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Unité de Recherche en Pneumologie, Centre de Recherche, Hôpital Laval, Institut Universitaire de Cardiologie et Pneumologie de l’Université, Laval, Quebec, Canada
Correspondence: Michel Laviolette, Hôpital Laval, 2725 Chemin Sainte-Foy, Sainte-Foy, Quebec G1V 4G5, Canada.. E-mail: medmla{at}hermes.ulaval.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10.4%, whereas inhibition of the
plasminogen-plasmin system decreased eotaxin’s effect by 44.4%
(P = 0.0002). Moreover, eotaxin-induced transmigration
was largely diminished in medium with low concentrations of serum
[0.5% FBS: 6.1 ± 2.4%; 10% FBS: 40.2 ± 5.8%
(P = 0.0001)] but returned to its initial level with
the addition of plasminogen (2 U/mL) to 0.5% FBS (43.1 ± 6.5%).
These data show that eotaxin is an efficient promoter of eosinophil
transmigration in vitro, that it is more potent with cells
from asthmatics than with normal cells, and that its effect depends
predominantly on the activation of the plasminogen-plasmin
system.
Key Words: asthma • 5-oxo-ETE • metalloproteinases • urokinase plasminogen activator receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The MMPs include a large family of calcium- and zinc-dependent neutral proteases, both types having more or less specific matrix component substrates [3 ]. Lymphocytes and neutrophils use both MMP-2 and MMP-9 for their transmigration [5 6 7 ]. The urokinase plasminogen activator (uPA) and the uPA receptor (uPAR) (CD87) promote matrix degradation by generating plasmin, a serine protease, from the abundant zymogen plasminogen [4 ]. Plasmin can digest fibrin, fibrinogen, and laminin and convert inactive zymogen pro-MMP into active MMP [4 , 8 ]. The uPAR plays an important role in the invasion of melanoma cells through an artificial basement membrane [9 ]. This effect is largely inhibited by an anti-CD87 monoclonal antibody (mAb) [9 ]. Overall, these data suggest that MMPs and the uPA-uPAR system could play an important role in leukocyte basement membrane transmigration.
Eosinophils express both MMP-9 and uPAR [10 11 12 13 ]. Okada et al. [14 , 15 ] showed that the combination of platelet activating factor (PAF) and interleukin (IL)-5 induces eosinophil basement membrane transmigration via the activation of MMP-9 and serine proteases. We recently showed that 5-oxo-6,-8,-11,-14-eicosatetraenoic acid (5-oxo-ETE), an arachidonic acid metabolite, induces a much more important eosinophil transmigration than PAF at similar concentrations [16 ]. 5-Oxo-ETE is an even more potent and specific stimulator of human eosinophil migration than PAF [17 ]. It likely acts through a specific receptor that has not yet been characterized [18 , 19 ]. 5-Oxo-ETE is produced in vitro by neutrophils, monocytes, macrophages, and eosinophils [17 18 19 20 ]. The inhibition of either MMP or uPAR decreases the 5-oxo-ETE-induced transmigration, suggesting that both protease families are significantly involved in eosinophil transmigration [16 ]. Eotaxin, a CC-chemokine, is also a potent and selective chemotactic factor for eosinophils [reviewed in references 21–23]. It is expressed by structural cells—mainly epithelial cells in normal subjects—and inflammatory cells [24 ], and it acts via CCR3, a chemokine receptor highly expressed on eosinophils [25 , 26 ].
