U.S. patent application number 10/738478 was filed with the patent office on 2004-07-08 for methods of detecting disorders involving defective p-selectin glycoprotein ligand or defective p-selectin.
Invention is credited to Cummings, Richard D., McEver, Rodger P., Moore, Kevin L..
Application Number | 20040132105 10/738478 |
Document ID | / |
Family ID | 27539559 |
Filed Date | 2004-07-08 |
United States Patent
Application |
20040132105 |
Kind Code |
A1 |
Cummings, Richard D. ; et
al. |
July 8, 2004 |
Methods of detecting disorders involving defective p-selectin
glycoprotein ligand or defective p-selectin
Abstract
P-selectin has been demonstrated to bind primarily to a single
major glycoprotein ligand on neutrophils and HL-60 cells, when
assessed by blotting assays and by affinity chromatography of
[.sup.3H]glucosamine-la- beled HL-60 cell extracts on immobilized
P-selectin. This molecule was characterized and distinguished from
other well-characterized neutrophil membrane proteins with similar
apparent molecular mass. The purified ligand, or fragments thereof
(including both the carbohydrate and protein components), or
antibodies to the ligand, or fragments thereof, can be used as
inhibitors of binding of P-selectin to cells, and to treat various
conditions involving leukocyte binding via P-selectin glycoprotein
ligand.
Inventors: |
Cummings, Richard D.;
(Edmond, OK) ; Moore, Kevin L.; (Oklahoma City,
OK) ; McEver, Rodger P.; (Oklahoma City, OK) |
Correspondence
Address: |
DUNLAP, CODDING & ROGERS P.C.
PO BOX 16370
OKLAHOMA CITY
OK
73113
US
|
Family ID: |
27539559 |
Appl. No.: |
10/738478 |
Filed: |
December 17, 2003 |
Related U.S. Patent Documents
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Application
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10738478 |
Dec 17, 2003 |
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10299917 |
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6667036 |
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10299917 |
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10039729 |
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6506382 |
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10039729 |
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09635297 |
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6309639 |
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09635297 |
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09207375 |
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6177547 |
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09207375 |
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08438280 |
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5852175 |
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08438280 |
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08278551 |
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5464778 |
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08278551 |
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07976552 |
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07650484 |
Feb 5, 1991 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
C07K 16/2896 20130101;
A61K 2039/505 20130101; A61K 38/00 20130101; C07K 16/2854 20130101;
C07K 14/70564 20130101; C07K 14/70596 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53 |
Goverment Interests
[0002] The United States government has rights in this invention as
a result of National Institutes of Health grants HI. 34363 (R. P.
McEver) and HL 45510 (R. P. McEver and K. L. Moore), CA 38701 (A.
Varki), IT4 RR 05351 (R. D. Cummings), and GM 45914 (D. F. Smith).
Claims
What is claimed is:
1. A method of detecting a disorder involving defective P-selectin
in a patient comprising: providing a quantity of P-selectin
glycoprotein ligand, or a P-selectin binding portion thereof, the
P-selectin glycoprotein ligand comprising a fucosylated sialylated
glycoprotein containing sialyl Lewis.sup.x antigen and the
glycoprotein ligand having an apparent relative molecular weight of
about 120,000 D as assessed by SDS-PAGE under reducing conditions
and having binding specific for P-selectin; and testing the binding
of the P-selectin glycoprotein ligand, or the P-selectin binding
portion thereof, to activated platelets or activated endothelial
cells derived from the patient.
2. A method of detecting a disorder involving defective binding of
P-selectin glycoprotein ligand in a patient, comprising: providing
an antibody to a protein component or to a carbohydrate-protein
component of P-selectin glycoprotein ligand, the glycoprotein
ligand comprising a fucosylated sialylated glycoprotein containing
sialyl Lewisx antigen and the glycoprotein ligand having an
apparent relative molecular weight of about 120,000 D as assessed
by SDS-PAGE under reducing conditions and wherein the antibody has
binding specific for P-selectin glycoprotein ligand and wherein the
antibody inhibits binding of P-selectin glycoprotein ligand to
P-selectin; and testing the binding of the antibody to leukocytes
obtained from the patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/299,917, filed Nov. 18, 2002, now U.S. Pat. No. 6,667,036,
issued on Dec. 23, 2003; which is a continuation of U.S. Ser. No.
