not restricted to PMN, and both soluble and membrane-bound forms were also reported on monocytes and lymphocytes (4, 5, 6). HLE received the closest attention over the last two decades, since it was found to participate in tissue destruction in a number of inflammatory disorders, including rheumatoid arthritis, cystic fibrosis (CF), glomerulonephritis, and emphysema. In addition to its capacity to digest numerous macromolecules of the extracellular matrix, HLE can also cleave specific leukocyte Ags, such as CD4 and CD8 (7), CD43 (8), and thrombin receptor (9). However, to cleave these receptors in vivo, favorable microenvironmental conditions are needed, so as to limit the rapid inhibition of HLE by naturally occurring protease inhibitors. Indeed, it was demonstrated in vitro that extracellular proteolysis may occur at physiologic protease inhibitor concentrations during close contact between PMN and a substrate-coated surface, therefore confining the proteolytic activity to the immediate pericellular zone of PMN contact, where this temporary microenvironment protects the catalytic activity of HLE from inhibition (10, 11, 12). The importance of proteases in intercellular adhesion is also supported by the recent observations that receptors of adhesion molecules are capable of binding HLE. Thus, Cai and Wright (13) demonstrated that Mac-1 was one of the ligands present on the cell surface of PMN that could bind HLE. They also showed that the expression level of HLE on PMN was inversely proportional to their degree of adhesiveness, and that a mAb against HLE prevented their detachment from fibrinogen-coated surfaces, indicating that HLE was involved in PMN-mediated interactions and could potentially act as a regulating factor on Mac-1-mediated adhesion. It is thus possible that proteases such as HLE could be important for the detachment of PMN, by cleaving Mac-1 ligand. A role for HLE during the infiltration of neutrophils was reported by Woodman et al. (14), in which the infiltration of PMN into inflamed vessels, observed by in vivo intravital microscopy, was significantly abrogated with a low m.w. inhibitor of HLE. Since ICAM-1-mediated interactions were shown to be critical for the firm attachment of PMN to endothelial cells, and since Mac-1 was able to bind HLE, we have, in the present work, tested the sensitivity of ICAM-1 to HLE-mediated proteolytic cleavage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HLE was obtained from Calbiochem (La Jolla, CA). The
1-antitrypsin (1-AT) and
N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone
(MSAAPVCK) were obtained from Sigma (St. Louis, MO). MMP-9 (gelatinase
B) was purified from supernatants of the human monocytic cell line
THP-1 by affinity chromatography on gelatin-Sepharose, and its
proteolytic activity was confirmed by fluorescent-activated substrate
conversion, as previously described (15). Flavobacterium
meningosepticum recombinant N-glycosidase F and
Vibrio cholerae neuraminidase were obtained from Boehringer
Mannheim (Laval, Quebec, Canada). Purified human sICAM-1 was kindly
provided by Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT).
Sputum samples were obtained from six patients with CF (four female,
two male; age 17–31 yr) during stable disease. All patients had
Pseudomonas aeruginosa in their sputum. None of the patients
were receiving antibiotics at the time of sputum collection. Sputum
samples were mixed 1:1 (weight:volume) with PBS, vortexed, and
centrifuged at 15,000 x g for 15 min. The supernatants
were stored at -70°C until analysis. SYPRO-RED was obtained from
Molecular Probes (Eugene, OR). Tissue culture reagents were obtained
from Life Technologies (Mississauga, Ontario, Canada). The
FITC-conjugated anti-ICAM-1 (B-H17 mAb) was obtained from BioSource
International (Camarillo, CA). The phycoerythrin-conjugated
anti-CD4 (clone MT-310) was obtained from Dako (Carpenteria, CA),
and the anti-HLA-DR (clone CR3/43) was obtained from Boehringer
Mannheim (Laval, Canada). The RR1/1 (anti-ICAM-1) was a gift from
Dr. T. A. Springer (Center for Blood Research, Boston, MA). CR3/43
(anti-HLA-DR) and FITC-conjugated goat anti-mouse IgG were
obtained from Boehringer Mannheim and Sigma, respectively.
Cell cultures
The U937 promonocytic and Raji B lymphocyte cell lines were obtained from Dr. D. Oth (Institut Armand-Frappier). MT-2 and MT-4 T lymphocyte cell lines were obtained from Dr. P. Talbot (Institut Armand-Frappier). Cells were maintained by in vitro culture in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES buffer, 100 U/ml penicillin, and 100 µg/ml streptomycin.