In asthma, the number of activated eosinophils is increased in blood and in the bronchial mucosa [1 , 27 ]. Consequently, blood eosinophils of asthmatics may better respond to chemotactic factors and migrate more efficiently through the vascular basement membrane barrier than eosinophils from normal subjects. This could explain, at least in part, the large number of eosinophils in the bronchial mucosa of asthmatics. PAF and 5-oxo-ETE induce similar eosinophil transmigration in both normal and asthmatic subjects [14 , 16 ]. Recently, Kawashima et al. [28 ] suggested that the affinity of CCR3 was up-regulated in asthma, increasing the chemotactic response of eosinophils to eotaxin. This observation suggests that eotaxin could induce a greater transmigration with eosinophils from asthmatics than with eosinophils from normal subjects. In this study, we evaluated the effect of eotaxin, compared with that of 5-oxo-ETE, on eosinophil transmigration and the roles of MMP and uPAR in eotaxin-induced eosinophil migration. The responses to eotaxin of eosinophils of normal and asthmatic subjects were compared.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Selection of subjects
Normal subjects without a history of allergy or asthma and
asthmatics meeting the criteria of the American Thoracic Society for
the diagnosis of asthma [29
] were recruited for this
study. The asthmatics had mild asthma defined by a morning
prebronchodilator forced expiratory volume in 1 s
(FEV1) that was 
85% of the predicted volume and the
requirement of only a short-acting ß2-agonist on demand.
The inclusion criteria were as follows: stable asthma for more than 3
months, no inhaled steroids or asthma medication other than
ß2-agonist over the 3 months preceding the study, the use
of no other drugs, and no disease other than asthma. Approval from the
local ethics committee was obtained, and subjects signed
informed-consent forms. All subjects underwent blood sampling early in
the morning. FEV1 were measured in the morning at least
8 h after any ß2-agonist inhalation with a PFT II
Vitalograph Spirometer® (Vitalograph Ltd., Buckingham, UK), and
results were expressed as percentages of predicted values
[30
]. Blood eosinophil counts were measured with a
Coulter STKS® electronic cell counter (model 809; Coulter
Electronics, Hialeah, FL).
Blood cell processing and eosinophil purification
Blood eosinophils were purified as previously described
[16
, 31
]. Briefly, venous blood (150 mL)
was centrifuged to remove platelet-rich plasma, and the resulting
pellet was sedimented for 30 min on 6% Dextran. The leukocytes were
resuspended and centrifuged on Ficoll-Paque (1.077 g/mL) for 20 min at
100 x g. No eosinophils were observed in the lymphocyte layer.
The granulocyte layer was resuspended briefly in distilled water,
resulting in lysis of the red cells. Eosinophils were purified from
neutrophils by negative selection using bead-conjugated anti-CD16
(Fc
R III) mAb and a magnetic cell sorter. Total cell counts and cell
viabilities of the resulting suspensions were determined with a
hemacytometer and by trypan blue exclusion, respectively. Differential
cell counts (Diff-Quik®; Dade Diagnostics, Aguada, Puerto Rico) of
cytospin preparations were performed. The purity of the eosinophil
preparations was always >98%. The few contaminating cells were
neutrophils and/or lymphocytes. Eosinophils were resuspended in RPMI
1640 medium containing 25 mM
4-(2-hydroxy-ethyl)-1-piperazine-N'-2-ethanesulfonic acid, 1%
penicillin-streptomycin, and 10% FBS.
Transmigration assay and protease inhibition
The transmigration of eosinophils through the basement membrane
components was evaluated in 24-well Biocoat Matrigel Invasion
Chambers® (Becton Dickinson, Bedford, MA) as previously described
[16
]. Briefly, eosinophils (0.5 x 106
in 0.5 mL) were incubated with or without IL-5 (10 ng/mL) for 30 min at
37°C in an atmosphere of 5% CO2, placed in the upper
chamber of the Matrigel Invasion Chamber®, and incubated at 37°C in
5% CO2 for 18 h, except for the kinetic study, in
which cells were incubated for 1–24 h. Eotaxin, 5-oxo-ETE, or PAF was
used as a chemotactic factor and added separately (all in 1 µL) in
the lower chambers to the following final concentrations: eotaxin,
0.001–0.1 µM; 5-oxo-ETE, 0.01–1 µM; and PAF, 1 µM. At the end
of the incubation, cells in both the upper and lower chambers were
removed by aspiration. The remaining cells on both sides of the
membranes were gently washed and harvested twice with cold RPMI
containing 5 mM ethylenediamine tetraacetic acid but no FBS. Cells of
each chamber were counted on a hemacytometer. For each condition, the
percentage of transmigration was calculated by dividing the number of
cells in the lower chamber of the Matrigel Invasion Chamber® by the
number of cells in the lower chamber of a control invasion chamber
without the Matrigel® membrane and then multiplying by 100. In the
presence of IL-5, most eosinophils (>95%) migrated through the
uncoated control invasion chambers. The recovery efficacy of the
washing procedures after transmigration was evaluated by calculating
the number of cells recovered in both chambers after transmigration and
comparing it with the number of cells added to the upper chamber of the
Matrigel Invasion Chamber®. The proportion of cells recovered was
always >95% of the number of added cells.
