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*
Laboratory of Immunology and Vascular Biology, La Jolla Institute for Experimental Medicine, La Jolla, CA 92037;
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92937; and
Department of Medicine, University of California at San Diego, La Jolla, CA 92122
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Mß2 integrin expression on eosinophils but
not neutrophils, while C5a induced shedding of L-selectin and
up-regulation of Mß2 integrin on both
eosinophils and neutrophils. Both C3a- and C5a-dependent adhesion to
venular endothelium was blocked by ex vivo treatment of eosinophils
with anti-4 and anti-ß2 integrin
mAbs. In vitro, both C3a (but not C3adesArg) and C5a
(including C5adesArg)-dependent transmigration of
eosinophils across IL-1ß-stimulated endothelial monolayer was
mediated by 4ß1 and
Mß2 integrins. Overall these studies
suggest that C3a is eosinophil-specific chemotactic mediator that
influences selectively eosinophil adhesion but not transmigration in
vivo. C5a in contrast is a complete activator of integrin-dependent
adhesion as well as transmigration of eosinophils and
neutrophils. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, receptors for C3a and C5a have been cloned and sequenced. These receptors are members of the G-coupled receptor family and are found in a variety of cell types (7, 8, 9, 10, 11). The C3a and C5a receptors are membrane constituents of both neutrophils and eosinophils; however, differing outcomes result from engagement of these receptors with their respective ligands.
Whereas C3a can evoke neutrophil calcium ion mobilization and a respiratory burst but is inactive chemotactically for these cells, C5a can mediate a greater range of neutrophil responses such as a respiratory burst, degranulation, and chemotaxis (6, 12, 13). However, the situation differs for eosinophils as both C3a and C5a are reported to be chemotaxins as well as mediators for degranulation and oxidant secretion (5, 14, 15, 16).
Most published findings demonstrating the chemotactic potential of C3a and C5a for eosinophils were based on static in vitro assays (5, 16). Because a greater range of complicating effects occurs in vivo including the physical influence of shear forces in blood vessels, it remains important to demonstrate the ability of the anaphylatoxins to mediate eosinophil adhesion and recruitment under in vivo conditions.
Although our current understanding of the recruitment of
eosinophils to allergic inflammatory sites is far from complete, a
general multistep paradigm has been described for leukocyte
mobilization that provides insight into this phenomenon. According to
this paradigm, the three sequential steps involve leukocyte rolling
along the vascular wall, followed by activation-dependent firm adhesion
of the leukocytes to the endothelium, and finally
chemoattractant-directed transmigration of the leukocytes into the
extravascular space (1, 17, 18, 19). For eosinophils, rolling is the first
and rate-limiting step of the multistep adhesion cascade in vivo and is
mediated by eosinophil L-selectin (CD62L) interaction (2, 44) with
endothelial sialylated and fucosylated glycoconjugates. In addition
4ß1 and 4ß7
integrins mediate eosinophil rolling on VCAM-1 (CD106) (20, 21, 22, 23, 24, 44).
Moreover, eosinophil rolling in blood vessels is also mediated by
endothelial P-selectin (CD62P) (25) but not E-selectin (CD62E) (21, 26). Leukocyte rolling is followed by a stimulus dependent firm
adhesion, a process that is accompanied by rapid of activation of
ß2 integrins (27). In case of eosinophils,
4 integrins in addition could contribute to the adhesion
process by interacting with inducible VCAM-1 on vascular endothelium
(23, 24).
Transmigration of adherent leukocytes including eosinophils across
cultured endothelial cells is dependent on the interaction of
Lß2 and Mß2
integrins with their endothelial counterreceptors, ICAM-1 (CD54) and
ICAM-2 (CD102) (17, 28, 29, 30, 31). In addition, homophilic interactions
between molecules of platelet endothelial cell adhesion molecule-1
(CD31) on leukocytes with those on endothelial cells have also been
reported to be essential for leukocyte motility (32).
Although these studies define a plausible scheme for eosinophil-
endothelial cell interactions in general, the underlining mechanisms by
which rolling eosinophils respond to a specific chemotactic stimulus
and sequester to sites of inflammation are not well understood.
