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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Dipartimento di Fisiologia e Patologia, the
Dipartimento di Medicina Clinica e Neurologia, Università di
Trieste, the IRCCS Burlo Garofolo, Trieste, Italy; the IRCCS H. San
Raffaele, the Istituto di Patologia Generale, and the Istituto di
Ricerche Farmacologiche Mario Negri, Milan, Italy.
Intravital microscopy was used to monitor leukocyte traffic across
rat mesenteric postcapillary venules induced by the inactive terminal
complement (C) complex (iTCC) topically applied to ileal mesentery.
Leukocytes started rolling within 15 minutes from the administration of
iTCC, and by 1 hour they adhered almost completely to the endothelium
emigrating from the vessels in the next 3 hours. C5a caused a similar,
though less marked, effect, whereas boiled iTCC was inactive, excluding
the contribution of contaminating lipopolysaccharide. The complex
stimulated the migration of polymorphonuclear neutrophils (PMNs) across
endothelial cells (ECs) in a transwell system after a 4-hour incubation
of ECs with iTCC added to the lower chamber of the transwell, whereas a
30-minute incubation was sufficient for C5a and interleukin (IL)-8 to
induce the passage of PMNs. C5a was not responsible for the effect of
iTCC because this complex had no chemotactic activity and contained too
small an amount of C5a to account for the transendothelial migration of
PMNs. Similarly, the effect of iTCC was not mediated by IL-8 released
by stimulated ECs because anti-IL-8 failed to inhibit the migration of
PMNs induced by the complex. Unlike tumor necrosis factor- Terminal complement (C) complex (TCC) represents
the end product of the C-activation cascade and is formed by the
assembly of the 5 late components of the C system. The best-known
function of this complex is to insert into the phospholipid bilayer of the cell target as membrane attack complex (MAC) causing cytolysis. However, evidence collected in the last 2 decades has convincingly proved that MAC in sublytic amounts is also able to induce noncytolytic effects on several target cells.1,2 An early manifestation of cell activation induced by MAC is the rise in Ca++
caused by the rapid influx of these ions from an extracellular source
and their cytoplasmic redistribution from intracellular stores.1
The endothelium is a potential target of MAC that can be formed
directly on the cell membrane as a result of C activation triggered by
cell-bound antibodies. Alternatively, MAC starts assembling in the
circulation and subsequently interacts with endothelial cells (ECs).
Cells attacked by MAC may undergo cytolysis, but, more frequently, they
release and express on their surfaces molecules involved in important
biologic functions. Mobilization of P-selectin,3
enhancement of tumor necrosis factor (TNF)- These noncytotoxic effects on ECs require the complex to insert into
the cytoplasmic membrane to signal the cell, and they have been
obtained in model systems using ECs coated with C-fixing antibodies.
The chances that this may happen in vivo are limited to those clinical
situations characterized by the presence of cytotoxic anti-endothelial
antibodies, such as hyperacute graft rejection. More often, the
endothelium is exposed to TCC that starts assembling in the bloodstream
in the vicinity of ECs, and that completes its formation on the cell
surface. However, the ability of this complex to insert into the EC
membrane as MAC is restricted by the rapid functional decay of nascent
TCC from the inactivating effect of inhibitors acting both in the fluid phase (S protein and clusterin12,13) and on the cell
surface (CD59).14 In many clinical conditions associated
with massive C activation, a cytolytically inactive TCC (iTCC)
accumulates in plasma as an apparently irrelevant byproduct of the C
sequence.15 We have previously shown that the inactive
complex not only binds to ECs, it stimulates these cells to express
adhesion molecules and tissue factor.16
Data accumulated in recent years have shown that terminal C
components are synthesized at extravascular sites by various cell types17,18 and that TCC can be formed in biologic fluids
other than blood.19,20 These findings led us to
investigate whether the inactive complex present at extravascular sites
may interact with ECs on the abluminal side promoting inflammation.
Evidence will be presented indicating that iTCC induces
transendothelial migration of polymorphonuclear neutrophils (PMNs) both
in vitro and in vivo.