10/039,729, filed Oct. 29, 2001, now U.S. Pat. No. 6,506,382,
issued on Jan. 14, 2003; which is a continuation of U.S. Ser. No.
09/635,297, filed Aug. 9, 2000, now U.S. Pat. No. 6,309,639, issued
on Oct. 30, 2001; which is a divisional of U.S. Ser. No.
09/207,375, filed Dec. 8, 1998, now U.S. Pat. No. 6,177,547, issued
on Jan. 23, 2001; which is a continuation of 08/438,280, filed May
10, 1995, now U.S. Pat. No. 5,852,175, issued on Dec. 22, 1998;
which is a divisional of U.S. Ser. No. 08/278,551, filed Jul. 21,
1994, now U.S. Pat. No. 5,464,778, issued on Nov. 7, 1995; which is
a continuation of U.S. Ser. No. 07/976,552, filed Nov. 16, 1992,
now abandoned; which is a continuation-in-part of U.S. Ser. No.
07/650,484, filed Feb. 5, 1991, now abandoned. The specification of
each of the patents and patent applications listed above is
expressly incorporated by reference herein in its entirety.
[0003] P-selectin (CD62, GMP-140, PADGEM), a Ca.sup.2+-dependent
lectin on activated platelets and endothelium, functions as a
receptor for myeloid cells by interacting with sialylated,
fucosylated lactosaminoglycans. P-selectin binds to a limited
number of protease-sensitive sites on myeloid cells, but the
protein(s) that carry the glycans recognized by P-selectin are
unknown. Blotting of neutrophil or HL-60 cell membrane extracts
with [125I] P-selectin and affinity chromatography of [.sup.3H]
glucosamine-labeled HL-60 cell extracts were used to identify
P-selectin ligands. A major ligand was identified with an
approximately 250,000 M.sub.r under nonreducing conditions and
approximately 120,000 under reducing conditions. Binding of
P-selectin to the ligand was Ca.sup.2+ dependent and was blocked by
mAbs to P-selectin. Brief sialidase digestion of the ligand
increased its apparent molecular weight; however, prolonged
digestion abolished binding of P-selectin. Peptide:N-glycosidase F
treatment reduced the apparent molecular weight of the ligand by
approximately 3,000 but did not affect P-selectin binding. Western
blot and immunodepletion experiments indicated that the ligand was
not lamp-1, lamp-2, or L-selectin, which carry sialyl Le.sup.x, nor
was it leukosialin, a heavy sialylated glycoprotein of similar
molecular weight. The preferential interaction of the ligand with
P-selectin suggests that it may play a role in adhesion of myeloid
cells to activated platelets and endothelial cells.
[0004] The selectins are three structurally related membrane
glycoproteins that participate in leukocyte adhesion to vascular
endothelium and platelets, as reviewed by McEver, "Leukocyte
interactions mediated by selectins" Thromb. Haemostas. 66:80-87
(1991). P-selectin (CD62), previously known as GMP-140 or PAD-GEM
protein, is a receptor for neutrophils, monocytes and subsets of
lymphocytes that is rapidly translocated from secretory granule
membranes to the plasma membrane of activated platelets, as
reported by Hamburger and McEver, "GMP 140 mediates adhesion of
stimulated platelets to neutrophils" Blood 75:550-554 (1990);
Larsen et al., "PADGEM protein: a receptor that mediates the
interaction of activated platelets with neutrophils and monocytes"
Cell 59:305-312 (1989) and endothelial cells, as reported by Geng
et al., "Rapid neutrophil adhesion to activated endothelium
mediated by GMP-140" Nature 343:757-760 (1990); Lorant et al.,
"Coexpression of GMP 140 and PAF by endothelium stimulated by
histamine or thrombin: A juxtacrine system for adhesion and
activation of neutrophils"J. Cell Biol. 115:223-234 (1991).
