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The Journal of Immunology, 2003, 171: 4786-4791.
Copyright © 2003 by The American Association of Immunologists

Complement Regulatory Protein C1 Inhibitor Binds to Selectins and Interferes with Endothelial-Leukocyte Adhesion 1

Shenghe Cai and Alvin E. Davis, III2

Center for Blood Research, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
C1 inhibitor (C1INH), a member of the serine proteinase inhibitor (serpin) family, is an inhibitor of proteases in the complement system, the contact system of kinin generation, and the intrinsic coagulation pathway. It is the most heavily glycosylated plasma protein, containing 13 definitively identified glycosylation sites as well as an additional 7 potential glycosylation sites. C1INH consists of two distinct domains: a serpin domain and an amino-terminal domain. The serpin domain retains all the protease-inhibitory function, while the amino-terminal domain bears most of the glycosylation sites. The present studies test the hypothesis that plasma C1INH bears sialyl Lewisx-related moieties and therefore binds to selectin adhesion molecules. We demonstrated that plasma C1INH does express sialyl Lewisx-related moieties on its N-glycan as detected using mAb HECA-452 and CSLEX1. The data also show that plasma C1INH can bind to P- and E-selectins by FACS and immunoprecipitation experiments. In a tissue culture model of endothelial-leukocyte adhesion, C1INH showed inhibition in a dose-dependent manner. Significant inhibition (>50%) was achieved at a concentration of 250 µg/ml or higher. This discovery may suggest that C1INH plays a role in the endothelial-leukocyte interaction during inflammation. It may also provide another example of the multifaceted anti-inflammatory effects of C1INH in various animal models and human diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C1 inhibitor (C1INH), 3 a member of the serine proteinase inhibitor (serpin) family, regulates all three pathways of complement activation. It is the sole natural inhibitor of C1r and C1s, is an inhibitor of the lectin pathway via inactivation of mannan-binding lectin-associated serine proteinase-1 and 2, and inhibits the alternative pathway of activation by binding to C3b (1). It is also the major regulator of coagulation factors XI and XII, and of plasma kallikrein (for review, see Ref.2). Therefore, C1INH is an inhibitory protein in the complement system, the contact system of kinin generation, and the intrinsic coagulation pathway. Heterozygosity for C1INH deficiency results in hereditary angioedema, characterized by recurrent episodes of enhanced vascular permeability (3). Clinical data, in vitro data, and data from C1INH-deficient mice indicate that angioedema in hereditary angioedema is mediated by bradykinin (4).

C1INH is the most heavily glycosylated plasma protein (2). Of its 104 kDa apparent molecular mass, the protein moiety of 478 aa accounts for only 52,869 Da. Carbohydrate, therefore, contributes ~35% of the total apparent molecular mass (5, 6, 7). C1INH contains 13 definitively identified glycosylation sites (7 O-linked and 6 N-linked), as well as an additional 7 potential O-linked glycosylation sites. Ten of the 13 glycosylation sites are located in the amino-terminal domain (first 100 residues), which is the longest amino-terminal extension among the known serpins. Carbohydrate analysis of C1INH indicated that O-linked oligosaccharides are predominantly NeuNAc2–3(6)Gal{beta}1–3GalNAc, and 27.1% of the N-glycans are fucosylated (7, 8).

The role of carbohydrate in the function of C1INH remains unknown, although it may contribute to its clearance from plasma (9). Although it has been suggested that carbohydrate may contribute to conformational stability and binding kinetics toward target proteases (10), the data currently available indicate that carbohydrate does not play a major role in inhibitory activity (11, 12). Based on the composition of the carbohydrate groups, the characteristic structure of the long amino-terminal extension, and the presence of {alpha}1,3-fucosyltransferase-VI in human liver (13), which is capable of producing sialyl Lewisx tetrasaccharide (NeuNAc{alpha}2–3Gal{beta}1–4(Fuc{alpha}1–3)GlcNAc)-related moieties on appropriate glycoproteins (14), we hypothesized that human plasma C1INH could bear sialyl Lewisx-related moieties and therefore could bind to selectin adhesion molecules. C1INH, therefore, might inhibit endothelial-leukocyte adhesion.