C reagents
Other reagents
Antibodies Neutralizing rabbit antibodies anti-human IL-8 were obtained from Peprotech (London, England). Monoclonal antibody (mAb) M89D3 anti-platelet-endothelial cell adhesion molecule-1 (PECAM-1) inhibiting leukocyte migration across ECs was generously provided by Dr M. R. Zocchi (San Raffaele Hospital, Milan, Italy). Two mAb anti-C5a G25/2 and C17/5, the latter of which recognized a neo-epitope
exposed on this fragmentwere a kind gift of Prof O. Götze
(Göttingen, Germany), and mAb aE11 directed against a C9
neo-antigen was kindly provided by Prof T. Lea (Oslo, Norway). This mAb
was linked to cyanogen bromide-activated Sepharose 4B (Pharmacia
Biotech, Milan, Italy) to prepare an affinity column. Goat antiserum to
C5 was purchased from Quidel.
Enzyme-linked immunosorbent assay TCC was quantitated using solid phase-bound mAb aE11 and biotin-labeled goat immunoglobulin (Ig)G anti-C5 as previously described.21 The levels of C5a, IL-8, and MCP-1 were measured according to published methods.22,23Intravital microscopy Male Wistar Kyoto rats, each weighing 250 to 270 g, were anesthetized intraperitoneally with sodium thiobarbital (100 mg/kg). A polyethylene catheter (PE50; Intramedic Clay-Adams, Sparks, MD) was inserted into the left carotid artery, and a Statham P23AC pressure transducer (Gould, Cleveland, OH) connected to a physiograph (Ote-Biomedica, Florence, Italy) was used to monitor the mean arterial pressure and the heart rate. Another catheter (PE20) was inserted into the left femoral vein and was connected to a micropump injector (Harvard Apparatus, South Natick, MA). The fluorescent marker acridine orange (AO) (Sigma Chemical) diluted in sterile saline was slowly infused through this device over the first 3 hours of the experimental procedure at a concentration of 0.025 mg/kg per minute and at a rate of 0.5 mL/h.The rats were placed on an adjustable stage of an upright microscope (model BX50WI; Olympus Optical, Tokyo, Japan). A 2-cm midline incision was made through the abdominal wall, and a loop of ileal mesentery was exteriorized and carefully draped over a transparent pedestal immersed in a plexiglas chamber containing temperature-controlled and sterile modified Krebs-Henseleit solution (118 mM NaCl, 4.74 mM KCl, 2.45 mM CaCl2, 1.19 mM KH2PO4, 1.19 mM MgSO4, 12.5 mM NaHCO3). Exposed tissue was superfused throughout the study with sterile buffered saline warmed at 37°C and bubbled with a mixture of 95% N2 and 5% CO2. The integrity of the microvasculature was checked by intravenous injection of fluorescein-labeled dextran 70 kd (Molecular Probes, Eugene, OR). The mesenteric microcirculation was transilluminated with a 12-V, 100-W direct current-stabilized light source and was viewed through 10× and 40× saltwater dipping objectives and a 10× ocular lens. AO-labeled leukocytes were made visible by epifluorescence transillumination with a UIF550 filter for excitation light (Olympus Optical, Tokyo, Japan). Images were recorded by a charge-coupled device camera connected through a peripheral component interconnect interface board (SensiCam PCO, Kelheim, Germany) to a computer device, where they were stored and analyzed off-line using dedicated imaging software (Analytica Lite, Milan, Italy). In vivo experimental procedure The rats were allowed to rest for 15 minutes after surgery, and segments of 3 to 5 unbranched postcapillary venules (25-40 µm diameter, 200 µm length) were selected for analysis. Venular diameter and center-line red blood cell velocity were evaluated off-line using a video caliper (Image Research, Ontario, Canada) and customized frame-by-frame analogic image analysis.24 Red blood cell velocity (VRBC) and venular diameter (D) were used to calculate venular wall shear rate (g) through the formula g = 8(Vmean/D), where Vmean = VRBC/1.6.25 Sterile saline containing either 10 7 C5a or
1.5 × 108 M iTCC, assuming that each complex contained
4 molecules of C9, were topically applied to the mesentery for 10 minutes after baseline evaluation, and image sequences were recorded
intermittently up to 240 minutes. Control animals were treated with
sterile saline or boiled iTCC for 15 minutes.