[0005] E-selectin (ELAM-1) is a cytokine-inducible endothelial cell
receptor for neutrophils, as reported by Bevilacqua et al.,
"Identification of an inducible endothelial leukocyte adhesion
molecule" Proc. Natl. Acad. Sci. USA 84:9238-9242 (1987),
monocytes, as reported by Hession et al., "Endothelial leukocyte
adhesion molecule 1: Direct expression cloning and functional
interactions" Proc. Natl. Acad. Sci. USA 87:1673-1677 (1990), and
memory T cells, as reported by Picker et al., "ELAM-1 is an
adhesion molecule for skin homing T cells" Nature (London)
349:796-799 (1991); Shimizu et al., "Activation-independent binding
of human memory T cells to adhesion molecule ELAM 1" Nature
(London) 349:799-802 (1991). L-selectin (LAM-1, LECAM-1), a protein
expressed on myeloid cells and most lymphocytes, participates in
neutrophil extravasation into inflammatory sites and homing of
lymphocytes to peripheral lymph nodes, as reported by Lasky et al.,
"Cloning of a lymphocyte homing receptor reveals a lectin domain"
Cell 56:1045-1055 (1989); Siegelman et al., "Mouse lymph node
homing receptor cDNA clone encodes a glycoprotein revealing tandem
interaction domains" Science (Wash. DC) 243:1165-1172 (1989);
Kishimoto et al., "Neutrophil Mac-1 and MEL-1 adhesion proteins
inversely regulated by chemotactic factors" Science (Wash. DC) 245:
1238-1241 (1989); Watson et al., "Neutrophil influx into an
inflammatory site inhibited by a soluble homing receptor IgG
chimaera" Nature (London) 349:164-167 (1991).
[0006] Each selectin functions as a CA.sup.2+-dependent lectin by
recognition of sialylated glycans. Both E- and P-selectin interact
with sialylated, fucosylated lactosaminoglycans on opposing cells,
including the sialyl Le.sup.x tetrasaccharide, as reported by
Phillips et al., "ELAM 1 mediates cell adhesion by recognition of a
carbohydrate ligand, sialy-Le.sup.x" Science (Wash DC)
250:1130-1132 (1990); Walz et al., "Recognition by ELAM-1 of the
sialyl-Le.sup.x determinant on myeloid and tumor cells" Science
(Wash. DC) 250:1132-1135 (1990); Lowe et al., "ELAM 1 dependent
cell adhesion to vascular endothelium determined by a transfected
human fucosyltransferase cDNA" Cell 63:475-484 (1990); Tiemeyer et
al., "Carbohydrate ligands for endothelial-leukocyte adhesion
molecule 1" Proc. Natl. Acad. Sci. USA 88:1138-1142 (1991); Goelz
et al., "ELFT: a gene that directs the expression of an ELAM-1
ligand" Cell 63:1349-1356 (1990); Polley et al., "CD62 and
endothelial cell-leukocyte adhesion molecule 1 (ELAM 1) recognize
the same carbohydrate ligand sialyl-Lewis x" Proc. Natl. Acad. Sci.
USA 88:6224-6228 (1991); Zhou et al., "The selectin GMP-140 binds
to sialyated, fucosylated lactosaminoglycans on both myeloid and
nonmyeloid cells" J. Cell Biol. 115:557-564 (1991). However, the
precise carbohydrate structures on myeloid cells recognized by
these two selectins under physiologic conditions are not known.
Such ligands might have unique structural features that enhance the
binding specificity and/or affinity for their respective
receptors.
[0007] P-selectin isolated from human platelets binds with apparent
high affinity to a limited number of sites on neutrophils (Moore et
al., "GMP 140 binds to a glycoprotein receptor on human
neutrophils: evidence for a lectin-like interaction" J. Cell Biol.
112:491-499 (1991); Skinner et al., "GMP-140 binding to neutrophils
is inhibited by sulfated glycans" J. Biol. Chem. 266:5371-5374
(1991) and HL-60 cells (Zhou et al., "The selectin GMP-140 binds to
sialyated, fucosylated lactosaminoglycans on both myeloid and
nonmyeloid cells" J. Cell Biol. 115:557-564 (1991)). Binding is
abolished by treatment of the cells with proteases (Moore et al.,
1991), suggesting that the glycans on myeloid cells recognized
preferentially by P-selectin are on glycoprotein(s) rather than on
glycolipids. The number of binding sites for platelet P-selectin on
neutrophils has been estimated at 10,000-20,000 per cell (Moore et
al., 1991; Skinner et al., 1991), suggesting that these sites
constitute a small component of the total cell surface protein. The
protein portion of this ligand(s) may be crucial for binding by
presenting the glycan in an optimal configuration, clustering
glycans to enhance avidity, favoring the formation of specific
oligosaccharide structures by cellular glycosyltransferases or
modifying enzymes, and/or stabilizing the lectin-carbohydrate
interaction through protein-protein interactions with
P-selectin.