In this study, we report that C1INH does express a sialyl Lewisx-related moiety, as defined by the mAbs HECA-452 and CSLEX1, and can bind to both P- and E-selectin adhesion molecules. We also show that plasma-derived C1INH can inhibit endothelial-leukocyte adhesion in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid, protein, and cell

The plasmids encoding human P- or E-selectin-IgG chimeric proteins were provided by B. Seed (Massachusetts General Hospital, Boston, MA) (15, 16). The selectin portion of these two constructs includes the signal sequence, the lectin domain, the epidermal growth factor-like repeat, and the first two consensus repeats fused to the hinge region, followed by the CH2 and CH3 domain of human IgG1. Plasma C1INH and C1s were obtained from Advanced Research Technologies (San Diego, CA). Recombinant E-selectin was purchased from Calbiochem, EMD Biosciences (San Diego, CA). The Chinese hamster ovary (CHO) cell lines that stably express P- or E-selectin were a generous gift from D. Wagner (Center for Blood Research, Boston, MA) (17, 18), and named CHO/P and CHO/E, respectively, throughout this work. CHO/P and CHO/E were cultured in {alpha} DMEM plus 10% dialyzed FCS (Invitrogen, Carlsbad, CA), containing 175 nM (for CHO/P) or 20 nM (for CHO/E) methotrexate (Sigma-Aldrich, St. Louis, MO). The cell line PRO-LEC11.E7 (generic name LEC11 throughout this work) (19) was obtained from P. Stanley (Albert Einstein College of Medicine, New York, NY). It is a CHO cell mutant that has an active {alpha}1,3-fucosyltransferase that can add fucose to certain sialylated glycoproteins and makes possible the biosynthesis of the sialyl Lewisx tetrasaccharide during posttranslational glycosylation. LEC11 was cultured in {alpha} MEM (Invitrogen). All other cell lines were from American Type Culture Collection (ATCC, Manassas, VA) and cultured according to ATCC protocols. These included CHO-K1, the human monocytic cell line U937, and HUVEC.

Antibodies

Rabbit anti-human C1INH antiserum was from DAKO (Glostrup, Denmark), and mouse anti-human P- and E-selectin mAbs were from BD PharMingen (San Diego, CA). Peroxidase-conjugated secondary Abs against rabbit IgG, mouse IgG, rat IgM, and mouse IgM were from Pierce (Rockford, IL).

The mAb HECA-452- and CSLEX1-producing hybridomas were cultured in RPMI 1640. The mAb, HECA-452, a rat IgM, can recognize sialyl Lewisx-related carbohydrate ligands (sialyl Lewisx and its isoform sialyl Lewisa) for human E-selectin, including the T cell E-selectin ligand cutaneous lymphocyte Ag (20, 21, 22). The mAb CSLEX1 is a mouse IgM that can recognize sialyl Lewisx (23). Both Abs have been used extensively in identification of selectin ligands (24, 25, 26). Rabbit and mouse IgM were from Antigenix America (Huntington Station, NY) and used as isotype controls for the mAb CSLEX1 and HECA-452, respectively.

Expression and purification of P-, E-selectin-IgG chimeric protein

P- and E-selectin-IgG chimeric plasmids were cotransfected into CHO-K1 cells with pcDNA3.1. The clones with the highest expression level were selected with G418 sulfate (1 mg/ml). P- and E-selectin expression was confirmed by Western blot analysis using mAbs against human P- or E-selectin. P- and E-selectin-IgG chimeric proteins were purified from the culture medium using protein G-agarose and eluted with 4 M imidazole.

Expression and purification of rC1INH

The full-length C1INH construct, including the signal sequence, the coding region, and partial transcriptional initiation and termination sequences in the expression vector pcDNA3.1 (11), was used to transfect CHO-K1 and LEC11 cells, respectively. The high-expressing clones were selected in growth medium containing G418 (1 mg/ml).

After centrifugation at 15,000 x g for 30 min to remove cell debris, the conditioned medium was concentrated using a Centricon Plus-80 (Millipore, Bedford, MA) and diluted with PBS, pH 7.4, containing 10 mM EDTA, 25 µM p-nitrophenyl-p-guanidino benzoate, and 1 mM PMSF, and applied to a jacalin-agarose (Vector, Burlingame, CA) column, which was pre-equilibrated with the same buffer. After washing with 10-column volumes of the starting buffer containing 0.5 M NaCl, C1INH was eluted with 10-column volumes of 0.125 M melibiose in the same buffer. The C1INH pool from the jacalin-agarose column was concentrated; (NH4)2SO4 was added to a final concentration of 0.4 M and applied to a phenyl-Sepharose column in an ÄKTA fast protein liquid chromatography system (Amersham, Piscataway, NJ). The flowthrough, containing C1INH, was collected and thereafter changed to PBS, pH 7.4, using a desalting column. C1INH concentration was determined by ELISA (11).