HLE treatments
Cells were washed three times in complete RPMI without serum
(RPMI-S-) and seeded in 96-well plates. The wells
contained 80 µl of cells (2 x 105 total)
resuspended in RPMI-S- and 10 µl of protease-containing
solution. When specified, 10 µl of 1-AT (1 mg/ml) was preincubated
15 min (37°C) with 10 µl of HLE (200 µg/ml) before addition to
cells. The volume was completed with RPMI-S- to 100 µl
and cells incubated at 37°C for the indicated times. After
incubation, cell surface expression of ICAM-1 was measured by
flow-cytometric analysis using specific fluorescent-labeled mAbs. In
some experiments, U-937 cells (2 x 105/well) were
preincubated for 1.5 h at 37°C with neuraminidase (0.2 U/ml), or
for 30 min at 4°C with a saturating concentration of RR1/1 mAb, and
then washed before HLE treatments.
Reexpression of ICAM-1 following proteolytic cleavage
U-937 cells were incubated 2 h at 37°C with or without HLE (20 µg/ml) in RPMI-S- medium. The enzymatic reaction was stopped by three washes of cells with RPMI. The cells were then washed extensively and resuspended in complete RPMI medium supplemented with 10% FCS. Aliquots of these cells (2 x 105) were added in 24-cluster-well plates and incubated at 37°C in 5% CO2. At the indicated time intervals, cells were analyzed for ICAM-1 expression by flow cytometry using an anti-ICAM-1 mAb, as described above.
In vitro analysis of proteolytic cleavage of sICAM-1
Purified human sICAM-1 (5 µg) was incubated with various concentrations of HLE (4–100 µg/ml) in a final volume of 10 µl for 1 h at 37°C in Eppendorf tubes. Proteolysis of ICAM-1 was determined by SDS-PAGE (10%) under denaturing conditions using a computerized image densitometer (Bio-Rad, Model GS-670, Mississauga, Ontario, Canada). In some experiments, purified ICAM-1 (1 µg) was preincubated for 20 h with N-glycosidase F (80 U/ml) or for 1 h with neuraminidase (0.4 U/ml) in a final volume of 10 µl at 37°C in sealed Eppendorf tubes before HLE treatment. The apparent molecular mass of ICAM-1 and its proteolytic fragments were determined by electrophoretic mobility relative to known m.w. standards (Bio-Rad). Proteins were visualized by staining with Coomassie blue, or using SYPRO-RED (Molecular Probes, Eugene, OR) under UV transillumination. When indicated, the protein bands were amplified by nonlinear amplification using a densitometric image analysis software (Bio-Rad). Amplification was used solely to ascertain the presence of bands with low intensity. Nonlinear amplification is a tool for increasing the foreground (i.e., protein bands) intensity by reducing the coamplification of the background. It is important to note that the semiquantitative aspect between bands is lost during nonlinear amplification of digitized gel images.
Cleavage of ICAM-1 in sputum samples
U-937 cells (2 x 105) were incubated with HLE
(20 µg/ml) or with dilution (1/4 or 1/40) of sputum samples from
patients with CF in presence or in absence of 1-AT (133 µg/ml) or
MSAAPVCK (10 mM) in a final volume of 15 µl at 37°C for 4 h in
Eppendorf tubes. After incubation, cell surface expression of ICAM-1
was quantitated by flow-cytometric analysis.
Flow-cytometric analysis
Cells were washed twice with ice-cold PBS containing 1% (v/v) BSA and 0.01% (v/v) sodium azide (PBA) and then incubated for 25 min on ice with predetermined concentrations of Abs. For indirect staining, cells were washed twice after binding of the first mAb and incubated again with a saturating concentration of fluorescein-conjugated goat anti-mouse IgG for 30 min on ice. After mAb binding, cells were washed twice with PBA and resuspended in 400 µl of PBS containing 0.01% of sodium azide. Samples were kept at 4°C in the dark and analyzed using a Coulter XL-MCL (Coulter Electronics, Hialeah, FL). Between 5,000 and 10,000 cellular events were analyzed for each sample.