Although studies demonstrating that C3a and C5a can function as
eosinophil active chemotaxins in vitro have also been published (5, 16), the comparative abilities of the anaphylatoxins to direct
eosinophil adhesion and subsequent transmigration across the vascular
endothelium have not been investigated. In the present report, we have
examined the ability of 4 and ß2 integrins
to mediate C3a and C5a dependent stable adhesion of rolling eosinophils
and their subsequent transmigration across inflamed postcapillary
venules of the rabbit mesentery in vivo and across cultured endothelial
cells in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dextran 70 was supplied by Baxter Diagnostics Inc. (McGaw Park, IL). Ficoll-Paque PLUS (endotoxin-free) was purchased from Pharmacia Biotech (Piscataway, NJ). Earle’s balanced salt solution was supplied by Life Technologies (Grand Island, NY). HEPES and Eosin Y were obtained from Sigma (St. Louis, MO). Goat anti-mouse IgG-coated magnetic beads and a magnetic separation unit were purchased from PerSeptive Biosystems (Framingham, MA). Unconjugated mouse anti-human mAbs against CD16 were purchased from Biosource International (Camarillo, CA). RPMI 1640 and FCS were from Bio-Whittaker (Walkersville, MD). IL-1ß was purchased from R&D systems (Minneapolis, MN). Endothelial cell basal media (EBM),3 endothelial cell growth media (EGM), and primary cultures of HUVECs were purchased from Clonetics (Walkersville, MD). Biocoat inserts (containing 3.0-µm pores) were obtained from Becton Dickinson Labware (Bedford, MA). The fluorescent compound, 5- and 6-carboxyfluorescein diacetate (CFDA), was from Molecular Probes (Eugene, OR). Loperamide hydrochloride (Immodium) was from McNeil-PPC (Fort Washington, PA), while xylazine hydrochloride and lidocaine were from Butler (Columbus, OH).
Isolation of human C3a, C5a, and carboxypeptidase N (CPN)
Human C3a and C5a were generated from complement components C3 and C5 using fluid phase C3 or C5 convertases. C3a and C5a were isolated subsequently by ion exchange chromatography (33). CPN was purified from the same batch of human plasma used to isolate >20 other plasma proteins (33). Briefly, the steps included 4.5–12% polyethylene glycol precipitation of barium citrate-treated plasma, DEAE-Sephadex column chromatography, gel filtration through Sephacryl S300, and affinity chromatography using p-aminobenzoyl-L-arginine-Sepharose.
Monoclonal Abs
Blocking mAbs with specificities to CD11a (L
integrin) (clone TS1/22.1.1.13) and CD11b (M integrin)
(clone 44aacb) were isolated from hybridoma cells obtained from
American Type Culture Collection (Manassas, VA) (34, 35). Anti-CD11c
(X integrin) (clone CBR-p150/4G1) was from Biosource
International. mAbs to CD29 (ß1 integrin chain) (clone
P4C10) (36) were from Life Technologies (Gaithersburg, MD). mAbs with
specificity to the integrin chain CD49d (4 integrin)
(clones P4G9 and P4C2) and those directed against CD51/CD61
(Vß3 integrin) (clone LM609) and CD51/ß5
(Vß5 integrin) (clone P1F6) were from Chemicon
(Temecula, CA) (37, 38). mAbs against CD18 (ß2 integrin)
(clone IB4) (39) and human tenascin 81C6 (40) were also used. A mAb
preparation against mouse ß7 integrin (clone FIB504) with
cross-reactivity to human ß7 integrin (41) and
anti-CD62L (L-selectin; clone DREG200) (42) were obtained from Dr.
Eugene Butcher (Stanford University, Stanford, CA). Preparations of
mAbs with endotoxin levels in range of 1–10 ng/mg as determined by
amebocyte Limulus test were used. Phycoerythrin-conjugated
secondary Abs to mouse and rat IgG were purchased from Jackson Research
Laboratories (West Grove, PA). FITC-conjugated Ab to CD9 was obtained
from PharMingen (San Diego, CA).
Eosinophil preparation
Eosinophils were prepared from the peripheral blood of normal donors, having at least 3% peripheral blood eosinophilia, using essentially a two-step procedure. First, an eosinophil-enriched fraction was obtained by centrifugation through Ficoll-Paque PLUS. The second step involved removal of residual neutrophils using negative selection of CD16-positive cells by a magnetic bead separation technique (43). Eosinophils were >99% pure as assessed by eosin staining.
For both in vitro and in vivo experiments, eosinophils were labeled fluorescently using CFDA similarly to a procedure previously described (44). Eosinophils (1.5 x 107 cells) were suspended in 5 ml of buffer A: 10 mM HEPES, pH 7.35, 0.145 M NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2 and reacted with a solution (100 µl) of CFDA (2 mg/ml) for 30 min in the dark. The cells were washed once with buffer containing 1 mg/ml human serum albumin and suspended in the same buffer or in EBM at a concentration of 106 cells/ml.