Fluorescence-activated cell sorting (FACS)

E-selectin expression on the cell surface of the CHO/E cells was confirmed using anti-E-selectin Abs. Cell surface localization of P-selectin in CHO/P cells was induced by H2O2 treatment (250 µM, 10 min) and confirmed by FACS using anti-P-selectin Ab. CHO/P, CHO/E, and untransfected CHO-K1 cells (1 x 106) were trypsinized and washed with PBS. Cells were incubated with human plasma-derived C1INH at 250 µg/ml in PBS containing 1 mM MgCl2 and 1 mM CaCl2 at 37°C for 60 min. After washing three times with the same buffer, cells were incubated with rabbit anti-human C1INH antiserum (1/100 dilution) at 37°C for 60 min and washed as above. Cells then were incubated with goat anti-rabbit IgG FITC (Caltag Laboratories, Burlingame, CA) (1/1000 dilution of 0.8 mg/ml) at 37°C for 60 min and washed five times. Cells were analyzed on a FACScan instrument using CellQuest software (BD Immunocytometry Systems, San Jose, CA). Normal rabbit serum (Sigma-Aldrich) and mouse IgG1 (Pierce) were used as isotype controls in FACS experiments.

Deglycosylation

To determine whether HECA-452 epitopes on C1INH were dependent on either N- or O-linked carbohydrate, C1INH (20 µg) was incubated with 2.5 mU of O-glycosidase and neuraminidase (Roche, Basel, Switzerland) or 5 U of N-glycosidase F (New England Biolabs, Beverly, MA) at 37°C overnight in a buffer containing 50 mM sodium phosphate, pH 7.5, and 1% Nonidet P-40. Deglycosylated C1INH was subjected to Western blot analysis, as described below.

Western blot

To determine whether C1INH bears sialyl Lewisx-related moieties, C1INH, ranging from 1 to 8 µg, was separated on 6% SDS-PAGE. BSA (20 µg) and CHO-K1 lysate (1 x 106 cells) were included as negative controls, while LEC11 and U937 lysate (1 x 106 cells) were used as positive controls. Proteins were transferred onto a nitrocellulose membrane. After blocking with PBS containing 0.05% Tween 20 and 5% nonfat dry milk, the blot was probed with mAb HECA-452 or CSLEX1 (concentrated conditioned culture medium). Blots were stripped with 0.2 N NaOH, blocked, and reprobed with anti-C1INH antiserum. Secondary Abs were HRP-conjugated goat anti-rat IgM (1/5,000 dilution), anti-mouse IgM, or anti-rabbit IgG (1/10,000 dilution), respectively. The proteins were detected with a SuperSignal Chemiluminescent Substrate kit (Pierce), and signals were developed using X-OMAT AR film (Eastman Kodak, Rochester, NY). In separate blots, rabbit and mouse IgM were used as isotype controls to replace the mAb CSLEX1 and HECA-452, respectively, and the Western blot was performed, as described above.

To determine whether N-linked glycosylation contributed to the HECA-452-reactive epitope on C1INH, O- and N-glycosidase-treated plasma-derived C1INH was subjected to SDS-PAGE and probed with HECA-452 and anti-C1INH antiserum, respectively, as described above.

To determine whether the HECA-452 reactivity of C1INH is defined by a sialyl Lewisx moiety as a result of the presence of active {alpha}1,3-fucosyltransferase, rC1INH from LEC11 and CHO-K1 cells was separated by SDS-PAGE, blotted, and probed with HECA-452. The same blot, after stripping, was reprobed with anti-C1INH antiserum, as described above.

Complex-formation assay

A C1INH-C1s complex-formation assay was used to determine whether the C1INH-selectin interaction interfered with the proteinase-inhibitory function of C1INH. C1INH (1 µg) was incubated with varying amount of C1s (1, 2, 4 µg) in the absence or presence of E-selectin (1 µg) at 37°C for 60 min in PBS containing 1 mM CaCl2 and 1 mM MgCl2. Samples then were subjected to SDS-PAGE and stained with Coomassie blue.

