Departments of 1 Surgery and 2 Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston 02115; and 3 Naval Blood Research Laboratory and 4 Biological Science Center, Boston University, Boston, Massachusetts 02215
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ABSTRACT |
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P-selectin is an adhesion molecule expressed on activated endothelial and platelet surfaces. The function of the short consensus repeats (SCRs) of P-selectin, homologous with the SCRs of complement regulatory proteins is largely unknown. In a model of murine hindlimb ischemia where local reperfusion injury is partly mediated by IgM natural antibody and classical complement pathway activation, we hypothesized that human soluble P-selectin (sP-sel) would moderate the complement component of the inflammatory response. Infusion of sP-sel supernatant or purified (p) sP-sel prepared from activated human platelets, reduced ischemic muscle vascular permeability by 48% and 43%, respectively, following reperfusion. Hindlimb immunohistochemistry demonstrated negligible C3 staining colocalized with IgM in these groups compared with intense staining in the untreated injured mice. In vitro studies of mouse serum complement hemolytic activity showed that psP-sel inhibited the classical but not alternative complement pathway. Flow cytometry demonstrated that psP-sel inhibited C1q adherence to sensitized red blood cells. From these data we conclude that sP-sel moderates skeletal muscle reperfusion injury by inhibition of the classical complement pathway.
murine; inflammation; complement activation; adhesion molecules; platelets
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INTRODUCTION |
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P-SELECTIN, A
MEMBER OF the selectin family of adhesion molecules, is stored in
the -granules of platelets and the Wiebel-Palade bodies of
endothelial cells (1, 4, 18,
25). After platelet or endothelial cell activation,
P-selectin is rapidly translocated and expressed on their
respective surfaces. The transient binding of endothelial P-selectin to
the P-selectin glycoprotein ligand-1 (PSGL-1) receptor of the
circulating polymorphonuclear leukocyte (PMN) leads to neutrophil
slowing and rolling along endothelial surfaces. This initial tethering
is followed by firm adherence of the neutrophil
2-integrin with the intercellular adhesion molecule 1 (5).
P-selectin has the same basic structure as both L- and E-selectin, consisting of a lectin head domain, an epidermal growth factor (EGF) domain, short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic domain (3). The lectin domain is made up of N-linked oligosaccharides, which allow P-selectin to bind to specific carbohydrate counterreceptors (20). The presence of the transmembrane domain allows full-length, 140-kDa soluble P-selectin (sP-sel) molecules to exist as dimers and three- to six-molecule oligomers. Absence of the transmembrane domain, as seen in the alternatively spliced form or following truncation of the molecule after the ninth SCR, results in monomeric sP-sel (29). sP-sel can also exist as the full-length molecule attached to platelet microparticles. The normal human plasma concentration of sP-sel is 36-250 ng/ml, comprised mainly of the truncated form with a molecular mass of ~100 kDa (11, 12). In disease conditions characterized by platelet and/or endothelial cell activation such as systemic lupus erythematosus, rheumatoid arthritis, and disseminated intravascular coagulation, sP-sel levels rise due to a systemic increase in its alternatively spliced isoform (6, 9, 26).
Human P-selectin, L-selectin, and E-selectin have 9, 5, and 2 SCRs, respectively. The function of these domains is unclear, but they may have a role in modifying ligand binding specificity (28). By extending the lectin/EGF domains away from the cell surface, the SCRs could function structurally as spacers, enabling molecular flexibility and optimal exposure to counterreceptor interaction (22). However, homology between the SCRs of P-selectin, complement proteins, and complement regulatory proteins suggests a more complex function, possibly involving the complement system.
A central role for the classical pathway of complement in mediating
local injury following ischemia and reperfusion has been demonstrated
in a number of studies. In experimental myocardial ischemia, C1
esterase inhibitor (C1-INH) administered before reperfusion prevented
deposition of C1q and significantly reduced the area of cardiac muscle
necrosis (2). Similarly, Weiser et al. (32) and Williams et al. (33), utilizing genetic knockout mice
in IgM (RAG1/
) and mice deficient in either C3 or C4 in a model of
hindlimb and gut ischemia, were able to demonstrate that local reperfusion injury is in part mediated by natural IgM antibody and
activation of the classical complement pathway. Preliminary work
undertaken in our laboratory utilizing gene-targeted
P-selectin-deficient mice showed that infusion of P-selectin-sufficient
platelets from wild-type mice significantly reduced ischemic muscle
reperfusion injury. In addition, complement deposition on ischemic
skeletal muscle as assessed by semiquantitative immunostaining for C3
and colocalization with IgM was negligible, compared with untreated animals (16).