Animal preparation and superfusion of the mesenteric vascular bed with C3a and C5a
The circulation of CFDA-labeled human eosinophils in postcapillary venules of IL-1ß-stimulated mesenteric blood vessels of New Zealand White rabbits was examined by intravital microscopy as described earlier (26, 44). The ability of C3a or C5a to mediate adhesion and transmigration of rolling eosinophils across IL-1ß-stimulated mesenteric venules was determined. Between 6 and 10 h after IL-1ß stimulation (i.p.), portions of the exposed mesentery were superfused with 10-9–10-6 M C3a or C5a using a constant flow infusion syringe pump (Harvard Apparatus, South Natick, MA). The flow of the syringe pump was adjusted to match the flow of the superfusion buffer. CFDA-labeled eosinophils (0.2–0.5 x 107 cells) were administered into the mesenteric circulation 5 min after the superfusion had ensued. These cells were injected successively through the side branch of the terminal superior mesenteric artery cannulated with a PE10 polyethylene catheter as previously described (27).
Intravital microscopy and image analysis
The passage of CFDA-labeled eosinophils in the inflamed venules was made visible by stroboscopic epiillumination as previously described, and the images were recorded with an SVHS video recorder (27). The video images were analyzed offline by manually counting the total number of CFDA-labeled eosinophils passing through a reference point in a venular segment (total flux). The tapes were rewound, and only those cells found to be visibly rolling along the venular wall were counted (rolling flux). Rolling fraction (RF) was calculated as the percentage of rolling cells in the total flux of eosinophils passing through a venular segment during a given injection. Adherent eosinophils were defined as cells that remained stationary in the venule for at least 30 s, and data are expressed as adherent cells/250-µm length of the vessel. Eosinophil transmigration was quantified by enumerating the number of adherent cells that had emigrated away from the vessel wall in parallel with a 250-µm length of a vessel segment.
Inhibition of in vivo adhesion and transendothelial migration by anti-integrin mAbs
The abilities of anti-4,
anti-ß1, anti-ß2, or
anti-ß7 integrin mAbs to block eosinophil adhesion
and transmigration across IL-1ß-stimulated venular endothelium were
determined as follows. Eosinophils were incubated ex vivo with
functional blocking anti-4 integrin (mAb P4G9),
anti-ß1 integrin (mAb P4C10),
anti-ß2 integrin (mAb IB4), anti-ß7
integrin (mAb FIB504) Abs, or combinations of these at a concentration
of 50 µg/ml for 20 min at room temperature before injection into the
rabbit mesentery, which was superfused with either C3a or C5a
(10-9–10-6 M). As a control a mAb with a
specificity vs human tenascin (clone 81C6) was used. Statistical
analyses were made using analysis of variance and multiple comparisons
tests. For all tests, p values <5% were considered
significant. Data are presented as mean ± SD. Statistical
calculations were computed with a statistical software package
(SigmaStat, Jandel Scientific, San Rafael, CA).
Flow cytometry
Granulocyte preparations, which were enriched for eosinophils (>10% eosinophils), were obtained according to the eosinophil isolation procedure using centrifugation through Ficoll-Paque PLUS (45). Hypotonic lysis to remove erythrocytes was not performed. Granulocytes were stimulated for 15 min at 37°C with either C3a (100 nM) or C5a (10 nM). The cells were immediately placed on ice and were incubated first with primary mAbs (5 µg/ml) in HBSS containing 10% FBS for 45 min. The cells were washed and incubated with appropriate phycoerythrin-conjugated secondary Abs to either mouse or rat IgG (FIB27) followed by incubation with FITC-conjugated Ab to CD9, an eosinophil-specific granulocyte marker. The cells were washed again and analyzed for receptor expression using Becton Dickinson FACScan or FACSort instruments with CellQuest software (Becton Dickinson, San Jose, CA). A minimum of 5000 eosinophils was collected and analyzed for each determination. Data from three experiments were normalized to the staining with nonspecific mouse IgG as a negative control and are expressed as arbitrary fluorescence units.
Eosinophil chemotaxis assays
Chemotaxis of eosinophils was performed using a two-chamber system. The upper chamber consisted of an insert that was separated from the lower chamber by a membrane of polyethylene terephthalate containing 3-µm pores. For each determination, a total of 105 labeled eosinophils was placed in the upper insert. The lower well contained 700 µl of buffer A containing 1 mg/ml human serum albumin with various concentrations of C3a (10-9–10-5 M) or C5a (10-10–10-6 M). The plates were incubated at 37°C for 2 h. Then EDTA was added to a final concentration of 10 mM to the bottom well, and the plates were allowed to stand for 10 min at room temperature before the inserts were removed from the wells. The cells, which had passed through the membrane and which were contained within a 1.1-mm2 central area on the bottom well, were counted using an inverted fluorescence microscope (Leitz Fluovert FS, Wetzlar, Germany).