Intravascular circulation of leukocytes in postcapillary venules and their extravascular emigration were analyzed off-line during playback of the digital file sequences. Labeled leukocytes were classified as rolling (if they moved more slowly than red blood cells, thus becoming visible) or adherent (if they remained stationary for more than 30 seconds). Rolling flux was expressed as the number of leukocytes seen moving past a reference point per minute, and adherence was measured by counting the number of adherent leukocytes per 200 µm venule length. Leukocyte emigration from the vessel was evaluated by counting the cells that had extravasated up to 100 µm away from the wall in parallel with 200-µm vessel segments. Endothelial cell culture The procedure for isolation of human umbilical vein ECs (HUVECs) from 3 to 5 normal umbilical cord veins by collagenase digestion and culture of pooled cells in tissue culture plates (Costar, Cambridge, MA) coated with 2% endotoxin-free gelatin has been reported.16,26 HUVECs were used at their first passage.Transendothelial migration and chemotaxis assays HUVECs (2 × 104) were seeded onto 2% gelatin- or 0.2 µM fibronectin-coated polycarbonate inserts of a 24-well Transwell system (6.5-mm diameter, 5-µm pores; Costar) and were used 5 days after plating.27 Transendothelial electrical resistance across the inserts was evaluated daily from the second day after seeding using Millicell-ERS voltammeter (Millipore, Bedford, MA). The value of resistance ( × cm2)
increased progressively and reached a plateau at day 5, when formation
of the EC monolayer was complete.
ECs were exposed for 4 hours at 37°C to the various stimuli added either to the lower or the upper compartment of the transwell. Twenty microliters PMN suspension (2.5 × 106/mL) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin prepared as previously reported16 was added to the upper chamber to a final volume of 100 µL and was further incubated at 37°C for 30 minutes. Cells that had migrated to the lower chamber and those that were detached from the lower surface of the filters by gentle washing were then harvested and counted in a ZBI Coulter Counter (Coulter Electronics, Luton, United Kingdom). A similar approach was followed to assess chemotaxis of PMNs except that the inserts of the transwell were uncoated. PMN adhesion assay HUVECs grown to confluence in 96-well tissue culture plates were stimulated for 4 hours at 37°C with iTCC or TNF- , and, after 3 washings with serum-free medium, 100 µL PMN suspension
(105 cells) was added to each well and was further
incubated at 37°C for 25 minutes. Nonadherent PMNs were removed by
washing the wells twice, and the number of adherent leukocytes was
evaluated by a colorimetric assay using tetramethyl benzidine (Sigma
Chemical) as a substrate for myeloperoxidase, as previously
described.26
Scanning electron microscopy An approach similar to that described for the PMN adhesion assay was followed except that HUVECs were grown to confluence on gelatin-coated glass coverslips (22 mm) and the adherent PMNs were fixed with 2.5% glutaraldehyde (Boehringer Ingelheim Bioproducts, Milan, Italy) in 0.2 M cacodylate buffer, pH 7.4. After dehydration in graded ethanol from 70% to 100%, the specimens were critically point-dried under liquid CO2, mounted on aluminum stubs, and sputter coated with gold (10 nm) using sputter pulse control. Cells were examined with a scanning electron microscope (Stereoscan 430I; Leica, Milan, Italy). Five to 7 microphotographs, each showing 15 to 20 PMNs, were digitally recorded, and the surface area of at least 100 cells was analyzed using a computer device with dedicated imaging software (Analytica Lite).Immunofluorescence HUVECs grown to confluence on coverslips coated with 1% gelatin in PBS were treated for 2 hours with iTCC (5 nM), TNF- (200 U/mL),
or control medium, washed with PBS and fixed with 1% paraformaldehyde (Merck, Darmstadt, Germany) in Dulbecco-PBS (Sigma Chemical) for 20 minutes. The cells were incubated first with mAb M89D3 anti-PECAM-1 (10 µg/mL) and then with fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgG (FITC-GAM; Zymed Laboratories,
San Francisco, CA). Actin morphology was examined on HUVEC monolayer
fixed with 1% formaldehyde and permeabilized with 0.05% Triton X-100
(Merck) for 1 minute at 0°C, as previously described.26
After they were washed with PBS, the coverslips were stained for
F-actin with rhodamine tetraisothiocyanate phalloidin (Sigma) for 20 minutes at room temperature and were mounted in 50% glycerol-PBS.