[0008] The potential importance of protein components in enhancing
ligand affinity is supported by studies of CHO cells transfected
with a specific fucosyltransferase (Zhou et al., 1991). These cells
express higher amounts of the sialyl Le.sup.x antigen than do HL-60
cells and have protease-sensitive binding sites for P-selectin.
However, the interaction of P-selectin with these sites is of much
lower apparent affinity than with those on myeloid cells, and
adhesion of transfected CHO cells to immobilized P-selectin is
weaker than that of neutrophils and HL-60 cells (Zhou et al.,
1991). These observations suggest that myeloid cells express one or
more membrane glycoproteins not found on CHO cells that enhance the
lectin-mediated interaction with P-selectin. Alternatively, myeloid
cells may express a glycosyltransferase or modifying enzyme not
present in CHO cells.
[0009] It is therefore an object of the present invention to
identify and characterize a specific glycoprotein ligand for
P-selectin (CD62).
[0010] It is a further object of the present invention to provide
methods and compositions derived from the characterization of a
specific glycoprotein ligand for P-selectin for use in modifying
inflammatory processes and in diagnostic assays.
SUMMARY OF THE INVENTION
[0011] P-selectin has been demonstrated to bind primarily to a
single major glycoprotein ligand on neutrophils and HL-60 cells,
when assessed by blotting assays and by affinity chromatography of
[.sup.3H] glucosamine-labeled HL-60 cell extracts on immobilized
P-selectin. This molecule was characterized and distinguished from
other well-characterized neutrophil membrane proteins with similar
apparent molecular mass.
[0012] The purified ligand, or fragments thereof (including both
the carbohydrate and protein components), or antibodies to the
ligand, or fragments thereof, can be used as inhibitors of binding
of P-selectin to cells.
DETAILED DESCRIPTION OF THE INVENTION
[0013] U.S. Ser. No. 07/554,199 filed Jul. 17, 1990 entitled
"Peptides Selectively Interacting with Selectins" by Rodger P.
McEver, described the ability of P-selectin (GMP-140) to mediate
cell-cell contact by binding to carbohydrate ligands on target
cells and specific binding to protease-sensitive sites on human
neutrophils. Studies with antibodies and with neuraminidase
indicated that P-selectin bound to carbohydrate structures related
to sialylated, fucosylated lactosaminoglycans. As described in U.S.
Ser. No. 07/650,484 entitled "Ligand for GMP-140 Selectin and
Methods of Use Thereof" filed Feb. 5, 1991 by Rodger P. McEver,
P-selectin was also demonstrated to bind to sialylated, fucosylated
lactosaminoglycans (including the tetrasaccharide sialyl Lewis x
(sLe.sup.x)) on both myeloid and nonmyeloid cells.
[0014] The ability of proteases to abolish P-selectin binding to
neutrophils indicated that high affinity binding of P-selectin to
myeloid cells occurred through interactions with cell surface
glycoprotein(s) rather than with glycolipids. As also described in
U.S. Ser. No. 07/650,484, P-selectin bound preferentially to a
glycoprotein in human neutrophil extracts of M.sub.r 120,000, as
analyzed by SDS-PAGE under reducing conditions. The glycoprotein
was partially purified on a P-selectin affinity column. It appeared
to be heavily glycosylated because it stained poorly with silver
and Coomassie blue. It appeared to be heavily sialylated because it
bound to a wheat germ agglutinin affinity column. Treatment of the
glycoprotein ligand with low doses of sialidase slowed its mobility
on SDS gels, a pattern consistent with partial desialylation of
heavily O-glycosylated proteins. Binding of P-selectin to the
glycoprotein ligand was Ca.sup.2+-dependent, blocked by monoclonal
antibodies to P-selectin that also block P-selectin binding to
leukocytes, and abolished by extensive treatment of the ligand with
sialidase.