Measure of HLE activity in sputum samples
Elastase-specific proteolytic activity in sputum samples was measured by standard colorimetric assays using the methoxysuccinyl-Ala-Ala-Pro-Val-nitroaniline as substrate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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U-937 cells were incubated in serum-free condition with exogenous
HLE, and ICAM-1 expression was measured by flow cytometry with a
FITC-labeled anti-ICAM-1 mAb. Treatment of U-937 cells for 2 h
with 20 µg/ml of HLE significantly reduced ICAM-1 expression at the
cell surface, as indicated by the reduced intensity of staining of
HLE-treated cells (MFI = 3.7) compared with untreated cells
(MFI = 13.7) (Fig. 1
, A
and B). The effect of HLE treatment on ICAM-1 expression was
dose dependent (Fig. 1C). At higher doses, up to 90%
reduction of ICAM-1 expression could be achieved within 2 h.
Addition of 1-AT, a potent inhibitor of elastase activity,
completely inhibited the ability of elastase to reduce ICAM-1
expression, strongly arguing in favor of a catalytic mechanism.
Furthermore, complete inhibition of HLE-reduced ICAM-1 expression was
observed in the presence of human serum (10% v/v), a rich source of
protease inhibitors, including 1-AT (data not shown). The
sensitivity of ICAM-1 to HLE was further confirmed using other cell
lines expressing ICAM-1, such as the MT-2 (Figs. 2B and
3A) and MT-4 T-lymphocyte cell
lines (data not shown), and on the RAJI B-lymphocyte cell line (Fig. 2A).
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B). We found no evidence of cleavage of ICAM-1 by MMP-9
when it was tested at equivalent molar concentrations (i.e., 60
µg/ml) used for HLE (i.e., 20 µg/ml) (Fig. 2A). In a
second series of experiments, we compared the sensitivity of ICAM-1 and
HLA-DR, both expressed at high levels on the surface of MT-2 cells. We
found that HLE treatment (20 µg/ml) reduced the expression of ICAM-1
by 50–60%, but not that of HLA-DR (Fig. 2C). Finally, we
found that preincubation of U-937 cells with anti-ICAM-1 RR1/1 mAb,
specific for the first Ig domain of ICAM-1, inhibited the ability of
HLE to cleave ICAM-1 (Fig. 2D).
To determine the kinetic characteristics of ICAM-1 cleavage by HLE,
MT-2 cells were incubated with HLE (20 µg/ml) for different periods
of time (15 min to 4 h). We found that cleavage of ICAM-1 by HLE
was time dependent (Fig. 3A). When HLE-treated cells were
seeded back in culture after removal of HLE from culture medium, ICAM-1
was gradually reexpressed upon normal culture condition (Fig. 3B).
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To establish that ICAM-1 is cleaved directly by HLE, we
investigated the proteolysis of purified human ICAM-1 in vitro. This
soluble form ICAM-1 (sICAM-1) contains the five extracellular Ig-like
domains. SDS-PAGE analysis confirmed that sICAM-1 was cleaved by HLE in
a dose-dependent manner. As shown in Fig. 4, HLE treatment of ICAM-1 resulted in
the appearance of a major diffuse band of approximately 45 kDa. The
diffuse pattern of the bands was due to extensive glycosylation of
ICAM-1 (16, 17). The 30-kDa band could be HLE as it corresponded to its
molecular mass of 29.5 kDa. The fact that the 30-kDa band
appeared less intense in the sample containing HLE alone compared with
that coincubated with ICAM-1 may be the consequence of an autocatalysis
of elastase in absence of ICAM-1. A longer HLE treatment (3 h) resulted
in complete digestion of sICAM-1, as no proteolytic fragments were
detected (data not shown). Repeated attempts to identify the cleavage
site(s) by amino acid analysis of proteolytic fragment bands were,
however, inconclusive, probably because of the heavy glycosylation of
ICAM-1.
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ICAM-1 is differently glycosylated depending on cell type, and
this is reflected in molecular mass that varies between 76 and 114 kDa.