Platelets possess C1q receptors, which may play a role in their activation with subsequent clot formation. This has been described in experimental settings where there are circulating immune complexes and the classical pathway is activated (23). Considering that platelets possess the ability to bind C1q, we wondered whether the SCRs of sP-sel were operative in this regard. This study examines the role of human sP-sel as a moderator of complement-mediated skeletal muscle reperfusion injury in mice. In addition, we examined the ability of human sP-sel to inhibit complement activity in vitro.
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METHODS |
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Animals. Male C57BL/6 mice purchased from Taconic Farms, (Germantown, NY) were used for all experiments.
Hindlimb model of ischemia-reperfusion injury. Mice aged 6-8 wk, weighing 20-25g, were anesthetized with intraperitoneal pentobarbital sodium (60 mg/kg) and underwent 2 h of hindlimb ischemia followed by 3 h of reperfusion. After a 2-min period of hindlimb elevation to minimize retained blood, bilateral rubber bands (Latex O-Rings) were applied above the greater trochanter, using the McGivney Hemorrhoidal Ligator (Miltex Instrument). Sham mice did not undergo ischemia. Five minutes before rubber band release, animals received 1 µCi of 125I-labeled bovine albumin (ICN, Irving, CA) in 0.3 ml of phosphate-buffered saline (PBS) via tail vein injection. Hydration was maintained by intravenous infusion of 0.1 ml of 0.9% saline during each hour of reperfusion. Mice were maintained in a supine position and kept anesthetized by intermittent intraperitoneal pentobarbital sodium injections. They were covered throughout the experiment to maintain body temperature. After euthanasia by an intraperitoneal pentobarbital sodium overdose (90 mg/kg), blood was aspirated from the right ventricle through a midline sternotomy, and its gamma radioactivity counted (Packard, Downes Grove, IL). Muscle was harvested from both hindlimbs, its radioactivity measured, and then was dried to a constant weight in a gravity convection oven (Precision Scientific Group, Chicago, IL) at 90°C for 72 h. Extravasation of 125I-albumin was used to assess the hindlimb vascular permeability index (PI), which was determined by the ratio of radioactivity per gram of dry muscle to radioactivity per gram of blood. In addition, hindlimb muscle harvested from these experiments was used for immunohistochemical analysis of IgM and complement deposition.
Animals in this study were maintained in accordance with the guidelines of the Committee on Animals of Harvard Medical School and those prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council [Department of Health, Education and Human Services, Publication no. 85-23 (National Institute of Health), revised 1985].Treatment with sP-sel supernatant. Mice were treated with sP-sel supernatant (SN) prepared from activated human platelets (as described below), administered intravenously 5 min before reperfusion. Each animal received a bolus of 180 ng (9 µg/kg) of sP-sel in the supernatant. Another group of mice received the same supernatant after extraction of sP-sel. These animals received 12 ng (0.6 µg/kg) of sP-sel SN and were used as negative controls.
Treatment with the full-length 140-kDa purified sP-sel. Mice were treated with sP-sel extracted from the supernatant of activated human platelets (as described below), administered intravenously 5 min before reperfusion. Each animal received a bolus of 225 ng (11 µg/kg) of purified sP-sel (psP-sel).
Treatment with truncated sP-sel standard. Mice were treated with the 80-kDa truncated form of sP-sel standard (tsP-sel; sCD62P; R & D Systems, Minneapolis, MN) administered intravenously 5 min before reperfusion. The truncated form of the molecule has no alterations in the lectin domain and the fourth SCR is present. Each animal received a bolus of 225 ng (11 µg/kg) of tsP-sel.
Bioviability of sP-sel. Mice that did not undergo hindlimb ischemia received a tail vein injection of 225 ng (11 µg/kg) of psP-sel. Saline in the same volume was injected in controls. Animals were euthanized at different time intervals, and blood was aspirated from the right ventricle through a midline sternotomy. The sera were separated from whole blood and tested at serial dilutions with a human P-selectin ELISA kit (Biosource International, Camarillo, CA).
Immunohistological analysis and histological injury evaluation. Immunoperoxidase labeling of IgM and C3 was performed on paraformaldehyde-fixed cryostat sections of hindlimb muscle using goat anti-mouse IgM (Sigma Chemical, St. Louis, MO) or goat anti-mouse C3 (5 mg/ml; Organon Teknia, Durham, NC) and a standard avidin-biotin protocol (15). Immunostaining of hindlimb muscle samples from sham animals was used as controls. Injury was assessed semiquantitatively by evaluating the presence of edema, disruption of normal muscle architecture, and individual myocyte detachment by a pathologist (L. Kobzik) in a blinded fashion. A score was assigned from 0 to 3, corresponding to normal, mild (<25% of sample area showing injury), moderate (25-50% of sample showing injury), and severe (>50% of sample area injured), respectively.
Activation of human platelets with ADP.
PIPES, NaCl, KCl, dextrose, ADP, epinephrine, CaCl2,
glycine, diethylamine, Tris (Trizma base), BSA, and PBS were purchased from Sigma. Ethylene glycol was obtained from Pierce (Rockford, IL).