Immunoprecipitation

P-selectin human IgG (P/IgG), E-selectin human IgG (E/IgG) chimeric protein, or human IgG (Sigma-Aldrich) was incubated without or with C1INH (200 µg/ml) at 37°C for 60 min, and further incubated with protein G-agarose (Sigma-Aldrich) at 4°C for 4 h. Unbound C1INH was washed away by centrifugation with serum-free culture medium containing 0.05% Tween 20. The bound C1INH was eluted from protein G-agarose and detected by Western blot using anti-C1INH antiserum.

Endothelial-Leukocyte adhesion assay

HUVEC (under 10 generations) were plated into 96-well flat-bottom fibronectin-coated plates (BD Biosciences, Franklin Lakes, NJ) at 3 x 104 cells/well 2 days before the assay. The cells were treated with human TNF-{alpha} (50 ng/ml) for 4 h and with H2O2 (250 µM) for 5 min at 37°C. Human plasma-derived C1INH in 0.5x HUVEC culture medium was added at the indicated concentration and incubated for 1 h at 37°C. A control containing EDTA (1 mM) and a control without C1INH added were included. The human monocytic cell line U937 was labeled by adding 10 µM of 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene, OR) and incubated at 37°C for 30 min. Labeled cells (100 µl of 5 x 106) were added into each well and incubated for 45 min at 37°C. Cells then were washed with HUVEC culture medium by gentle swirling, followed by inverting the plate and blotting the unbound cells four times. PBS (100 µl) containing BSA (100 µg/ml) was added into each well, and the fluorescence was measured using a fluorescence reader (Dynex Technologies, Chantilly, VA) at an excitation peak of 485 nm and an emission peak of 530 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C1INH bears a sialyl Lewisx-related moiety on its N-linked carbohydrate

To test whether plasma C1INH bears a sialyl Lewisx-related moiety, we tested whether C1INH reacted with mAbs HECA-452 and CSLEX1. Western blot analysis indicated that plasma C1INH bears a HECA-452 and a CSLEX1-reactive epitope (Fig. 1, A and B). Although untransfected CHO-K1 cells and BSA show no signal, cell lysates from U937 and LEC11 cells show distinct reactivity. The differences in reactivity of the two mAbs with U937 cells are consistent with previous observations (27). The specificity of the reactivity of HECA-452 and CSLEX1 with C1INH was confirmed using the isotype controls rabbit IgM (for HECA-452) and mouse IgM (for CSLEX-1), which did not react with C1INH on the blots (data not shown).



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  		FIGURE 1. Plasma C1INH bears a sialyl Lewisx-related moiety. Human plasma C1INH was separated on SDS-PAGE and subjected to Western blot analysis with mAb HECA-452 (A) and CSLEX1 (B). The same blots, after stripping, were reprobed with anti-C1INH antiserum. A, Lanes 1–4 are plasma-derived C1INH, 8, 4, 2, and 1 µg, respectively. Lanes 5–8 are U937 lysate, LEC11 lysate, CHO-K1 lysate, and BSA, respectively. B, Lane 1 is BSA (20 µg), lanes 2 and 3 are plasma-derived C1INH (5 and 2.5 µg, respectively), and lane 4 is U937 lysate. All cell lysates are 1 x 106 cells. Protein size markers are labeled in kDa. C, Western blots of rC1INH preparations isolated from transfected CHO-K1 and LEC11 cells were probed with HECA-452 and anti-C1INH antiserum. The rC1INH expressed in LEC11 cells can be detected with HECA-452 (lane 1), while that in CHO-K1 cells showed no signal (lane 2).

 
To test whether the HECA-452 reactivity of C1INH is specifically determined by the sialyl Lewisx moiety, the synthesis of which is catalyzed by {alpha}1,3-fucosyltransferase, we expressed C1INH in LEC11 and CHO-K1 cells. The isolated recombinant protein from the LEC11 cells was detected with HECA-452. This protein also reacted with anti-C1INH antiserum. However, the recombinant protein from CHO-K1 cells did not react with HECA-452 (Fig. 1C). These results indicate that the HECA-452 reactivity of C1INH is dependent on the {alpha}1,3-fucosyltransferase that is present in LEC11 cells, but absent in CHO-K1 cells.