Preparation of HUVECs
Individual growth factor supplements were added to EBM to formulate the growth media EGM. These supplements include bovine brain extract, human epidermal growth factor, hydrocortisone, gentamicin, and FBS (final concentration, 2%). EGM (4 ml) containing 1–2 x 105 cells/ml of HUVEC was transferred to tissue culture flasks and incubated in a humidified atmosphere at 37°C and 5% CO2. Medium was changed every 2 days until reaching confluence between 6 and 10 days after plating. Confluent cells were trypsinized, washed, and resuspended in EGM. Aliquots of 250 µl were added to the upper fibronectin-coated inserts of the transwell assemblies, and 700-µl portions of EGM were added to the lower wells. The medium was changed every 2 days. FITC-labeled albumin was added to the upper well of both HUVEC-containing and empty Transwell (Corning Costar, Cambridge, MA) chambers to guide assessment of confluency. When confluent, EBM containing 103 U/ml IL-1ß was added to both upper and lower chambers, and cells were cultured for an additional 18 h before transmigration assays.
Eosinophil transendothelial migration assays
Transmigration of eosinophils through endothelial cells, which were grown as confluent monolayers on fibronectin-coated inserts, was performed similarly to the assays for chemotaxis through pores in polyethylene terephthalate membranes, except that EBM was used instead of buffer A. The inhibition of C3a- or C5a-mediated transmigration of eosinophils by various anti-integrin mAbs was performed by mixing the eosinophils with mAbs (5 µg/ml) for 10 min at 23°C before these cells were used in transmigration assays. To examine the potential role of CPN on eosinophil transmigration evoked by C3a and C5a, CPN (10 µg/ml) was placed into the bottom wells just before the addition of eosinophils, and transmigration assays were conducted as before.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the possible effects of C3a and C5a on the behavior of
circulating eosinophils and neutrophils, CFDA-labeled cells were
administered into the rabbit mesentery microcirculation, and their
ability to interact with IL-1ß-stimulated postcapillary venules was
visualized by intravital microscopy. CFDA-labeled eosinophils were
observed to roll avidly (RF; range, 20–80%) along the walls of
cytokine-stimulated postcapillary venules. In the absence of further
activation, spontaneous adhesion of rolling eosinophils was not
frequently observed in the majority of the venules examined. Although
firm adhesion of a few eosinophils was occasionally noticed, this
represented only 1–5% of the total number of rolling eosinophils. We
next determined whether rolling eosinophils would firmly adhere in the
postcapillary venules in response to extravascular stimulation by the
complement anaphylatoxins. To examine this question, the
microcirculation within the exteriorized mesentery, which had been
pretreated for 6–10 h with IL-1ß, was constantly superfused with C3a
or C5a (10-6–10-9 M), and the interaction of
the infused labeled eosinophils in the inflamed blood vessels was
examined microscopically (Fig. 1
). In
contrast to a lack of spontaneous adhesion of eosinophils observed in
majority of the blood vessels, administration of CFDA-labeled
eosinophils into the mesentery superfused with C3a
(10-6–10-8 M) resulted in the rapid and
stable adhesion of rolling eosinophils (range, 2–4 adherent
cells/250-µm length at 10-6 M) (Fig. 2). Adhesion was observed in venules but
not arterioles (Fig. 1). In comparison with eosinophils, neutrophils
failed to adhere in response to C3a, suggesting that this
chemoattractant selectively activates rolling eosinophils, but not
neutrophils (Fig. 2).