Microscopic analysis was performed with a Bio-Rad MRC 1000 confocal
scanning microscope (Bio-Rad Laboratories, Milan, Italy), and
fluorescence images were recorded on Kodak T-Max 100 film using a Focus
Imagecorder Plus (Focus Graphics, Foster City, CA).
Reverse transcription-polymerase chain reaction Total RNA was extracted from resting and activated HUVECs with a commercially available solution (RNAzol B; Tel-Test, Friendswood, TX). The RNA was reverse transcribed with M-MLV reverse transcriptase (Gibco-BRL, Milan, Italy) using 1 µg total RNA and random hexanucleotide primers (Boehringer Mannheim, Germany) according to the manufacturer's instructions. Polymerase chain reaction (PCR) was performed with 0.2 mM each dNTP, 1.4 U Taq DNA polymerase (Finnzymes OY, Espoo, Finland), 0.5 µM specific 20-mer oligonucleotide primers (Amersham Pharmacia-Biotech, Milan, Italy), and cDNA in a PTC-100 thermal cycler (MJ Research, Watertown, MA). PCR products were run through a 1.5% agarose gel containing ethidium bromide for UV detection. EMBL accession number, amplified sequences, and amplified products were as follows: IL-8: XM 003501, 74-373, 299 bp28; MCP-1: Y 18933, 30 + 324, 354 bp29;
 -actin: M 10278, 311-838, 528 bp.30
Statistical analysis Results are expressed as mean ± SD. Data were compared by analysis of variance using posthoc analysis for paired multiple comparisons with Fisher corrected t test. P [le] .05 was considered statistically significant.
Leukocytes adhere to endothelium and transmigrate across mesenteric venules in response to iTCC In our initial experiments, we sought to determine whether iTCC was able to recruit leukocytes into tissue by using intravital microscopy analysis to monitor cell mobilization. Based on preliminary results obtained from a dose-response curve, a group of rats was treated with a dose of complex found to be effective in recruiting leukocytes. Included in the experimental protocol were 3 additional groups of rats that received C5a at a dose similar to that selected for iTCC treatment and saline or boiled iTCC used to exclude the effects caused by contaminating LPS.None of the rats showed overt modifications of systemic blood pressure
levels and local hemodynamic parameters during the entire period of
observation. Initial shear rate values in animals treated with C5a
(403.7 ± 31 s
Both iTCC and C5a induced progressive increases in the number of
leukocytes stably adherent to the endothelium in the examined microvessels (Figure 1B). The mean number of leukocytes that adhered to
a vessel segment 200 µm long at 3 hours after exposure to the various
stimuli was significantly higher in rats treated with iTCC (22 ± 6
leukocytes) or C5a (35 ± 9 leukocytes) than in rats receiving saline
(6 ± 5 leukocytes) or boiled iTCC (4 ± 3 leukocytes). Representative photomicrographs of leukocytes adherent to ECs are shown
in Figure 2.