Plasma C1INH contains both N- and O-glycans. To determine whether N- or O-linked carbohydrate contributed the HECA reactivity, we treated C1INH with O-glycosidase and N-glycosidase F. Deglycosylation was confirmed by the size decrease on SDS-PAGE (~90 kDa) (Fig. 2). Deglycosylation with N-glycanase or O-glycanase, or both, had no major effect on the functional activity of C1INH (12). C1INH deglycosylated with N-glycosidase F lost its HECA reactivity (Fig. 2), which indicated that the sialyl Lewisx-related moiety is located on the N-glycan of C1INH.



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  		FIGURE 2. The C1INH sialyl Lewisx-related moiety is located on N-linked carbohydrate. To determine whether HECA-452 epitopes on C1INH were dependent on sialic acid and/or O-linked sialoglycoproteins, C1INH was treated with O-glycosidase and neuraminidase, or N-glycosidase F, respectively. Deglycosylated C1INH was subjected to Western blot analysis with HECA-452 (A) and anti-C1INH antiserum (B). Lane 1 is untreated plasma-derived C1INH (5 µg), lane 2 is C1INH treated with N-glycosidase F (5 µg), and lane 3 is C1INH treated with O-glycosidase and neuraminidase (15 µg). Protein size markers are labeled in kDa.

 
C1INH can bind to P- and E-selectin adhesion molecules

We used FACS to test whether C1INH can bind to P- and/or E-selectin on the cell surface. Untransfected CHO/K1 cells do not react with either anti P-selectin or anti-E-selectin Abs, nor does C1INH bind to the cells, as demonstrated using FACS (data not shown). Expression of E-selectin by the CHO/E cells was confirmed by FACS using specific anti-E-selectin Ab (Fig. 3A). Localization of P-selectin on the cell surface of the CHO/P cells was induced by H2O2 treatment and was confirmed using specific anti-P-selectin Ab (Fig. 3B). FACS analysis showed that plasma-derived C1INH could bind P- and E-selectin adhesion molecules expressed on the transfected cell surfaces (Fig. 3, C and D). The binding of C1INH to the P- and E-selectin molecules does not appear to be strong, but is similar to that of the soluble complement receptor 1 modified to express the sialyl Lewisx-related moiety in LEC11 cells (28). It is also consistent with the observation that binding of selectins and their ligands characteristically is of low affinity. It has been hypothesized that low affinity binding is a general feature of cell adhesion receptors (29). To further investigate whether plasma C1INH can bind to P- and E-selectin, we incubated C1INH with fluid-phase P/IgG or E/IgG chimeric protein and precipitated the P/IgG or E/IgG chimera together with any bound C1INH using protein G-agarose. Western blot analysis of this material clearly demonstrated the presence of C1INH. The specificity of the binding was confirmed by the demonstration that human IgG alone does not bind C1INH (Fig. 4).



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  		FIGURE 3. C1INH binds to P- and E-selectin on transfected cells. CHO/P cells were pretreated with H2O2 (250 µM, 5 min) to induce cell surface localization. The surface expression of P- and E-selectin on the transfected cells was confirmed by FACS using specific mAb to human P- and E-selectin, respectively. CHO/E and pretreated CHO/P cells were incubated with human C1INH (250 µg/ml) at 37°C for 60 min. The bound C1INH was detected with rabbit anti-human C1INH antiserum and FITC-conjugated goat anti-rabbit IgG; normal rabbit serum was used as an isotype control. A, Expression of E-selectin on the CHO/E cell surface. CHO/E cells were stained with mAb to E-selectin (unshaded) compared with the isotype control (shaded). B, Expression of P-selectin on the CHO/E cell surface. Pretreated CHO/P cells were stained with mAb to P-selectin (unshaded) compared with the isotype control (shaded). C, C1INH binding to E-selectin on the surface of CHO/E cells. Fluorescent staining with anti-C1INH antiserum (unshaded) compared with the isotype control (shaded). D, C1INH binding to P-selectin on the surface of CHO/P cells. Fluorescent staining with anti-C1INH antiserum (unshaded) compared with the isotype control (shaded). Experiments were performed three times, yielding similar results. Representative results are shown.

 


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  		FIGURE 4. Coimmunoprecipitation of P- and E-selectin with C1INH. P/IgG, E/IgG, or human IgG was incubated in the absence or presence of C1INH (100 µg/ml). Unbound C1INH was removed by extensive washing, and the bound C1INH was precipitated using protein G-agarose and detected on Western blot using anti-C1INH antiserum. Protein size markers are labeled in kDa.