To study the roles of the various proteinases in transmigration, eosinophil suspensions (0.5 x 106 cells/0.5 mL in complete RPMI medium) were preincubated with IL-5 with or without anti-MMP-9 mAb (2 µg/mL), anti-CD87 mAb (10 µg/mL), or PAI-1 (0.1 µg/mL) for 30 min at 37°C in 5% CO2 before incubation in the invasion chamber. A dose-response analysis was performed with PAI-1 at concentrations up to 10 µg/mL, and its effect was determined to be optimal at 0.01 µg/mL. When BB-3103 (5 µM), an MMP inhibitor, was used, it was added directly to the wells after the 30-min incubation with IL-5. The appropriate concentrations of mAbs and of BB-3103 were chosen from previous reports, including ours on 5-oxo-ETE [9 , 15 , 16 ]. Three different sets of experiments were done to evaluate the role of plasminogen and proteins, which are present in FBS added to cell medium, in eotaxin-induced eosinophil transmigration. First, transmigration was measured with cell medium supplemented with a low concentration of FBS (0.5%), and plasminogen was added or not to this medium. Second, transmigration was measured in the presence of a protein concentration of BSA (3.5%) equivalent to the concentration of 10% FBS, and plasminogen (2 U/mL) was added or not to BSA. Finally, autologous serum was depleted of plasminogen and transmigration were measured in the presence of either complete autologous or plasminogen-depleted autologous serum. In some wells, plasminogen was added to plasminogen-depleted autologous serum.
Depletion of plasminogen from human serum.
Venous blood, collected in anticoagulant-free tubes, was
centrifuged for 20 min at 1,000 x g. The resulting
serum was collected and incubated for 30 min at 4°C with a mouse mAb
against human plasminogen in the presence of a 2:1 molar excess of
plasminogen. A goat anti-mouse IgG conjugated to magnetic beads was
added in a 1.5:1 molar excess of the primary antibody, and the serum
was incubated at 4°C for 20 min. Then the serum was incubated in a
type A2 magnetic cell sorter separation column (Miltenyi Biotec) under
a magnetic field for 15 min and eluted with RPMI 1640 medium. The
quantity of RPMI 1640 used for elution was adjusted to contain serum at
a final concentration of 10%. The serum was tested for plasminogen
biological activity [32
] and concentration by
radioimmunodiffusion (The Binding Site LTD, Birmingham, UK), which
were, respectively, 1.4 and 1.5 U/mL in untreated serum and 0.7 and 0.5
U/mL in plasminogen-depleted serum.
Flow-cytometric analysis of eosinophils
For the expression of CD87, eosinophils (106
cells/mL) were incubated in RPMI 1640 and stimulated or not with
recombinant IL-5 (10 ng/mL) or eotaxin (0.01 µM) for 30 min at 37°C
in a 5% CO2 atmosphere. They were then washed once with
1x phosphate-buffered saline (PBS) supplemented with 0.1% BSA.