The extravascular appearance of AO-labeled cells served as a useful marker in evaluating the effects of iTCC and C5a on leukocyte extravasation (Figure 2, bottom panel). Numbers of adherent cells migrating across the wall of the mesenteric venules in the iTCC-treated mesenteric tissue started rising at 60 minutes after topical application of the complex, to a mean value of 8 ± 4 (P < .05 vs control) over a surface area of 200 × 200 µm, and it progressively increased with time to reach a mean value of 15 ± 6 at 4 hours (Figure 1C). Migration of leukocytes to the extravascular space was also seen in rats receiving C5a, but the extent of C5a-induced migration was less marked than in rats treated with iTCC, though the value of 6 ± 3.5 observed at 60 minutes was significantly different from the control value observed in the saline-treated group (P < .05 vs control). iTCC promotes transendothelial migration of PMNs in vitro These in vivo data did not clarify whether iTCC induced transendothelial migration acting directly on ECs or through an intermediate effect on macrophages and other cell types in the peritoneal cavity, which, in turn, release cytokines and chemokines. To address this issue, HUVECs were grown to confluence on the polycarbonate insert of transwell cell culture chambers and were incubated with iTCC added to the lower compartment. PMNs were then introduced into the upper chamber and were allowed to migrate into the lower compartment. As shown in Figure 3A, the exposure of HUVECs to iTCC for 4 hours resulted in active diapedesis of a proportion of PMNs that was related to the dose of iTCC used. Attempts to induce similar effects by incubating ECs with iTCC for 30 minutes or by stimulating the cells with either LPS or boiled iTCC, as a source of contaminating endotoxin, failed. The complex, prepared with limited amounts of C9, also proved to be ineffective, as was the preparation depleted of iTCC by affinity chromatography with mAb aE11 to poly C9 (data not shown). Interestingly, the complex, like TNF-, promoted the migration of PMNs even when added to the upper
chamber of the transwell to stimulate ECs (Figure 3B).
ECs exposed to concentrations of the C complex causing PMN
migration exhibited increased adhesiveness for these cells (Figure 4). Scanning electron microscopy analysis
of adherent PMNs revealed that a large number of cells maintained a
rounded shape, whereas PMNs bound to HUVECs treated with TNF-
iTCC is not chemotactic for PMNs To investigate whether iTCC exhibited chemotactic activity for PMNs, which could account, at least in part, for its ability to induce leukocyte transendothelial migration, we followed an approach similar to that used for the experiment presented in Figure 3, except that PMNs were allowed to migrate toward iTCC through the polycarbonate insert of the transwell chamber not coated with ECs. As shown in Figure 7, iTCC was unable to attract PMNs chemotactically at the concentration of 5 nM, and it was inactive when tested within a concentration range between 0.5 and 10 nM. By contrast, both C5a and IL-8 caused PMN migration across transwell filters, irrespective of whether they were coated with ECs. PMN response was dose related and was no longer seen at concentrations lower than 500 nM for C5a and IL-8 (5 × 108 M and
0.5 × 109 M, respectively). Interestingly, exposure of
ECs to either C5a or IL-8 for 30 minutes was sufficient to induce cell
migration, whereas a longer incubation time of 4 hours was required for
iTCC to be similarly effective. To exclude that C5a as a contaminant in
our iTCC was responsible for the effect of the C complex, we measured
C5a level using a sensitive enzyme-linked immunosorbent assay (ELISA)
and found that it was approximately 1 nM. This concentration was far
below the amount shown to induce the migration of PMNs across
HUVECs.
HUVECs stimulated by iTCC release IL-8 and MCP-1 The possibility that chemokines released by HUVECs could contribute to the transendothelial migration of PMNs promoted by iTCC was first explored by evaluating the ability of iTCC to stimulate the secretion of IL-8 and MCP-1 by ECs. To this end, the cells were incubated overnight with the complex, and ELISA was used to measure the amounts of the 2 chemokines released in the culture supernatant. The data presented in Figure 8A show that HUVECs treated with iTCC were able to secrete levels of IL-8 and MCP-1 significantly higher than those released by unstimulated cells, though they were lower than the amount produced by TNF--stimulated ECs. To
confirm this observation, we analyzed the levels of mRNA for IL-8 and
MCP-1, expressed by HUVECs treated with iTCC or TNF-, as a positive
control. As shown in Figure 8B, low amounts of the 2 transcripts were
already detected in unstimulated HUVECs, but their levels increased
substantially in cells stimulated by iTCC or TNF-.