Washed human platelets were suspended in PIPES buffer (25 mM PIPES, 137 mM NaCl, 4 mM KCl, 0.1% dextrose, pH = 7.0) at a platelet count
of 109/ml. The sample was treated with
ADP/epinephrine/CaCl2 at a final concentration of 20 µM
ADP, 20 µM epinephrine, and 3 mM CaCl2. The platelets
were allowed to stand for 15 min at room temperature and then
centrifuged at 500 g for 20 min at 4°C. The supernatant was again centrifuged at 5,000 g for 20 min at 4°C and
filtered via 0.2-µm membranes, lyophilized, and stored at 70°C.
Preparation of sP-sel supernatant from activated platelets. The above lyophilized samples were reconstituted with PBS and dialyzed against PBS at 4°C for 24 h with a 10-kDa cutoff membrane (Pierce). Dialyzed samples were concentrated using Centriplus concentrator tubes (30-kDa cutoff; Amicon, Beverly, MA). The samples were analyzed with ELISA kits (Biosource). Quantified supernatant samples were used in animal experiments as well as in the subsequent P-selectin purification.
Extraction of psP-sel from supernatant. Streptavidin immobilized magnetic beads (Dynabeads M-280 Streptavidin; Dynal, Lake Success, NY) and biotinated mouse monoclonal anti-human P-selectin antibody (Southern Biotechnology Associates, Birmingham, AL) were allowed to come to room temperature. Dynabeads were washed four times with a solution of 0.1% BSA/PBS. One milligram of antibody was mixed with 10 ml of Dynabeads and shaken slowly for 1 h at room temperature. The beads were then washed four times with BSA/PBS. Above dialyzed samples (10 ml) were incubated with the beads for 45 min at room temperature. The beads were collected by a magnetic particle concentrator and the supernatant was aspirated. The supernatant after extraction contained only small amounts of sP-sel and was used in animal experiments as a negative control. The beads were washed six times with a total of 600 ml of PBS. A solution of 50 mM diethylamine, pH = 11.2, was shaken with the beads for 30 min at room temperature to elute sP-sel. The eluate was immediately neutralized with crystallized glycine and the sample dialyzed against PBS at 4°C for 24 h with a 10-kDa cutoff membrane. Dialyzed samples were concentrated using Centriplus concentrator tubes (30-kDa cutoff). The immunoaffinity beads could be reused several times by washing with BSA/PBS. The purified samples were characterized by ELISA for P-selectin, Western blotting, and total protein (Micro BCA protein assay kit; Pierce).
Murine serum complement activity assay. Both the classical and alternative complement pathways were examined. Most of the assays developed to measure complement activity in serum make use of sensitized sheep red blood cells. However, antibody-sensitized sheep red blood cells are resistant to hemolysis by the mouse classical complement pathway (13), thus not sensitive enough to determine mouse complement activity (7). To overcome these problems, a new assay specific to mouse classical complement pathway was developed using purified mouse IgG2 antibody to rabbit red blood cells (27).
Classical complement pathway hemolytic activity (CH50).
Fresh citrated whole rabbit blood was washed three times with 0.9%
saline and suspended in gelatin veronal buffer (GVB++;
Sigma), resulting in a concentration of 1.2 × 108 red
blood cells/ml. Mouse blood was collected as previously described and
allowed to clot for 10 min at room temperature before being centrifuged
at 4°C, 850 g for 10 min. The extracted serum was frozen
in dry ice immediately and stored at 70°C. At a later date the
serum was defrosted and centrifuged at 2,500 g for 5 min
before assay. Forty microliters of serially diluted serum in
GVB++ were added to 25 µl of rabbit red blood cell
solution and 25 µl of purified mouse IgG2 (diluted 64 times). The plate was shaken for 30 s and incubated for 1.5 h
at 37°C. After centrifugation for 10 min at 850 g, 60 µl
of each supernatant were collected. Absorbance was determined at 405 nm. To estimate the medium control and 100% lysis value, 65 µl of
GVB++ and deionized water were added into 25 µl of rabbit
red blood cell solution, respectively.
Alternative complement pathway hemolytic activity (APCH50). Sera were diluted in Mg-EGTA (7 mM Mg2+, 10 mM EGTA). EGTA selectively chelates Ca2+ ions, preventing activation of the classical pathway, while allowing activation of the alternative pathway in the presence of Mg2+. Forty microliters of serially diluted serum were added to 25 µl of rabbit red blood cell solution and 25 µl of Mg-EGTA. The plate was shaken for 30 s and incubated for 1.5 h at 37°C. The rest of the procedure was performed as described above for the classical pathway.
Calculation.