 
C1INH, in the presence of selectin adhesion molecules, retains its full protease-inhibitory function

C1INH, like other serpins, forms a SDS-resistant complex with target proteases. The protease within this complex is inactivated. Complex-formation assays, therefore, can provide information about the protease-inhibitory capacity of C1INH (11). As shown in Fig. 5, C1INH in the presence of E-selectin retains its ability to complex with C1s. Therefore, E-selectin does not appear to have any significant effect on the protease-inhibitory function of C1INH.



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  		FIGURE 5. E-selectin has no effect on C1INH complex formation with C1s. C1INH (1 µg) was incubated without or with E-selectin (1 µg) at 37°C for 60 min in PBS containing 1 mM CaCl2 and 1 mM MgCl2. Varying amounts of C1s were added and incubated at 37°C for an additional 60 min. Samples then were subjected to SDS-PAGE and stained with Coomassie blue. Lane 1 is a prestained protein molecular mass marker (in kDa); lanes 2–4 are untreated C1INH, C1s, and E-selectin, respectively. Lane 5 is C1INH (1 µg) incubated with C1s (1 µg) without E-selectin; lanes 6–8 are C1INH incubated with C1s (1, 2, and 4 µg, respectively) in the presence of E-selectin (1 µg).

 
C1INH mediates leukocyte-endothelial cell adhesion

We investigated whether plasma C1INH can inhibit leukocyte-endothelial cell adhesion under static conditions. Adhesion of fluorescent-labeled U937 cells to HUVEC was inhibited by C1INH in a dose-dependent manner (Fig. 6). The experiment was repeated three times, and a representative result is shown in Fig. 6. Significant inhibition (>50% inhibition compared with the control) can be achieved with 250 µg/ml of C1INH, which is within the concentration range of circulating C1INH during inflammation.



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  		FIGURE 6. Plasma C1INH inhibits U937-endothelial cell adhesion. HUVEC coated onto a flat-bottom fibronectin-coated plate were treated with human TNF-{alpha} and H2O2 and incubated without or with plasma C1INH, ranging from 500 to 31.25 µg/ml. The mean concentration of C1INH in the normal plasma is ~100 µg/ml. The concentration in acute-phase plasma is ~250 µg/ml. The BCECF-AM-labeled U937 cells were added and incubated at 37°C for 60 min. The bound cells were analyzed for fluorescence intensity at an excitation peak of 485 nm and an emission peak of 530 nm. Representative results from one of three experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
C1INH was first identified in 1956 (30), and in 1963 it was shown that its deficiency resulted in the inherited disease, hereditary angioedema (31). C1INH is a single-chain glycoprotein of 500 aa, including a 22-residue signal sequence. The molecule can be divided into two distinct domains that may be observed by electron microscopy (32): a serpin domain that is responsible for protease-inhibitory function, and a rod-like amino-terminal domain of ~100 aa that is the longest amino-terminal extension of all the known serpins. The amino-terminal domain shows no significant homology with any known protein, although its amino acid composition and glycosylation pattern are mucin-like. Its most striking characteristic is the presence of 10 glycosylation sites, of which 7 are O-linked and 3 are N-linked. Carbohydrate analysis of C1INH indicated that O-linked oligosaccharides are predominantly NeuNAc2–3(6)Gal{beta}1–3GalNAc, and 27.1% of the N-glycans are fucosylated (7, 8).

C1INH is an acute-phase protein (APP) with a normal plasma level of 80–195 µg/ml (33) that may increase up to 2.5-fold during inflammation (34). Among APPs, sialyl Lewisx can be detected on {alpha}1-acid glycoprotein (35), and to a lesser extent on {alpha}1-antichymotrypsin and haptoglobin (36), while there is no expression of sialyl LewisX detectable on {alpha}1-antitrypsin. Interestingly, in sharp contrast to the situation in the human, sialyl Lewisx epitopes do not occur on APPs in the mouse, probably because of the absence of {alpha}1,3-fucosyltransferase in mouse liver, thereby making the mouse incapable of producing the sialyl Lewisx epitope on APPs (37). Liver is the major source of human C1INH, although a large variety of other cells can also produce limited amounts (38, 39). It has been demonstrated that {alpha}1,3-fucosyltransferase-VI is expressed in human liver (40) as well as in hepatocyte-derived HepG2 cells (41). {alpha}1,3-Fucosyltransferase-VI is essential for peripheral fucosylation of APPs and is capable of synthesizing the sialyl Lewisx moiety on N-glycans of appropriate glycoproteins (14). C1INH can also be produced by HUVEC (42). {alpha}1,3-Fucosyltransferase-VI is the major form of {alpha}1,3-fucosyltransferases in HUVEC (43). C1INH, with its long amino-terminal extension, may provide a proper scaffold for attachment of the sialyl Lewisx moiety. The fact that C1INH is fucosylated is consistent with this hypothesis. In addition, the data presented in this work clearly show that C1INH reacts with two different mAbs that recognize the sialyl Lewisx epitope.