These results suggest but do not prove that the 2 chemokines released
by iTCC-activated ECs are actually involved in the transendothelial migration of PMNs. To address specifically this question, we stimulated diapedesis of PMNs across HUVECs with the complex in the presence of a
neutralizing antibody directed against IL-8. The data shown in Figure
9 clearly indicate that this antibody is
effective in inhibiting PMN migration induced by IL-8 and TNF-
Contribution of PECAM-1 to the iTCC-dependent transendothelial migration of PMN PECAM-1 molecules are constitutively expressed on endothelial cells, and their localization at the borders of adjacent cells makes them a good candidate for playing an active role in the process of cell adhesion and migration. To examine whether the transmigration of PMNs across HUVECs caused by iTCC was associated with changes in the distribution of PECAM-1 at the interendothelial junctions, confluent HUVEC monolayers were incubated with either iTCC or TNF- or control
medium, and the distribution of PECAM-1 stained with a specific mAb was
analyzed by confocal scanning microscopy. As shown in Figure
10, unstimulated confluent cells were
brightly stained for PECAM-1 molecules that showed a linear and
homogeneous distribution at the cell borders (Figure 10A). Conversely,
staining for PECAM-1 was less intense in TNF-stimulated HUVECs, and
these molecules appeared to be redistributed on the cell surface from a
compact junctional to a punctate, speckled pattern (Figure 10B). HUVECs
stimulated with iTCC exhibited a pattern similar to that of the control
cells, with no overt changes in the staining and distribution of
PECAM-1 molecules (Figure 10C). However, iTCC-treated ECs differed from
unstimulated cells in the distribution of F-actin filaments stained
with rhodamine-labeled phalloidin. In particular, the latter cells were
characterized by a peripheral rim of fluorescent staining and by a lack
of prominent filament bundles in the central area, whereas a proportion
of HUVECs activated by either iTCC or TNF- showed a less distinct peripheral staining rim and a reorganization of F-actin into thick cables running parallel to the long axis of the cells as stress fibers
(Figure 10 insets).
To further investigate the involvement of PECAM-1 in the transmigration
of PMNs, we sought to determine whether this process could be
controlled by the presence of mAbs directed against the junctional
molecule in the assay system. Prior incubation of ECs with a mAb to
PECAM-1 resulted in a significantly reduced number of PMNs migrating
across HUVECs stimulated by iTCC, though inhibition was not complete,
whereas the same mAb had no effect on cell migration induced by either
IL-8 or TNF-
The C system has long been known to recruit leukocytes from the circulation through the action of the chemotactic fragment C5a, made available in inflamed tissues as a result of C activation by various stimulating agents.31 We now present in vivo and in vitro data indicating that TCC, even in a cytolytically inactive form, can contribute to mobilize leukocytes. Intravital microscopy, used in this study to analyze the
mobilization of leukocytes in response to iTCC, provides a useful tool
for observing leukocyte-endothelium interaction in vivo. The finding
that leukocytes circulating in the rat mesentery firmly adhered to the
endothelium of the postcapillary venules after the topical application
of iTCC was not surprising because we have previously shown that this
complex stimulates HUVECs to express molecules including endothelial
E-selectin, intercellular adhesion molecule-1 (ICAM-1), and
vascular cell adhesion molecule-1, involved in leukocyte
adhesion.16 iTCC differs in this regard from sublytic MAC
because the latter complex induces the expression of
P-selectin3 whereas it is ineffective in up-regulating the
cell surface appearance of E-selectin and ICAM-1 unless TNF- Both iTCC and C5a proved to be effective in promoting the transmigration of leukocytes across the mesenteric venules into extravascular sites in our in vivo model. Results obtained with C5a are compatible with the known ability of this fragment to induce the expression of adhesion molecules on endothelium33 and to exert a chemotactic attraction on leukocytes,34 and again they confirm similar observations made in rabbits by DiScipio et al.32 An interesting finding of this study was that iTCC was able to induce cell migration to an extent and with a kinetic pattern that did not differ from those seen in C5a-treated rats. The presence of C5a in our iTCC may explain the in vivo biologic effect of the complex. However, the level of C5a detected in the iTCC preparation used in all the experiments was below the amount that was effective in inducing leukocyte mobilization. Alternatively, the inflammatory reaction observed with iTCC might have been caused by contaminating LPS, but a significant contribution of endotoxin to the effect induced by the complex was excluded by the finding that the activity of iTCC was lost after boiling. The rat model has been extremely useful to ascertain the in vivo