The lysis percentage was calculated as [sample optical density
(OD) medium control OD] / (100% lysis OD
medium
control OD). The data were plotted according to the
transformation of the Von Krogh equation to determine the CH50 and
APCH50 titer per milliliter of undiluted mouse serum (13).
Selectin preparations as inhibitors of complement activation. The effect of human sP-sel on complement activation was studied by adding psP-sel in a 1:2 dilution with mouse serum and examining both CH50 and APCH50. To determine the site of inhibition within the molecule, the effects of the recombinant lectin/EGF domain fraction of P-selectin (L/E polypeptide) and recombinant (r) PSGL-Ig fusion protein (gifts from Genetics Institute, Cambridge, MA) were studied. If the site of complement inhibition were to be at the lectin/EGF domain, then the L/E polypeptide would be expected to decrease the CH50. On the other hand, by blocking the lectin domain of psP-sel with the addition of its ligand rPSGL-Ig, one would anticipate preventing the decrease in CH50.
L/E polypeptide, at a concentration of 200 ng/ml, was added in a 1:2 dilution with mouse serum. Also, 55 µl of 10 µg/ml rPSGL-Ig were added to 50 µl of 50 ng/ml psP-sel or 50 µl PBS buffer and incubated for 1.5 h at 37°C. One hundred microliters of diluted mouse serum were added into the above samples and incubated for a further 1.5 h at 37°C. The CH50 for each solution was determined as previously described.Effect of heat treated serum on the binding properties of psP-sel. One hundred microliters of 50 ng/ml of psP-sel were incubated for 1.5 h with 100 µl of serially diluted normal or heat-treated (HT) mouse serum. Heat treatment consisted of heating mouse serum to 60°C for 1 h, resulting in denaturation of the complement proteins. Twenty-five microliters of sensitized rabbit red blood cell solution were then added, and the plate was shaken for 30 s and incubated for 1.5 h at 37°C. In another set of wells psP-sel was incubated with either sensitized rabbit red blood cells or buffer. After incubation, the solutions were transferred to microtubes, centrifuged for 10 min at 850 g, and 60 µl of each supernatant were collected for psP-sel analysis. The percent recovery of the initial concentration of psP-sel was calculated.
Flow cytometry. Twenty-five microliters of 200 µg/ml purified human C1q [Advanced Research Technologies (ART), San Diego, CA] were incubated with 25 µl of psP-sel or PBS for 30 min at 37°C. Sensitized rabbit red blood cell solution also purchased from ART was washed twice with GVB++ and diluted to a final concentration of 1 × 107 cells/ml. Fifty microliters of sensitized red blood cells (sRBC) were added to the mixture and incubated for 1.5 h at 37°C. The samples were washed twice with GVB++. FITC-labeled antibody against human C1q (Accurate, Westbury NY) was added to washed sRBC at a 1:10 dilution in GVB++ and incubated for 1 h at 37°C. The red blood cells were washed twice again and reconstituted with GVB++. Samples were then diluted and run on an Ortho Cytofurograf. The fluorescent intensity of the cells was measured.
Statistical analysis. Results are presented as means ± SE in the text, Table 1, and Figs. 1-8. Groups were subjected to a one-way ANOVA, and, when significance was found, Student's t-test with the Bonferroni correction for multiple comparisons was applied. Percentage reduction in PI was calculated after subtraction of the background value was determined in animals that had not undergone ischemia (sham).
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RESULTS |
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Skeletal muscle PI.
Reperfusion of ischemic skeletal muscle resulted in vascular injury,
manifested by the extravasation of radiolabeled albumin (Fig.
1). PI in untreated mice
(n = 19) after 3 h of reperfusion was 2.11 ± 0.12, significantly higher than sham PI of 0.20 ± 0.03 (n = 10, P < 0.05). Mice treated with
180 ng of sP-sel SN (n = 16) had a PI of 1.20 ± 0.10, representing a 48% reduction in permeability (P < 0.05). On the other hand, animals treated with 12 ng of sP-sel SN
(n = 17) had a PI of 1.94 ± 0.10, similar to
untreated mice. Animals treated with 225 ng of psP-sel
(n = 17) had a PI of 1.28 ± 0.06, representing a
43% reduction in permeability (P < 0.05). Mice
treated with 225 ng of tsP-sel standard (n = 15) did
not exhibit a significant reduction in PI (2.05 ± 0.19).
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Bioviability of psP-sel.
The serum concentration of psP-sel was virtually unchanged at 30 min.
After 3 h a 24% reduction was observed (P < 0.05). sP-sel was undetectable in serum from control mice injected with
saline, indicating either no cross-reactivity of murine sP-sel with the human P-selectin ELISA assay or lack of sensitivity due to low circulating levels of mouse sP-sel (Fig.
2).
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Immunohistological analysis.
Hindlimb muscle samples snap-frozen in optimal cutting temperature
compound 3 h after reperfusion were stained for IgM and C3 (Fig.