[0015] The preferential binding of P-selectin to the 120,000 D
glycoprotein ligand in myeloid cell extracts suggested that it
contained special structural features that are recognized with high
affinity by P-selectin. Such structures might not be present on
every protein or lipid characterized by sialylated, fucosylated
structures such as sLe.sup.x. It has now been further demonstrated
that the adhesion of myeloid cells to immobilized P-selectin is
much stronger than that to NeoLewis CHO cells (a cell line
expressing sialylated, fucosylated lactosaminoglycans, described in
U.S. Ser. No. 07/650,484), even though the NeoLewis cells express
higher levels of sLe.sup.x antigen, as reported by Zhou et al., J.
Cell Biol. 115:557-564 (1991). Furthermore, fluid-phase
[.sup.125I]P-selectin binds with high affinity to a limited number
of sites on myeloid cells, whereas it binds with lower affinity to
a higher number of sites on NeoLewis CHO cells. The 120,000 D
glycoprotein ligand for P-selectin in neutrophil extracts is likely
to correspond to the limited number of protease-sensitive, high
affinity binding sites for P-selectin on intact neutrophils.
Interaction of P-selectin with these sites may be required for
efficient adhesion of leukocytes in flowing blood to P-selectin
expressed by activated platelets or endothelial cells.
[0016] Further structural features of the glycoprotein ligand for
P-selectin and a method for purifying the ligand are described
below. The purified ligand, or fragments thereof (including both
the carbohydrate and protein components), or antibodies to the
ligand, or fragments thereof, can be used as inhibitors of binding
of P-selectin to cells.
MATERIALS AND METHODS
[0017] Materials
[0018] Wheat germ agglutinin (WGA)-agarose, pepstatin, aprotinin,
N-acetylglucosamine, leupeptin, antipain, benzamidine, MOPS, Pipes,
BSA, EDTA, EGTA, and Ponceau S were purchased from Sigma Chemical
Co. (St. Louis, Mo.). Diisopropylfluorophosphate,
dichloroisocoumarin, Triton X-100 (protein grade), and sialidase
(neuraminidase) from Arthrobacter ureafaciens (75 U.mg, EC
3.2.1.18) were obtained from Calbiochem-Behring Corp. (La Jolla,
Calif.), Micro BCA protein assay kits and Lubrol PX (Surfact Amp
PX) were purchased from Pierce Chemical Company (Rockford, Ill.).
Enzymobeads.TM., Tween-20, Affigel.TM.-15, and high molecular
weight protein standards were from Bio Rad Laboratories (Richmond,
Calif.). Endo-.beta.-galactosidase (150 U/mg, EC 3.2.1.103) from
Bacteroides fragilis, 4-methyl-umbelliferyl
.alpha.-N-acetylneuraminic acid, and
2,3-dehydro-2,3-dideoxy-N-acetyineuraminic acid (Neu2en5Ac) were
obtained from Boehringer Mannheim Biochemicals (Indianapolis,
Ind.). Peptide:N glycosidase F (PNGaseF) from Flavobacterium
meningosepticum (EC 3.2.2.18, N-glycanase) and
endo-.alpha.-N-acetylgalactosaminidase form Diplococcus pneumoniae
(EC 3.2.1.97, O-glycanase.TM.) were purchased from Genzyme
(Cambridge, Mass.). HBSS was obtained from Gibco Laboratories
(Grand Island, N.Y.). Vecta-Stain ABC kits were purchased from
Vector Laboratories Inc. (Burlingame, Calif.).
Phycoerythrin-streptavidin was obtained from Becton Dickinson &
Co. (San Jose, Calif.) and phycoerythrin-conjugated anti-mouse
IgG.sub.1 was from Caltag (South San Francisco, Calif.). Rabbit
anti-mouse IgG was purchased from Organon Teknika (Durham, N.C.)
and protein A-Sepharose CL4B was from Pharmacia Fine Chemicals
(Piscataway, N.J.). [6-.sup.3H]glucosamine was obtained from
Dupont/New England Nuclear (Boston, Mass.). All other chemicals
were of the highest grade available.