The complete deglycosylation of ICAM-1 results in a protein of 55–60
kDa corresponding to the molecular mass predicted by its mRNA sequence
(18). We next investigated the possible influence of
N-linked carbohydrates on cleavage by HLE. For this purpose,
sICAM-1 was pretreated with N-glycosidase F (molecular mass
35.5 kDa), and then subjected to HLE treatment. As shown in Fig. 5, the N-glycosidase treatment
resulted in the appearance of a ladder of bands, consistent with the
heterogeneous deglycosylation pattern of ICAM-1. The thin band of
approximately 50–60 kDa most likely represented the complete
deglycosylated form of ICAM-1. After a 1-h treatment with HLE, both
native (glycosylated) and deglycosylated forms of ICAM-1 were sensitive
to proteolysis by HLE. At 4 h, however, although HLE treatment
resulted in complete digestion of native ICAM-1, a significant amount
of the deglycosylated forms of ICAM-1 was still detectable upon
incubation with HLE, suggesting that removal of negatively charged
residues from ICAM-1 may slightly slow the proteolysis of ICAM-1,
possibly by interfering with the binding of the positively charged HLE
to its substrate. The diffuse 45-kDa band, previously described on Fig. 5, appeared very lightly on this gel following a 1-h HLE treatment on
native forms of ICAM-1. Minor bands of low molecular mass (10–30 kDa),
which represent other ICAM-1 proteolytic fragments, were still
detectable after 1- and 4-h HLE treatments on native and deglycosylated
forms of ICAM-1. From these observations, we conclude that enzymatic
removal of N-linked sugar residues did not significantly
influence ICAM-1 sensitivity to HLE.
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A).
Similar results were obtained by flow cytometry on U-937 cells, where
enzymatic removal of sialic acid residues from the cell surface induced
a slight, but reproducible, decrease of ICAM-1 proteolysis (Fig. 6B). These data corroborate previous finding on CD43, in
which sialidase treatment did not increase CD43 sensitivity to HLE (8).
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It is well known that PMN activities, such as phagocytosis and
oxygen burst, are impaired in lungs of patients with CF (19). Sputum of
CF patients have been shown to contain very high concentrations of HLE,
which is not detectable in bronchoalveolar lavage of healthy
individuals (7). To determine whether the concentration of PMN elastase
is produced in sufficient amounts to significantly modulate the
cleavage of ICAM-1 from the cell surface of leukocytes, we incubated
U-937 cells with sputum samples obtained from patients with CF. We
found that the ICAM-1 expression was reduced significantly by
incubation of cells in sputum samples, even when they were diluted 1 in
40 (i.e., in unsaturating conditions) (Fig. 7A), and correlated with the
amount of elastase-specific activity found in these fluids (Fig. 7C). When diluted 1/4, almost complete cleavage of ICAM-1
was observed for all samples (i.e., >95%, data not shown). The
cleavage of ICAM-1 in the sputum was HLE specific since addition of
MSAAPVCK (a specific HLE inhibitor) completely inhibited the effect.
Moreover, the sensitivity of ICAM-1 to cleavage by HLE was concomitant
to that of CD4 (Fig. 7B), previously identified as a
substrate for HLE (7). Interestingly, the extent of cleavage of ICAM-1
and CD4 followed closely the severity of the disease, as sputum samples
SP-20 and 21 were obtained from patients with a mild condition; SP-40
was obtained from a patient with moderate clinical manifestations; and
SP-30, SP-4, and SP-27 were obtained from patients that are now
deceased, or were at the limit of respiratory failure. These data
provide direct evidence that sufficient concentrations of catalytically
active HLE are present in sputum of CF patients to rapidly
down-regulate the expression of cell surface ICAM-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-AT, a natural HLE protease inhibitor.
Furthermore, a mAb (RR1/1) directed against the N-terminal domain 1 of
ICAM-1 almost completely blocked ICAM-1 proteolysis by HLE, suggesting
that HLE must first bind the domain 1 of ICAM-1. The ability of HLE to
degrade ICAM-1 was confirmed by electrophoretic analyses using sICAM-1
(D1-D5). We also showed that HLE cleavage of ICAM-1 was not
significantly affected by the posttranslational modifications. Finally,
we showed that the sputum samples of CF patients were capable of
degrading cell surface ICAM-1, and that this cleavage was inhibited by
1-AT and MSAAPVCK. Over the years, several studies on ICAM-1 have revealed a complex structure-function relationship derived from posttranscriptional and posttranslational modifications that could potentially affect the cleavage of ICAM-1 by HLE. The ability of ICAM-1 to form homodimers (20), the expression of isoforms resulting from alternative splicing (21), and the presence of different cell-specific glycosylation patterns (16, 17) could all affect the processing of ICAM-1 by HLE. Furthermore, other extrinsic factors, such as heteropolymerization of ICAM-1 with other molecules, expression of membrane protease inhibitors to HLE, expression of HLE receptors, and the presence of other HLE substrates may limit the efficiency of ICAM-1 cleavage at the cell surface. The question as to whether the glycosylation of ICAM-1 significantly affects its proteolysis by HLE was considered in the present work. Since it has been shown that Mac-1 binding to ICAM-1 was regulated by N-linked glycosylation of ICAM-1 (16), the heterogeneity of glycosylation found on ICAM-1 may then shield specific preferential cleaving site(s) for HLE. Our in vitro experiments with differential glycosylated forms of sICAM-1, obtained by enzymatic removal of N-linked sugar residues, did not show any significant change in ICAM-1 sensitivity for HLE following deglycosylation. Although these experiments may not necessarily simulate natural differential glycosylation patterns in different cell types, other studies using purified ICAM-1 obtained from different cell types expressing distinct glycosylation patterns of ICAM-1 will be needed to further elucidate the influence of posttranscriptional modifications in a more physiologic context, including experiments that address the sensitivity of ICAM-1 isoforms to proteolytic cleavage by HLE.