3). Ischemic untreated mice and animals
treated with 12 ng of sP-sel SN or with tsP-sel demonstrated
colocalization of IgM and C3 on the endothelium. There was associated
edema and muscle cell detachment and deformation, indicative of severe
injury (Table 1). Tissue architectural
injury and C3 colocalization with IgM were negligible in ischemic
animals treated with 180 ng of sP-sel SN or psP-sel. There was no IgM
or C3 staining observed in the sham group.
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Characterization of psP-sel.
ELISA and total protein assay indicated the purity of the sample to be
>90%, as calculated by the sP-sel level divided by the total protein
level. Western blot identified 1 band with an approximate molecular
mass of 140 kDa (Fig. 4).
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CH50 analysis.
In vitro, human sP-sel inhibited the classical but not the alternative
pathway of complement activation (Fig.
5). The CH50 following incubation with
recombinant L/E polypeptide was 280.4 U/ml compared with a control
value of 253.6 U/ml, indicating that this part of the P-selectin
molecule did not inhibit complement. Similarly, incubation with
rPSGL-Ig fusion protein had no effect on the anti-complement activity
of psP-sel, indicating that the lectin domain of psP-sel is not
responsible for the observed complement inhibitory effects (Fig.
6). Incubation of psP-sel with sRBC or mouse serum heated to 60°C for 1 h failed to decrease its
concentration from the supernatant, as indicated by the high level of
psP-sel detected by ELISA (Fig. 7). These
data suggest lack of binding between psP-sel and antibody-coated red
blood cells or non-complement serum components. Incubation of psP-sel
with normal serum resulted in a significant reduction of psP-sel
detection in the supernatant, indicating complex formation with
complement components, which are presumably centrifuged to the pellet
(Fig. 7).
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Flow cytometry.
The data from the C1q FITC experiments showed that psP-sel reduced the
amount of C1q binding to sensitized sheep red blood cells (Fig.
8). The intensity of red blood cells
staining positively for C1q in the presence of 25 and 200 ng/ml of
sP-sel was decreased from 324 ± 3.3 to 266 ± 18.7 and
124 ± 1.3, respectively.
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DISCUSSION |
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This study examines the role of human sP-sel as a possible inhibitor of classical complement pathway activation in a murine model of hindlimb ischemia-reperfusion. Infusion of sP-sel SN at a dose of 180 ng reduced the local vascular permeability index by 48%. Conversely, extraction of sP-sel from the supernatant before infusion into the ischemic animals failed to significantly reduce local injury, as shown in mice treated with 12 ng of sP-sel in the supernatant, indicating sP-sel to be the active anti-inflammatory component. In addition, treatment with the final purified product demonstrated a 43% reduction in permeability, confirming the protective role of psP-sel in skeletal muscle reperfusion injury. Considering that the truncated form of the molecule failed to moderate local permeability, it would suggest that only the 140-kDa full length molecule is active in this setting.
The dependence of skeletal muscle reperfusion injury on complement activation has been confirmed in published reports, where experimental complement inhibition with the C3 and C4 convertase inhibitor soluble complement receptor 1 (sCR1) has ameliorated local reperfusion injury of the hindlimb in mice by 50%, as well as in other animal species (17, 32). The colocalization of IgM and C3 has been previously shown, and, together with a 39% protection in local skeletal muscle injury noted in C4 knockout mice, indicates a role for the classical pathway of complement (32). The ischemic event is hypothesized to cause binding of IgM antibody on exposed novel antigenic determinants in reperfused endothelial cells, as a result of alterations in the plasma membrane following ischemia (32). These membrane alterations could come about by a variety of mechanisms such as a reduction in phospholipid biosynthesis or by activation of Ca2+-dependent phospholipases and proteases, or by endothelial cell expression of new epitopes either preformed or newly synthesized (21). Formation of this pentameric IgM natural antibody-antigen complex facilitates activation of C1 and the classical complement pathway, leading to the formation and integration of the membrane attack complex into the membrane of the vascular endothelium and tissue injury.
Support for the proposed complement inhibitory role of sP-sel resulting in reduced vascular permeability is provided by the results of immunostaining sections of injured hindlimb. C3 deposition colocalized with IgM was present on the local vascular endothelium and myocytes of reperfused untreated mice and animals receiving a supernatant essentially depleted of sP-sel (12 ng) but was negligible in mice treated with either 180 ng sP-sel SN or psP-sel. In addition, the injury score was significantly less in these groups, suggestive of a protective effect. The most likely explanation for these observations in that the anti-inflammatory effects exhibited by sP-sel are by inhibition of complement activation. Furthermore, C3 deposition and its colocalization with IgM in mice treated with tsP-sel was similar to that of untreated animals, as was the tissue injury, suggesting absence of complement inhibitory activity of the truncated form of the molecule. Interestingly, levels of IgM staining in reperfused animals treated with either 180 ng sP-sel SN or psP-sel were reduced compared with the other injured groups. This may be because the amount of IgM binding reflects the overall injury and neoantigen exposure, which are greater in the animal groups where complement activity is not inhibited (33).