efficacy of iTCC in recruiting leukocytes into tissues, but it does not
clarify the mechanism responsible for this process. It is possible that
the complex elicits cell mobilization through the intermediate action
of peritoneal cells, in particular macrophages and mast cells,
stimulating these cells to release cytokines that, in turn, activate
endothelium and promote PMN chemotaxis. The involvement of macrophages
and mast cells in the migration of PMNs into the mouse peritoneal
cavity has recently been elucidated in an elegant study by Ajuebor et
al.35 Although this possibility cannot be excluded, we
favor the hypothesis that ECs may play a relevant role in the
mobilization of PMNs induced by iTCC, based on our previous finding of
increased expression of surface adhesion molecules on HUVECs treated
with this complex.16 To investigate this point more
directly, we set up an in vitro model of PMN migration across ECs grown
on the insert of a transwell cell culture chamber and found that iTCC
promotes PMN passage when added to the lower compartment. These results
led us to question whether iTCC was chemotactic for PMNs, but repeated
attempts to document this activity failed. This indicates that the
fully assembled terminal complex differs from the trimolecular complex
C5b67 shown to stimulate chemotaxis of PMNs in vitro and in
vivo.36-38 The presence of the trimolecular complex in our
iTCC preparation appeared unlikely. iTCC, to be active, must be
prepared with C9 in excess; it does not work when C9 is limited.
Several groups have reported data demonstrating that transendothelial
migration of PMNs can also be promoted by biologic factors that are not
necessarily chemotactic for PMNs, including IL-1 and
TNF- The 2 cytokines, IL-1 and TNF- Because C5a and IL-8 have been shown to induce diapedesis of PMNs
across ECs,42 and IL-8 is apparently implicated in
neutrophil migration across cytokine or LPS-activated
ECs,43 we wondered whether these chemotactic factors could
contribute to produce the effect caused by iTCC. As mentioned above,
direct intervention of C5a can be ruled out by the finding that the
iTCC used in these experiments had no chemotactic activity and
contained an amount of C5a unable to induce the transendothelial
migration of PMNs. One cannot exclude, however, that iTCC-stimulated
ECs produce chemokines that are ultimately responsible for the PMN
passage. The increased expression of mRNA for IL-8 and MCP-1 and the
secretion of these 2 chemokines in the culture supernatant of HUVECs
stimulated by iTCC are in line with this hypothesis and confirm similar
findings obtained by Kilgore et al5 using sublytic
concentrations of MAC to activate HUVECs. However, the amount of
chemokines produced by HUVECs at the time (4 hours) iTCC mobilized PMNs
was negligible and was, therefore, unlikely to account for the
iTCC-dependent leukocyte transmigration. This conclusion is further
supported by the failure of the anti-IL-8 antibody to inhibit the
migration of PMNs induced by iTCC, whereas it was effective in
neutralizing the biologic activity of IL-8. Conversely, anti-IL-8
antibody inhibited to some extent PMN migration through
TNF- HUVECs treated with iTCC maintain a regular distribution of
PECAM-1 on the intercellular junctions despite the rearrangement of
cytoskeletal microfilaments; thus, they behave differently than the
cells activated with TNF- In conclusion, the results of the current investigation disclose a yet unrecognized function of the cytolytically inactive TCC in promoting transendothelial migration and egression of PMNs from postcapillary venules. The novelty of our observation is that PMNs responded to the complex applied on the abluminal side of the EC monolayer in the in vitro model and on the external side of the vessel wall in the rat model, suggesting that iTCC present at the tissue level is involved in recruiting leukocytes at inflammatory sites. This possibility is not unlikely because the complex has been detected in various biologic fluids19-20 besides plasma, and recently it has been detected in human lymph (Tedesco et al, unpublished observations, 2001). Local formation of an excessive amount of iTCC may cause an unrestricted inflammatory process that is deleterious to tissue and that should be prevented by an appropriate therapeutic approach, such as the use of specific antibodies.