One of the most important roles for ICAM-1 was shown to be its ability to mediate PMN adhesion to vascular endothelium. Although the cleavage of ICAM-1 by elastase in vivo is likely to be inhibited by the presence of high concentrations of protease inhibitors, most notably in intravascular situations, the concentration of elastase may not have to be necessarily high to achieve cleavage of ICAM-1. As the excessive proteolytic activity encountered is primarily delimited by a high enzyme:inhibitor ratio, it is possible that the presence of HLE inhibitors is counterbalanced by a local concentration of HLE that outnumbers inhibitor molecules, allowing proteolysis to occur in evanescent pericellular zones (11). Moreover, during cell-cell adhesion, reorientation of the microtubule-organizing center polarizes and directs secretion of granule content to the region of the surface of the congener cell (22). Consistent with this hypothesis, ICAM-1 and Mac-1 have both been shown to cluster at the interface during cell-cell contact (23, 24). Several studies have also reported that HLE is expressed at the cell surface of many types of leukocytes as a noncovalently membrane-bound protease (5, 25). Recently, Cai and Wright (13) have shown that HLE was a ligand for Mac-1, and that an Ab against HLE prevented detachment of PMN from fibrinogen-coated surfaces, suggesting a new mechanism that could be used by cells to regulate Mac-1 adhesiveness. Our data bring support to this model. Interestingly, other studies have shown that ß2 integrins play a major role in the secretion of HLE by PMN. In fact, it has been reported that binding of sICAM-1 to PMN, or the cross-linking of ß2 with a mAb induced high secretion levels of HLE (26, 27). Mac-1/ICAM-1-mediated PMN cellular interactions could then induce the release of HLE via the cross-linking of CD18 to allow the cleavage of ICAM-1 when intercellular contacts are no longer required. Since D1 has previously been shown to be the region containing the binding site for LFA-1 (28), it appears likely that LFA-1/ICAM-1-mediated cell-cell adhesion would also be affected following the proteolysis of membrane-bound ICAM-1 by HLE. Similarly, binding of rhinoviruses to ICAM-1 will also be affected by proteolysis of ICAM-1 by HLE. Since our in vitro experiments with sICAM-1 revealed that HLE cleaved ICAM-1 at multiple sites, it is also likely that binding of Mac-1 to ICAM-1, which is mediated by the third domain of ICAM-1 (16), will also be affected by HLE treatment. The observation that cleavage of sICAM-1 by HLE first generates a large fragment of 45 kDa, corresponding approximately to the molecular mass of three Ig-like domains, supports the possibility that Mac-1 binding is also affected. Our observation, however, that binding of RR1 mAbs, specific for D1, inhibits cleavage of ICAM-1, suggests that contact between LFA-1 and ICAM-1 will prevent degradation of ICAM-1 by HLE. Identification of the putative cleavage sites of ICAM-1 will help to clarify this issue. Furthermore, the physical association of HLE to Mac-1 would potentially enhance and facilitate the proteolysis of ICAM-1. The extracellular expression of proteases such as HLE could be a general mechanism used for the locomotion and the deadhesion of cells bound to other cells and/or to macromolecules of the extracellular matrix. However, whether Mac-1-bound elastase is still enzymatically active toward ICAM-1, or whether HLE needs to be released from CD11b/CD18 to cleave ICAM-1 on the opposing cell also remains to be tested.