Our results also may shed light on the observation of Bergamaschini et al. (44), who used ELISA and immunovisualization methods to demonstrate that C1INH can bind to HUVEC following incubation in the cold. Takada et al. (45) demonstrated that E-selectin expression was up-regulated following in situ perfused cold ischemia. It, therefore, is possible that the findings described by Bergamaschini et al. were a result of increased expression of E-selectin, which is then available to bind C1INH.

The expression of endothelial E- and P-selectin, which results in rolling of leukocytes on the endothelial surface, is an important and early component of inflammation. Most selectin ligands contain a peripheral fucose that is attached by an {alpha}1,3-glycosidic linkage to the sialyl Lewisx oligosaccharide epitope (for review, see Refs. 46 and 47). The work described in this study presents evidence for an unusual and surprising feature of C1INH: inhibition of leukocyte-endothelial cell adhesion mediated by binding to selectins via a sialyl Lewisx-related moiety. This interaction may have important pathophysiological consequences. C1INH has multiple anti-inflammatory effects, including inhibition of all three pathways of complement activation, inhibition of contact system activation, and inhibition of the intrinsic coagulation pathway. Recently, we have shown that C1INH also inhibits the inflammatory and pathological effects of Gram-negative endotoxin via a direct interaction with LPS (48). The inhibition of the initial leukocyte-endothelial cell interaction by C1INH may provide another example of the multifaceted anti-inflammatory effects of C1INH. These diverse activities may explain the therapeutic effect of C1INH in a variety of human diseases and animal models, including sepsis, vascular leak syndrome, pancreatitis, acute respiratory distress syndrome, trauma, reperfusion injury, and hyperacute transplant rejection (for review, see Ref.49).

Taken together, these data suggest a model in which C1INH participates in the down-regulation of leukocyte migration from the vasculature during an inflammatory response. During the early stages of inflammation, endothelial E- and P-selectin are up-regulated, but C1INH levels remain normal and therefore are unlikely to interfere with leukocyte rolling. As the acute inflammatory response develops, the C1INH concentration increases up to 2.5-fold (200–480 µg/ml). At these concentrations, C1INH, very likely together with {alpha}1-acid glycoprotein, as well as other selectin ligands, would interfere with the leukocyte-selectin interaction, which would result in the inhibition of migration of cells to inflammatory sites.

The binding of C1INH to selectin molecules on the endothelial surface may also serve to localize and concentrate C1INH at these sites, which would result in more efficient local regulation of activation of the complement and contact systems. This would further suppress vascular permeability mediated by the contact system and the inflammatory effects mediated by complement system activation.


   Acknowledgments
 
We thank Dr. Brian Seed of Massachusetts General Hospital for providing the human P- and E-selectin-IgG plasmids; Dr. Denisa Wagner of the Center for Blood Research, Harvard Medical School, for the CHO cell lines that stably express P- or E-selectin; and Dr. Pamela Stanley of the Albert Einstein College of Medicine (New York, NY) for providing the cell line PRO-LEC11.E7. We also thank Jennifer Scafidi for her excellent technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants HD22082 and HD33727. Back

2 Address correspondence and reprint requests to Dr. Alvin E. Davis III, Center for Blood Research, Harvard Medical School, 800 Huntington Avenue, Boston, MA 02115. E-mail address: aldavis{at}cbr.med.harvard.edu Back

3 Abbreviations used in this paper: C1INH, C1 inhibitor; APP, acute-phase protein; BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; CHO, Chinese hamster ovary; CHO/E, CHO cell lines that stably express E-selectin; CHO/P, CHO cell lines that stably express P-selectin; E/IgG, E-selectin-human IgG chimeric protein; P/IgG, P-selectin-human IgG chimeric protein; serpin, serine proteinase inhibitor. Back

Received for publication March 27, 2003. Accepted for publication August 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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