Briefly, 250,000/100 µL were incubated for 45 min at 4°C with 1
µg/mL of mouse monoclonal anti-human CD87 or mouse IgG2a (isotype
control). Cells were washed once with PBS plus 0.1% BSA, resuspended
in 100 µL of the same diluent, and incubated for 45 min at 4°C with
1-µg/mL of fluorescein isothiocyanate-conjugated goat anti-mouse Ig.
Expression of CD87 on eosinophils was analyzed immediately by flow
cytometry (EPICS ELITE ESP; Coulter Electronics). For the expression of
CCR3, freshly purified eosinophils were fixed with 4% paraformaldehyde
for 10 min and then washed with PBS plus 0.1% BSA. Cells (250,000/100
µL) were incubated 45 min at 4°C with rat monoclonal anti-human
CCR3 (25 µg/mL) or rat IgG2a (isotype control, 25 µg/mL). They were
washed with PBS plus 1% BSA, resuspended in 100 µL of the same
diluent, and incubated for 45 min at 4°C with biotinylated goat
anti-rat Ig (0.5 µg/mL). They were washed twice and incubated with
streptavidin-phycoerythrin (0.1 µg/mL) for 30 min at 4°C. After two
washes, cells were analyzed by flow cytometry.
Statistical analyses
Means and SEs were determined for continuous
variables. For all analyses, normality and variance assumptions were
fulfilled. A posteriori comparisons were performed using Tukey’s
method. Kinetics and dose-response data were evaluated by a
repeated-measures analysis of variance (ANOVA). Comparisons between
eotaxin and eotaxin plus IL-5 were performed using the Student paired
t-test. The response of asthmatics to IL-5 was compared with
that of normal subjects by using a crossed-nested design. Mean values
of Fig. 5
were compared by two-way ANOVA (randomized block design). The
effect of plasminogen on eosinophil transmigration was analyzed by
three-way ANOVA using the subject as the blocking factor. The results
were considered significant if P values were <0.05. The
data were analyzed using the statistical package program SAS (6.12
version; SAS Institute Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Kinetics and dose-response of eotaxin on eosinophil transmigration
In the presence of IL-5, eotaxin induced a significant
transmigration through the Matrigel® of both normal and asthmatic
subjects’ purified blood eosinophils (Fig. 1
). The maximal effect of eotaxin was obtained at a concentration of
0.01 µM in both cell preparations and decreased with higher
concentrations (Fig. 1A)
. In the kinetic study, the effect of eotaxin
(0.01 µM) increased for up to 18 h (Fig. 1B)
. An additional
experiment, done at 18 and 24 h on eosinophils from six subjects,
showed that eotaxin-induced transmigration plateaued at 18 h
(44.2 ± 6.1% and 46.6 ± 4.1% at 18 and 24 h,
respectively). The difference between the eosinophil migration rates of
normal and asthmatic subjects reached statistical difference in the
kinetic study at 18 h [eosinophils from normal subjects,
50.7 ± 2.1%; eosinophils from asthmatic subjects, 73.9 ±
5.6% (P = 0.018)]. Comparison of eotaxin and
5-oxo-ETE at different concentrations shows that at a low concentration
(0.01 µM) eotaxin was a more potent inducer of eosinophil
transmigration than 5-oxo-ETE. However, at higher concentrations,
5-oxo-ETE was more effective at inducing eosinophil transmigration than
eotaxin (Fig. 2
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this experiment, eotaxin proved to promote significantly eosinophil transmigration through basement membrane components. Such an effect was not observed by Okada et al. [14 , 15 ], who supplemented their eosinophil incubation medium with albumin instead of complete serum. The absence of serum and the consequent absence of plasminogen, the precursor of plasmin, could explain the very low eosinophil transmigration rate that they observed. Our results show that, in the presence of plasminogen, eotaxin induces eosinophil transmigration in vitro. This is further demonstrated by the selective inhibition of eotaxin-induced transmigration by CD87 blocking and the presence of a serine proteinase inhibitor. Since the magnitude of eosinophil transmigration obtained with PAF in this study is comparable to the magnitude reported by Okada et al. [14 , 15 ], the difference observed with the use of eotaxin is unlikely due to other differences in transmigration assay techniques. The concentration of human plasminogen (2 U/mL) used in our experiments was slightly higher than the concentration of serum measured in humans (normal range: 0.88–1.37 U/mL)[32 ].