Several immunologic lung disorders are characterized by the incapacity of the local immune response to clear effectively bacterial infections, rendering patients highly prone to chronic infections and antibiotic treatments (19). In CF, the immunologic defect has been shown to be related to abnormal production of active HLE that cleaves Igs and the C3b receptor on neutrophils, thereby reducing phagocytosis of pathogens (29, 30). Our results now show that abnormal HLE expression will significantly affect ongoing intercellular adhesion between leukocytes that involves ICAM-1 and Mac-1. CD11b/CD18 receptor regulates cellular activation of PMN and their ability to mediate phagocytosis of particles that are bound via CR1 and the integrins on leukocytes (32). Interestingly, sICAM-1 has been reported in CF patients (33, 34). In CF, sICAM-1 is mainly found in clinically well patients, whereas during acute exacerbation, the level of sICAM-1 dropped significantly. Given our data on the capacity of elastase to cleave ICAM-1 at several sites, this sICAM-1 drop may be due to increased elastase activity during exacerbation (33). In other pathologic conditions, such as purulent peritonitis, abdominal exudate was shown to contain an average of 68 µg/ml of HLE (35). These peritoneal fluids have abnormally high levels of sICAM-1 that correlate with the number of PMN during episodes of infection. However, given the large number of conditions associated with abnormally high levels of sICAM-1, and given the recent isolation of alternative spliced mRNA encoding sICAM-1 (36), it is too early to conclude to what extent the activity of HLE is responsible for the presence of circulating forms of ICAM-1. Whether other proteases that are released by PMN, such as cathepsin G, can also cleave ICAM-1 remains an interesting possibility that will be addressed in future experiments. In CF, however, cathepsin G would probably not be involved in the cleavage of ICAM-1 since: 1) the concentration of cathepsin G in broncho-alveolar lavage (BAL) fluids is most likely too low to expect significant cleavage of ICAM-1 (37), and 2) our data showed that the specific HLE-inhibitor MSAAPVCK almost completely inhibited the cleavage of ICAM-1 in the sputum of CF patients.
As expected, we found that MHC class II molecules were resistant to cleavage by HLE. This was expected since MHC class II molecules, before being expressed at the surface of lymphocytes, must resist harsh environment along an intracellular pathway that encounters endosomal compartment that favors proteolysis of native Ags into antigenic peptides. The fact that ICAM-1, as well as CD4, are both cleaved, will significantly affect the functional recognition of MHC/peptide complexes by T cells. Our data showed indeed that cleavage of ICAM-1 and CD4 occurs at the surface of the same cell, at the same amplitude, and at any given concentrations of HLE. It remains possible that cleavage of other accessory molecules will further affect that delivery of coaccessory signals necessary for activation of T cells. Whether an anergic state is induced upon cleavage of these accessory signals involved in T cell-APC interactions remains an interesting possibility that is currently being investigated.
In summary, we showed that ICAM-1 is sensitive to proteolytic cleavage by the serine protease HLE, which is ubiquitously expressed on leukocytes. Because the ß2 integrin Mac-1 is able to bind HLE specifically and because the release of HLE seems to be regulated through ß2, our results suggest that intercellular binding mediated by Mac-1/ICAM-1 interactions could be regulated and abrogated by HLE-mediated proteolytic cleavage of ICAM-1. This regulatory pathway could be instrumental in various phenomena involving intercellular contacts, such as extravasation and leukocyte activation. Whether other receptors are also cleaved upon exposure to HLE remains to be tested, but the cleavages of CD4, CD8, and ICAM-1 most likely are sufficient to explain, in a large part, the severe immunologic disorders found in lung diseases. Our results will help to better understand the immunologic abnormalities in lung diseases, and bring support to novel approaches that could overcome damages induced by overproduction of HLE.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yves St-Pierre, Immunology Research Center, Institut Armand-Frappier, P.O. Box 100, Laval, Québec, Canada H7N 4Z3. E-mail address:
3 Abbreviations used in this paper: PMN, polymorphonuclear; 1-AT, 1-antitrypsin; CF, cystic fibrosis; HLE, human leukocyte elastase; MFI, mean fluorescent intensity; MMP-9, matrix metalloproteinase-9; MSAAPVCK, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone; sICAM-1, soluble ICAM-1.
Received for publication May 22, 1998. Accepted for publication July 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mß2) and modulates polymorphonuclear leukocyte adhesion. J. Exp. Med. 184:1213., IL-8, soluble ICAM-1, and neutrophils in sputum of cystic fibrosis patients. Pediatr. Pulmonol. 21:11.[Medline]
, and granulocyte elastase release in human secondary peritonitis. Cytokine 9:288.[Medline]
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