Further support for the proposed interaction of P-selectin with complement components is the observation that PB1.3 monoclonal antibody (CY1747; Cytel, San Diego, CA), directed against an unspecified epitope of P-selectin, was effective in reducing hindlimb and gut reperfusion injury in the rat by inhibition of complement deposition on the vascular endothelium and intestinal epithelium, respectively, as assessed by C5b-9 antibody immunochemistry (8, 29). Although PB1.3 might be expected to bind both soluble and endothelial P-selectin, the increase in circulating sP-sel observed following platelet activation, such as during skeletal muscle or intestinal reperfusion, is the neutrophil-cleaved truncated form that, according to our data, does not interact with complement (29). It might be hypothesized that endothelial P-selectin facilitates complement deposition on ischemic tissue while the soluble form of the full-length molecule would antagonize complement.
In vitro studies were in concert with this thesis, showing that psP-sel at a concentration of 125 ng/ml provided 100% inhibition of the classical but not the alternative pathway of complement. To try and ascertain which domain of psP-sel exhibits anti-complement activity, binding studies were performed. The lectin and epidermal growth factor domains of P-selectin are important with respect to ligand binding and neutrophil sequestration (10, 19). Our data show that these domains when tested in the form of the L/E peptide did not antagonize complement activity. Further, rPSGL-Ig, which ligates and blocks the lectin domain, did not reduce the anti-complement action of psP-sel. These data suggest that another part of the psP-sel molecule, likely the SCRs, is the functional site of complement inhibition. There are nine SCRs in P-selectin with several possible functions, none of which are mutually exclusive. First, these regions may act as spacers, permitting leukocyte-platelet or endothelial interactions in a sterically favorable geometry (22). Second, they may act more directly by promoting increased leukocyte binding. Thus one report describes the fourth SCR as playing a role in P-selectin-leukocyte interactions (24). Third, they may act to regulate the complement cascade as do homologous SCRs of the regulatory proteins involved in moderating classical complement pathway activity, such as C1-INH, C4 binding protein, and CR1 (14).
Our in vitro studies indicate that sP-sel binds to a heat-labile molecule, which we believe to be C1. Further, utilizing flow cytometry, we noted that sP-sel inhibited binding of C1q to sRBC, supporting the notion of interaction between these molecules. It is also possible that sP-sel acted on the next step of the complement cascade, limiting the interaction of C1q with C1r and C1s. Both C1r and C1s have two SCR domains per molecule, lending plausibility to this latter postulate. These putative modes of action are not mutually exclusive. As noted previously, the truncated form of the molecule failed to antagonize complement. A possible explanation for this observation is that, in the absence of the membranous and cytoplasmic domains from that sP-sel isotype, the changed conformation of the SCRs would hinder their ability to form dimers and oligomers, and this could inhibit effective interaction with C1q. This thesis is directly supported by electron microscopic data showing the full-length 140-kDa molecule forming oligomers while the neutrophil-cleaved truncated molecule of 100 kDa remains as a monomer (29).
A central role for the neutrophil in mediating reperfusion injury after tissue ischemia has been documented in a number of organs including skeletal muscle (31). Experimental PMN immunodepletion achieved a 36% reduction in local permeability in a rat model of hindlimb ischemia-reperfusion (31). A similar reduction in injury was observed when animals were treated with the oligosaccaride sialyl-Lewis X, which antagonizes the neutrophil receptor PSGL-1 by binding to the lectin domain of endothelial selectins (31). One could hypothesize that sP-sel may be operative in this setting by also binding to the PSGL-1 receptor and thus inhibiting PMN-endothelial cell adhesion and injury. In addition, the fourth SCR of sP-sel might interact with circulating neutrophils and block their tethering to the vascular endothelium. However, a similar effect would be anticipated with the truncated form of the molecule, possessing a normal lectin domain and a fourth SCR, which was demonstrated not to moderate vascular permeability and injury score. Thus we are led to conclude that the reduction in reperfusion injury observed in mice receiving this dose of psP-sel appears to be independent of any PMN inhibitory mechanism. It is likely that the relatively low dose of psP-sel given in these experiments is ineffective in blocking PMN-endothelial cell interaction. It is also possible that the SCRs of psP-sel have a high affinity for complement fragments.