We thank Paolo Macor and Tito Ubaldini for excellent technical assistance.
Submitted February 22, 2001; accepted August 30, 2001.
Supported by grants from the Italian MIUR, CNR (Target Project on Biotechnology), Ministry of Health (Progetto Finalizzato Cod. B08), and Region Friuli-Venezia Giulia.
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: Francesco Tedesco, Dipartimento di Fisiologia e Patologia, Università di Trieste, Via Fleming 22, 34127 Trieste, Italy; e-mail: tedesco{at}univ.trieste.it.
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  F. Corallini, F. Bossi, A. Gonelli, C. Tripodo, G. Castellino, T. E. Mollnes, F. Tedesco, L. Rizzi, F. Trotta, G. Zauli, et al. The soluble terminal complement complex (SC5b-9) up-regulates osteoprotegerin expression and release by endothelial cells: implications in rheumatoid arthritis Rheumatology, March 1, 2009; 48(3): 293 - 298. [Abstract] [Full Text] [PDF]
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A. Polenghi, F. Bossi, F. Fischetti, P. Durigutto, A. Cabrelle, N. Tamassia, M. A. Cassatella, C. Montecucco, F. Tedesco, and M. de Bernard The Neutrophil-Activating Protein of Helicobacter pylori Crosses Endothelia to Promote Neutrophil Adhesion In Vivo J. Immunol., February 1, 2007; 178(3): 1312 - 1320. [Abstract] [Full Text] [PDF]
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F. Fischetti, P. Durigutto, V. Pellis, A. Debeus, P. Macor, R. Bulla, F. Bossi, F. Ziller, D. Sblattero, P. Meroni, et al. Thrombus formation induced by antibodies to {beta}2-glycoprotein I is complement dependent and requires a priming factor Blood, October 1, 2005; 106(7): 2340 - 2346. [Abstract] [Full Text] [PDF]
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F. Bossi, F. Fischetti, V. Pellis, R. Bulla, E. Ferrero, T. E. Mollnes, D. Regoli, and F. Tedesco Platelet-Activating Factor and Kinin-Dependent Vascular Leakage as a Novel Functional Activity of the Soluble Terminal Complement Complex J. Immunol., December 1, 2004; 173(11): 6921 - 6927. [Abstract] [Full Text] [PDF]
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O. Manches, G. Lui, L. Chaperot, R. Gressin, J.-P. Molens, M.-C. Jacob, J.-J. Sotto, D. Leroux, J.-C. Bensa, and J. Plumas In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas Blood, February 1, 2003; 101(3): 949 - 954. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), C5a (10
), boiled iTCC
(1.5 × 10
), and control saline (
).
Measurements were performed on 4 to 6 different segments of unbranched
venules (25- to 40-µm diameter, 200-µm length). (A) Values of
rolling leukocyte flux defined as number of cells that become visible
as bright spheres if they travel inside the venules more slowly than
red blood cells. (B) Numbers of leukocytes stably adherent to the same
site of postcapillary vascular endothelium of rat mesentery for at
least 30 seconds. (C) Numbers of cells that migrated to the
extravascular space up to 100 µm away from the vessel wall, in
parallel with 200-µm vessel segments. *P < .01
versus control.
). The
percentage of migrated PMNs was determined after 30 minutes. Values are
means ± SD of triplicate determinations of 5 experiments.
*P < .01 versus control.
and TNF-