Although its maximal effect was observed at lower concentrations, eotaxin was less effective than 5-oxo-ETE in inducing cell migration at optimal concentrations. This could be explained, at least in part, by the greater capacity of 5-oxo-ETE to activate members of both eosinophil proteinase families. The proteinase activities of MMP and the plasminogen-plasmin system were not, however, evaluated in this study. Moreover, eotaxin, but not 5-oxo-ETE, was more active on eosinophils from asthmatics than on eosinophils from normal subjects. Since eosinophil CCR3 expression was significant and was similar in normal and asthmatic subjects, this difference could be explained by an increased affinity of CCR3 receptors for eotaxin in eosinophils from asthmatics, as suggested by Kawashima [28 ]. Our asthmatic subjects had increased blood eosinophil counts and did not take inhaled steroids. We can therefore conclude that their eosinophils were activated, and we can therefore postulate that they expressed CCR3 with high affinity, although this was not measured in the present study. These data suggest that, in asthma, the tissue recruitment of eosinophils in response to eotaxin is increased, contributing to the observed increase in tissue eosinophilia.
As described in relation to 5-oxo-ETE, eotaxin alone could induce eosinophil transmigration, an effect that was increased by IL-5. Since IL-5 by itself has no effect on cell transmigration, it likely amplifies the effect of eotaxin by priming eosinophils for an enhanced responsiveness to this chemokine, as has already been proposed [33 , 34 ]. We did not evaluate the mechanisms of this priming, which tended to be more important for eosinophils from asthmatic subjects than for those from normal subjects.
MMP-9 and the plasminogen-plasmin system represent the two major known proteinases expressed by eosinophils. As mentioned above, our data on the inhibition of MMP and the plasminogen-plasmin system support the conclusion that eotaxin and 5-oxo-ETE differ in their capacity to activate the different proteinases. Although the inhibition of MMPs and uPAR provided significant and similar inhibition of eosinophil transmigration, neither completely blocked eosinophil transmigration. We previously reported that the proteinase inhibition was not complete in the presence of inhibitors and/or mAbs [16 ]. We suggested that these enzymes are active on the cell membrane area that is in contact with the basement membrane and that this physically confined area remains, to a certain point, out of reach of the inhibitors. The large decrease of eosinophil transmigration in medium with a low concentration (0.5%) of FBS, in BSA, and in plasminogen-depleted autologous serum and its restoration by the addition of plasminogen to these media support the hypothesis that the efficacy of these inhibitors is limited. However, other molecules, such as the new family of membrane proteins containing a disintegrin and metalloproteinase domain (ADAM), could be involved in cell-mediated extracellular-matrix digestion [35 ]. To our knowledge, no ADAM has been observed on eosinophils.
In conclusion, this study shows that eotaxin is a potent promoter of eosinophil transmigration and that its effect is more important for eosinophils from asthmatic subjects than for those from normal subjects. These data suggest that eotaxin could play an important role in the increased bronchial eosinophil recruitment observed in asthma. Also, various proteinases, notably MMP-9 and the plasminogen-uPAR system, mediate eosinophil transmigration induced by chemotactic factors. The profiles of proteinase activation for eotaxin, which activates predominantly the plasminogen-plasmin system, and for 5-oxo-ETE, which activates both MMP-9 and the plasminogen-plasmin system, differ [16 ].
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ACKNOWLEDGEMENTS |
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The authors thank Luce Trépanier for her precious help in recruiting and evaluating the subjects, Joanne Milot and Francine Deschesnes for blood sampling, Sophie Lavigne for performing the flow cytometry analysis, and Serge Simard for doing the statistical analysis.
Received July 20, 2000; revised November 6, 2000; accepted December 27, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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