Complement-mediated skeletal muscle ischemia-reperfusion injury is a pathophysiological event occurring in elective and emergency clinical situations alike, resulting in severe illness. Therapy directed at limiting this key initial injury is of prime importance in reducing the systemic inflammatory response syndrome and associated multiple organ failure. Proposed therapy using complement antagonists such as sCR1 inhibit both the classical and alternative complement pathways. This complete blockade will likely enhance host susceptibility to bacterial sepsis, a target of the alternative pathway. We have described a novel property of sP-sel as a selective antagonist of classical complement pathway activation, with the potential benefit of not compromising the host to bacterial sepsis.
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ACKNOWLEDGEMENTS |
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We thank Gray Shaw of the Genetics Institute, Cambridge, MA, for the generous donation of L/E peptide and rPSGL-Ig used in the experiments and Amy C. Imrich for performing the immunohistochemistry.
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FOOTNOTES |
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This work was supported in part by National Institute of General Medical Sciences Grants GM-07560, GM-35141, and GM-52585 and by The Brigham Surgical Group, Inc., and The Trauma Research Foundation.
Address for reprint requests and other correspondence: H. B. Hechtman, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (E-mail: hhechtman{at}partners.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 20 September 1999; accepted in final form 13 April 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berman, CL,
Yeo EL,
Wencel-Drake JD,
Furie BC,
Ginsberg M,
and
Furie B.
A platelet alpha-granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein.
J Clin Invest
78:
130-137,
1986[ISI][Medline].
2.
Beurke, M,
Prufer D,
Dahm M,
Oelert H,
Meyer J,
and
Darius H.
Blocking of classical complement pathway inhibits endothelial adhesion molecule expression and preserves ischemic myocardium from reperfusion injury.
J Pharmacol Exp Ther
286:
429-438,
1998
3.
Bevilaccqua, MP,
and
Nelson RM. .
Selectins.
J Clin Invest
91:
379-387,
1993[ISI][Medline].
4.
Bonfanti, R,
Furie BC,
Furie B,
and
Wagner DD.
PADGEM (GMP-140) is a component of Weibel-Palade bodies of human endothelial cells.
Blood
73:
1109-1112,
1989[Abstract].
5.
Carlos, TM,
and
Harlan JM.
Leukocyte-endothelial adhesion molecules.
Blood
84:
2068-2101,
1994
6.
Chong, BH,
Murray B,
Berndt MC,
Dunlop LC,
Brighton T,
and
Chesterman CN.
Plasma P-selectin is increased in thrombotic consumptive platelet disorders.
Blood
83:
1535-1541,
1994
7.
Dewaal, RMW,
Schrijver G,
Bogman MJJT,
Assmann KJM,
and
Koene AP.
An improved sensitive and simple microassay of mouse complement.
J Immunol Methods
108:
213-221,
1988[ISI][Medline].
8.
Gibbs, SA,
Weiser MR,
Kobzik L,
Valeri CR,
Shepro D,
and
Hechtman HB.
P-selectin mediates intestinal ischemic injury by enhancing complement deposition.
Surgery
119:
652-656,
1996[ISI][Medline].
9.
Ishiwata, N,
Takios K,
Katayama M,
Watanabe K,
Titani K,
Ikeda Y,
and
Handa M.
Alternatively spliced isoform of P-selectin is present in-vivo as a soluble molecule.
J Biol Chem
269:
23708-23715,
1994
10.
Kansas, GS,
Saunders KB,
Ley K,
Zakrzewicz A,
Gibson RM,
Furie BC,
Furie B,
and
Tedder TF.
A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion.
J Cell Biol
124:
609-618,
1994[Abstract].
11.
Kansas, GS,
Spertini O,
Stolen LM,
and
Tedder TF.
Molecular mapping of functional domains of the leukocyte receptor for endothelium LAM-1.
J Cell Biol
114:
351-358,
1991[Abstract].
12.
Katayama, M,
Handa M,
Araki Y,
Ambo H,
Kawai Y,
Watanabe K,
and
Ikeda Y.
Soluble P-selectin is present in normal circulation and its plasma level is elevated in patients with thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome.
Br J Haematol
84:
702-710,
1993[ISI][Medline].
13.
Klerx, JP,
Beukelman CJ,
Van Dijk H,
and
Willers JMN
Microassay for colorimetric estimation of complement activity in guinea pig, human, and mouse serum.
J Immunol Methods
63:
215-220,
1983[ISI][Medline].
14.
Klickstein, LB,
Barbashov SF,
Liu T,
Jack RM,
and
Nicholson-Weller A.
Complement receptor type 1 (CR1, CD35) is a receptor for C1q.
Immunity
7:
345-355,
1997[ISI][Medline].
15.
Kobzik, L,
Bredt DS,
Lowenstein CJ,
Drazen J,
Gaston B,
Sugarbaker D,
and
Stamler JS.
Nitric oxide synthase in human and rat lung: immunohistochemical and histological localization.
Am J Respir Cell Mol Biol
9:
371-377,
1993[ISI][Medline].
16.