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and 2). Maximum adhesion was observed when rolling
eosinophils were superfused at a concentration of 10-6 M
C3a, while C5a-induced adhesion was maximally observed between
10-7 and 10-8 M. In all experiments that we
performed, C5a was more effective than C3a in inducing firm
adhesion of rolling eosinophils on cytokine-stimulated
venular endothelium. Effect of anti-integrin mAbs on C3a- and C5a-mediated firm adhesion in vivo
4
(4ß1/4ß7) and
ß2 integrins are known to mediate adhesion of leukocytes
in vitro. Therefore, we hypothesized that the observed
activation-dependent firm adhesion of eosinophils or neutrophils to
rabbit mesentery venules induced by C3a and C5a was dependent on the
engagement of one or several of these adhesion molecules. To examine
this, eosinophils and neutrophils were treated ex vivo with functional
blocking mAbs directed against 4, ß1,
ß2, and ß7 integrins before administration
into C3a- or C5a-stimulated rabbit mesentery, and the ability of
rolling eosinophils to adhere to the venular endothelium was determined
(Fig. 3). Pretreatment of eosinophils
with anti-4, anti-ß1, or
anti-ß2 integrin mAbs resulted in 
40–80%
inhibition of C3a-induced adhesion, and similarly these Abs inhibited
C5a-mediated adhesion 50–80%. A smaller effect was also observed
with anti-ß7 integrin, which inhibited C3a- and
C5a-induced adhesion 20%. No further inhibition of adhesion was
observed when eosinophils were pretreated with
anti-ß1 integrin and anti-ß7
integrin mAbs in combination. The nearly complete inhibition of
adhesion induced by preincubation of eosinophils with
anti-ß1 and anti-ß2 integrin mAbs
in combination is suggestive of the significant involvement of both
ß1 (4ß1) and
ß2 integrins in C3a- or C5a-mediated adhesion. An
irrelevant control mAb 81C6 had little inhibitory effect on C3a- and
C5a-induced adhesion of eosinophils. In contrast to eosinophils,
neutrophil adhesion to the venular endothelium induced by C5a was
inhibited only by mAbs against ß2 integrins and not by
those against ß1, 4, or ß7
integrins (Fig. 3).
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Although C3a evokes chemotaxis of eosinophils in vitro (5), C3a
was not observed to cause extravascular migration of the adherent
eosinophils in vivo after the mesentery was superfused with this
anaphylatoxin (10-7–10-6 M) (Fig. 4). It was not possible to evaluate the
effect of C3a at a superfusion concentration of >10-6 M
because at these levels of anaphylatoxin application to the rabbit
mesentery resulted in intravascular coagulation, platelet aggregation,
thrombus formation, and even cessation of blood flow. A clear evidence
of extravascular migration was not discernible after superfusion of the
mesentery with C3a (1 emigrated cell in n = 5 rabbits,
3–5 venules/experiment) (Fig. 5).
However, a careful inspection of the venular endothelium under higher
resolution (x40 objective) revealed that this anaphylatoxin did cause
eosinophil penetration between adjacent venular endothelial cells.
Nevertheless, these adherent eosinophils failed to emigrate from the
vessel wall into the extravascular space even after 30 min of C3a
exposure (Fig. 4).
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). The extent of C5a-induced eosinophil
migration was variable and ranged from 3 to 5 cells along a 250-µm
length of the venular wall (Fig. 5). Maximal transmigration of
eosinophils was observed when C5a was superfused at a concentration of
10-7–10-8 M. Likewise, the extent of
C5a-induced neutrophil emigration varied from donor to donor (range,
3–7 cells along a 250-µm length of the venular wall) (Fig. 5). Most
adherent cells were observed to emigrate into the extravascular space
after 10–15 min of exposure to C5a. Venular hemodynamics
In addition to adhesion, we examined the effects of C3a and C5a
superfusion on venular hemodynamics in the rabbit mesentery (Table I). Superfusion of C3a and C5a
(10-6 and 10-7 M, respectively) had no
significant effect on either the velocity of rolling eosinophils and
neutrophils or shear stress within the postcapillary venules.
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Since chemoattractants modulate activity and expression of cell
surface receptors, we used flow cytometry to examine whether or not C3a
and C5a affected the levels of several eosinophil and neutrophil
adhesion molecules (Table II). C3a
altered eosinophil but not neutrophil surface expression of several
adhesion molecules that we examined. Exposure of eosinophils to C3a
resulted in an 20% reduction of L-selectin, an appreciable
augmentation (>100%) of M integrin, and a smaller rise
(20–40%) in ß2- and X integrins. In
contrast to the selective effect of C3a on the expression of eosinophil
adhesion molecule expression, C5a had similar effects on both cell
types. For both eosinophils and neutrophils C5a evoked a 20–50%
reduction in L-selectin and a significant up-regulation (170–220%) of
M integrin. The up-regulation of ß2
integrin by C5a was the only adhesion molecule that showed any
appreciable cell type dependence, where the augmentation for
neutrophils (160%) exceeded that of eosinophils (30%). For both
cell types, C5a evoked moderately large increases (40–100%) in
X integrin expression. No significant changes in the
expression of L, 4, or ß1
integrins were observed after stimulation of either cell type with
either anaphylatoxin.