Kyriakides, C,
Austen WG, Jr,
Wang Y,
Favuzza J,
Kobzik L,
Moore FD, Jr,
Valeri CR,
Shepro D,
and
Hechtman HB.
Soluble (s) P-selectin moderates complement dependent reperfusion injury (Abstract).
FASEB J
13:
A21,
1999[ISI].
17.
Lindsay, TF,
Hill J,
Ortiz F,
Rudolph A,
Valeri CR,
Hechtman HB,
and
Moore FD, Jr.
Blockade of complement activation prevents local and pulmonary albumin leak after lower torso ischemia-reperfusion.
Ann Surg
216:
677-683,
1992[ISI][Medline].
18.
McEver, RP,
Beckstead JH,
Moore KL,
Marshall-Carlson L,
and
Bainton DF.
GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is located in Weibel-Palade bodies.
J Clin Invest
84:
92-99,
1989[ISI][Medline].
19.
Mehta, K,
Patel D,
Laue TM,
Erickson HP,
and
McEver RP.
Soluble monomeric P selectin containing only the lectin and epidermal growth factor domains binds to P selectin glycoprotein ligand-1 on leukocytes.
Blood
90:
2381-2389,
1997
20.
Moore, KL,
Eaton SF,
Lyons DE,
Lichenstein HS,
Cummings RD,
and
McEver RP.
The P-selectin glycoprotein ligand from human neutrophils displays sialylated, fucosylated, O-linked poly-N-acetyllactosamine.
J Biol Chem
269:
23318-23327,
1994
21.
Ogawa, S,
Clauss M,
Kuwabara K,
Shreeniwas R,
Butura C,
Koga S,
and
Stern D.
Hypoxia induces endothelial cell synthesis of membrane associated proteins.
Proc Natl Acad Sci USA
88:
9897-9901,
1991[Abstract].
22.
Patel, KD,
Nollert MU,
and
McEver RP.
P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils.
J Cell Biol
131:
1893-1902,
1995[Abstract].
23.
Peerschke, EI,
and
Ghebrehiwet B.
C1q augments platelet activation in response to aggregated Ig.
J Immunol
159:
5994-5598,
1997.
24.
Ruchaud-Sparagano, MH,
Malaud E,
Gayet O,
Chignier E,
Buchaland R,
and
McGregor JL.
Mapping the epitope of a functional P-selectin monoclonal antibody (LYP20) to a short complement-like repeat (SCR 4) domain: use of human-mouse chimera and homologue-replacement mutagenesis.
Biochem J
332:
309-314,
1998[ISI][Medline].
25.
Stenberg, PE,
McEver RP,
Shumen MA,
Jacques YV,
and
Bainton DF.
A platelet alpha-granule membrane protein (GMP 140) is expressed on the plasma membrane after activation.
J Cell Biol
101:
880-886,
1985[Abstract].
26.
Takeda, I,
Kaise S,
Nishimaki T,
and
Kasukawa R.
Soluble P-selectin in the plasma of patients with connective tissue diseases.
Int Arch Allergy Immunol
105:
128-134,
1994[ISI][Medline].
27.
Tanaka, S,
Suzuki T,
and
Nishioka K.
Assay of classical and alternative pathway activities of murine complement using antibody-sensitized rabbit erythrocytes.
J Immunol Methods
86:
161-170,
1986[ISI][Medline].
28.
Tu, L,
Chen A,
Delahunty MD,
Moore KL,
Watson SR,
McEver RP,
and
Tedder TF.
L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity.
J Immunol
157:
3995-4004,
1996[Abstract].
29.
Ushiyama, S,
Laue TM,
Moore KL,
Erickson HP,
and
McEver RP.
Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin.
J Biol Chem
269:
15229-15237,
1993
30.
Weiser, MR,
Gibbs SA,
Kobzik L,
Valeri CR,
Shepro D,
and
Hechtman HB.
P-selectin mediates local reperfusion injury after lower torso ischemia.
Surgical Forum
XLV:
389-391,
1994.
31.
Weiser, MR,
Gibbs SA,
Valeri CR,
Shepro D,
and
Hechtman HB.
Anti-selectin therapy modifies skeletal muscle ischemia and reperfusion injury.
Shock
5:
402-407,
1996[ISI][Medline].
32.
Weiser, MR,
Williams JP,
Moore FD, Jr,
Kobzik L,
Ma M,
Hechtman HB,
and
Carroll MC.
Reperfusion injury of ischemic skeletal muscle is mediated by natural antibody and complement.
J Exp Med
183:
2343-2348,
1996[Abstract].
33.
Williams, JP,
Pechet TVT,
Weiser MR,
Reid R,
Kobzik L,
Moore FD, Jr,
Carroll MC,
and
Hechtman HB.
Intestinal reperfusion injury is mediated by IgM and complement.
J Appl Physiol
86:
938-942,
1999