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Since C3a and C5a were observed to evoke eosinophil adhesion and
C5a induced transmigration in vivo, we proceeded further to elucidate
mechanisms and regulation of these two complement mediators for
eosinophil chemotaxis and transendothelial migration in vitro.
Eosinophils were observed to be more responsive chemotactically to C5a
than C3a when assayed for migration through 3-µm pores in
polyethylene terephthalate filters (Fig. 6). The effective range of concentration
for chemotaxis induced by C5a was 10-8–10-6
M, whereas that for C3a was 10-7–10-5 M.
Furthermore, at peak activities, about 1.5-fold more eosinophils
responded to C5a than C3a (Fig. 6).
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). In
these cases, at the optimal concentrations of the anaphylatoxins, more
than twice as many eosinophils transmigrated in response to C5a than
C3a. This effect occurred whether or not the endothelial cells were
primed with IL-1ß. Furthermore, the optimal concentration for
transendothelial migration was an order of magnitude lower for C5a than
C3a (10-7 M vs 10-6 M).
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and 7). Since the
in vivo observations suggested the engagement of eosinophil
4/ß1 and ß2 integrins (with
counterreceptors presented by activated endothelial cells), we examined
potential inhibition by anti-integrin mAbs on transmigration of
eosinophils through both unstimulated and IL-1ß-primed HUVECs. Effect of anti-integrin mAb on C3a- and C5a-mediated transmigration in vitro
The profiles for the inhibition by anti-integrin mAbs of C3a and
C5a induced transmigration of eosinophils through unstimulated
endothelial cells were similar (Fig. 8).
Both C3a- and C5a-induced eosinophil migration were inhibited strongly
by mAbs to L and ß2 integrins, but only
weak inhibition, if any, was caused by the other anti-integrin mAbs
tested (Fig. 8).
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and 9). When cytokine-stimulated endothelial
cells were used, inhibition was caused not only by mAbs to
L and ß2 integrins but also by those to
M, 4, and ß1 integrins, and
to a lesser extent ß7 integrin (Fig. 9). In contrast,
other mAbs failed to block appreciably eosinophil transmigration. This
spectrum of inhibition by mAbs was the same whether or not C3a or C5a
was the mediator for eosinophil migration (Fig. 9).
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To explore possible differences in eosinophil migration evoked by
C3a and C5a in vivo and in vitro, the influence of CPN, a plasma
inhibitor of the anaphylatoxins, was examined. When this
carboxypeptidase was present at a concentration of 10 µg/ml along
with the anaphylatoxins, all migration of eosinophils to C3a
(C3adesArg) was lost, whereas residual migration to C5a
(C5adesArg) was retained (Fig. 10). The optimal concentration of
C5adesArg for transmigratory activity was about 1–2 orders
of magnitude higher than that for intact C5a, and the number of cells
that migrated at the optimal concentration of C5adesArg was
almost one-half that at the optimal concentration of C5a.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
In contrast, C5a induces rapid activation-dependent adhesion of both
rolling neutrophils and eosinophils. Moreover, C5a-induced adhesion is
associated with subsequent chemotaxis and transmigration of both
eosinophils and neutrophils into tissue. These studies suggest that in
vivo C3a selectively influences rolling eosinophils to adhere at sites
of inflammation but may not be a potent chemoattractant in that it
fails to induce transmigration of the adherent cells under the
conditions reported. In contrast, C5a is a complete activator of
integrin-dependent adhesion as well as an effective chemoattractant for
eosinophils and neutrophils in vivo. The effects of C3a and C5a on
eosinophils and neutrophils are biochemical and are not a result of
alterations in hemodynamic properties (Table I). Parameters such as
rolling velocity and shear rate of eosinophils and neutrophils did not
vary appreciably to account for the effects of the anaphylatoxins.
Variations in the spectrum of adhesion molecules on the surface of the
eosinophil in response to C3a and C5a are expected to be important for
the transitions that this cell type undergoes during emigration as
described by the multistep paradigm. Alterations of the levels of
several eosinophil and neutrophil membrane adhesion molecules were
determined by flow cytometry after these cells were exposed to C3a or
C5a. For eosinophils both anaphylatoxins acted similarly by evoking a
shedding of L-selectin with a concomitant rise in M,
X, and ß2 integrins, while
L, 4, and ß1 integrin
profiles remained essentially unchanged (Table II). L-selectin is
utilized for eosinophil rolling along the vascular endothelium (44),
and its shedding corresponding with an up-regulation of eosinophil
Mß2 integrin was found to occur during
transendothelial migration (30). This suggests that alterations of
these adhesion molecules may be important for the accumulation of
eosinophils at sites of allergic inflammation. Accordingly, the
transition from rolling to adherent eosinophils that occurs in vivo
after application of either anaphylatoxin (Figs. 1 and 2) could be
brought about in part by altered levels of L-selectin and
Mß2 integrin.
The application of functional blocking mAbs to eosinophil adhesion
molecules in vivo indicated that for both anaphylatoxins the observed
transition from eosinophil rolling to firm adhesion is dependent not
only on ß2 integrins but also on 4
integrins as well. Although, the levels of 4 and
ß1 integrins are not up-regulated by the anaphylatoxins
(Table II), we suggest that the most likely interpretation is an
augmented functional adhesive state of 4ß1
integrin. This integrin is known to be able to modulate its functional
state and has been reported transition from a low avidity to high
avidity state after GM-CSF stimulation of eosinophils (46, 47). Our
data suggest that 4ß1 integrin is more
efficient than 4ß7 integrin as an adhesion
receptor during episodes of inflammation mediated by the anaphylatoxins
C3a or C5a, although both 4ß1 and
4ß7 integrins can support eosinophil
rolling on VCAM-1 in vitro and in
vivo.4
When eosinophil chemotaxis and transendothelial migration were examined
in vitro, we observed that the optimal concentration range of C3a
(10-7–10-5 M) was an order of magnitude
higher than that of C5a (10-8–10-6 M), and
the number of eosinophils migrating in response to C3a was less than
that to C5a (Figs. 6 and 7). Although the potential plasma
concentration of C3a is 20 times that of C5a, our results indicated
that whether chemotaxis through plastic membranes or transmigration
through endothelial cells was measured, C5a evoked the migration of
1.5–3-fold more cells than did C3a (Figs. 6 and 7). An explanation for
this could be rooted in eosinophil heterogeneity (48), and it is
conceivable that only a subpopulation of eosinophils may respond
chemotactically to C3a.
Both anaphylatoxins evoked greater motility for eosinophils though
IL-1ß-stimulated endothelial cells than through unstimulated
endothelial cells or through pores in naked plastic (Figs. 6 and 7).
The increased transmigration across IL-1ß endothelial monolayers, in
comparison with unstimulated cells, is likely to be a consequence of an
augmented adhesion of the eosinophils to cytokine-stimulated
endothelial cells. This effect is probably caused by an up-regulation
of endothelial ICAM-1 (CD54), and induction of VCAM-1 (CD106) (48, 49),
which interact with greater avidity with their eosinophil
counterreceptors, ß2 and 4 integrins,
respectively (20, 24, 50, 51, 52, 53). The augmented interaction should result
in greater traction, facilitating mobility of the
eosinophil.
Inhibition studies using mAbs directed against several eosinophil
adhesion molecules indicated that in the absence of cytokine
stimulation, C3a- or C5a-mediated eosinophil transmigration across
HUVEC monolayers was primarily dependent on
Lß2 integrin (CD11a/CD18), which interacts
with ICAM-1 and ICAM-2 (CD102), that are constitutively expressed at
low levels on unprimed endothelial cells (28, 54, 55). However, when
IL-1ß-treated endothelial cells were used, significant inhibition was
observed by mAbs with specificities against L and
ß2 integrins as well as those against M,
ß1, and 4 integrins, and to a lesser
degree against ß7 integrin (Figs. 8 and 9). It is thus
concluded that the engagement of several of the eosinophil integrins
including Lß2,
Mß2, and 4ß1
integrins is required for optimal eosinophil migration across
cytokine-stimulated endothelium induced by either C3a or C5a. Similar
results have been reported for the chemokine RANTES and for
platelet-activating factor. These agents were found to induce greater
eosinophil transendothelial migration through IL-1ß-primed
endothelial cells than through unprimed cells. Moreover, of
anti-integrin mAbs to eosinophils resulted in a pattern of inhibition
that highlighted the importance of 4 and
ß2 integrins, for RANTES- and platelet-activating
factor-mediated transmigration of this cell type (29, 56).
As noted, there is a correspondence in the inhibitory effects of
several mAbs to eosinophil integrins for adhesion and transmigration in
vitro and in vivo for both C3a and C5a stimulation (Figs. 3, 8, and 9).
The ability of several mAbs to block eosinophil transmigration in vivo
may be a consequence of prior blockade of the adhesion step or it may
reflect a function of 4 and ß2 integrins
to participate directly in the actual emigration process. Hence, the
ability of eosinophils to accumulate in an inflammatory site will
depend on the nature of the chemotaxin and the priming of the
endothelium. However, the actual machinery of mobility, which involves
the engagement of adhesion molecules, appears to be the same in all
cases examined, which include our results with C3a and C5a as well as
previously published findings for platelet-activating factor and RANTES
(29, 56).
Although in vitro experiments demonstrate clearly that C3a and C5a can
evoke chemotaxis and transmigration of eosinophils in buffer or
serum-free medium (5, 16) (Figs. 6 and 7), it is realized that in vivo
these anaphylatoxins could be regulated by blood components. Since
plasma contains the anaphylatoxin inhibitor CPN (57), we examined the
effect of this carboxypeptidase on C3a- and C5a-mediated eosinophil
transmigration. The outcome of in vitro experiments, in which CPN was
included with the anaphylatoxins in the chemotactic wells, was that
transmigration to C3a (C3adesArg) was lost completely;
however, residual activity to C5a (C5adesArg) was retained
(Fig. 10). The concentration of CPN (10 µg/ml), which was used for
these experiments, is comparable to the estimated plasma concentration
(15–30 µg/ml) (57). The differences in the retained
functional activity of C5adesArg but not
C3adesArg in inducing eosinophil migration are similar to
previously published results for a wide range of other functional
effects on eosinophils and neutrophils. For example,
C3adesArg, in contrast to intact C3a, was completely
inactive in inducing guinea pig ileum spasmogenic activity, neutrophil
respiratory burst, eosinophil enzyme release activity, and eosinophil
respiratory burst (5, 12, 15, 58). In contrast to the inertness of
C3adesArg, C5adesArg, although weaker than C5a,
is nevertheless an effective chemotaxin for neutrophils and eosinophils
(6, 58) (Fig. 10).
The lack of eosinophil transmigration induced by C3a across rabbit
mesenteric venules could possibly be attributable of the action of
regulatory molecules such as CPN. This carboxypeptidase could reduce
the effective concentration or the effective duration of C3a because in
vitro studies indicate that C3a is completely inactivated when in the
presence of CPN (Fig. 10). However, it is possible that
C3adesArg could support eosinophil adhesion but not
transmigration in vivo.
CPN is a large molecule and is expected to have limited access to
interstitial fluid (59). Hence it is conceivable that C3a could operate
in an extravascular microenvironment of an allergic inflammatory site
free of regulation by CPN. Since C5adesArg retains some
chemotactic activity but C3adesArg does not (Fig. 10), it
is concluded that C3a is designed and regulated to act as a more short
range or short term mediator than is C5a.
What is important is that C3a selectively causes stable adhesion of
rolling eosinophils on cytokine-stimulated venular endothelium. In
contrast to C5a, C3a had no effect on the stable adhesion of
neutrophils in postcapillary venules (Fig. 2). Thus C3a can join a
short list of mediators, which includes MCP-3, MCP-4, RANTES, and
eotaxin (60, 61, 62, 63), which are chemotactic for eosinophils and basophils
but not neutrophils.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Pragada Sriramarao, Laboratory of Immunology and Vascular Biology, La Jolla Institute for Experimental Medicine, 505 Coast Boulevard South, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: EBM, endothelial cell basal medium; CFDA, carboxyfluorescein diacetate; CPN, carboxypeptidase N; EGM, endothelial cell growth medium; RF, rolling fraction.
4 P. Sriramarao, R. G. DiScipio, G. H. Hughes, M. Elices, K. P. Sung, C. Cybulsky, and D. H. Brodie (1998). 4ß7 integrins support eosinophil rolling and adhesion on VCAM-1 in vitro and in vivo: influence of ß1-integrin activation on 4ß1 stable arrest of rolling eosinophil under conditions of flow. Submitted for publication.
Received for publication June 8, 1998. Accepted for publication October 7, 1998.
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References |
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4ß7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 and fibronectin. Immunology 89:112.[Medline]
4ß1 (very late antigen-4) dependent. J. Immunol. 146:3419.[Abstract]
2ß1 and 3ß1 in cell-cell and cell-substrate adhesion of human epidermal cells. J. Cell Biol. 110:1387.Vß3 and Vß5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. J. Cell Biol. 113:919.4ß7 mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation. J. Immunol. 153:3847.[Abstract]
4 integrin-mediated adhesion of human eosinophils to fibronectin and vascular cell adhesion molecule-1. J. Allergy Clin. Immunol. 99:648.[Medline]
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) were
determined. Cells were considered to be adherent if they remained
stationary along the vessel wall for at least 30 s. Data represent
mean ± SD adherent cells/250-µm length of venule derived from
2–5 venules/animal (n = 3–5 rabbits).