[0019] Antibodies and Proteins
[0020] The anti-P-selectin murine mAbs S12 and G1, and goat
anti-human P-selectin IgG were prepared and characterized as
described by McEver and Martin, "A monoclonal antibody to a
membrane glycoprotein binds only to activated platelets" J. Biol.
Chem. 259:9799-9804 (1984); Geng et al., (1990); Lorant et al.,
(1991). Rabbit polyclonal antisera and murine mAbs to human lamp-1
(CD3), described by Carlsson et al., "Isolation and
characterization of human lysosomal membrane glycoproteins,
h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying
polylactosaminoglycan" J. Biol. Chem. 263:18911-18919 (1988), and
lamp-2 (BB6), Carlsson and Fukuda, "Structure of human lysosomal
membrane glycoprotein 1. Assignment of disulfide bonds and
visualization of its domain arrangment" J. Biol. Chem.
264:20526-20531 (1989), and rabbit polyclonal anti-human
leukosialin antiserum, described by Carlsson and Fukuda, "Isolation
and characterization of leukosialin, a major sialoglycoprotein on
human leukocytes" J. Biol. Chem. 261:12779-12786 (1986) were
provided by Dr. Sven Carlsson (University of Umea, Umea, Sweden).
Anti-human leukosialin (CD43) mAb (Leu-22) was purchased from
Becton Dickinson & Co. (San Jose, Calif.). The anti-L-selectin
murine mAb antibodies DREG-56, DREG-55, and DREG-200, described by
Kishimoto et al., "Identification of a human peripheral lymph node
receptor: a rapidly down-regulated adhesion receptor" Proc. Natl.
Acad. Sci. USA 87:2244-2248 (1990) were provided by Dr. Takashi Kei
Kishimoto (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield,
Conn.). All mAbs are of the IgG.sub.1 subtype and were used in
purified form. Leukosialin purified from HL 60 cells (Carlsson and
Fukuda, 1986) was provided by Dr. Sven Carlsson (University of
Umea). P-selectin was purified from human platelets as described by
Moore et al., (1991). The teachings of these references are
specifically incorporated herein.
[0021] Preparation of Membranes
[0022] Erythrocyte membranes were isolated from leukocyte-depleted
human erythrocytes as described by Rollins and Sims, "The
complement-inhibitory activity of CD59 resides in its ability to
block incorporation of C9 into membrane C5b9" J. Immunol.
144:3478-3483 (1990) and extracted with 0.1 M NaCl, 10 mM MOPS, pH
7.5, 1% Lubrol.TM. PX. Detergent-insoluble material was removed by
centrifugation at 16,000 g for 10 min.
[0023] Human neutrophils isolated by discontinuous leukopheresis
from volunteer donors were purchased from the Oklahoma Blood
Institute (Oklahoma City, Okla.). Each product contained
1.5-3.3.times.10.sup.10 leukocytes (approximately 85% neutrophils).
The neutrophil product was centrifuged at 200 g for 20 min and the
platelet-rich plasma removed. Erythrocytes were lysed by
resuspending the pellets with 5 mM EDTA, pH 7.5, in H.sub.2O for 20
s. An equal volume of 1.8% NaCl, 5 mN EDTA, pH 7.5, was then added
to restore isotonicity. The cells were centrifuged at 500 g for 5
min and resuspended in ice-cold HBSS containing 5 mM EDTA and 10 mM
MOPS, pH 7.5. Diisopropylfluorophosphate was then added to a final
concentration of 2 mM and the cell suspension incubated for 10 min
on ice. The cells were centrifuged at 500 g for 5 min at 4.degree.
C. and resuspended in ice-cold 100 mM KCl, 3 mM NaCl, 1 mM
Na.sub.2ATP, 3.5 mM MgCl.sub.2, 10 mM Pipes pH 7.3 (relaxation
buffer). To this suspension the following protease inhibitors were
added at the indicated final concentrations: 2 mM
diisopropylfluorophosphate, 20 .mu.M leupeptin, 30 .mu.M antipain,
and 1 mM benzamidine. The cell suspension was pressurized with
N.sub.2 at 350 psi in a cell disruption bomb (model 4635; Parr
Instrument Company, Moline, Ill.) for 40 min at 4.degree. C. with
constant stirring as described by Borregaard et al., "Subcellular
localization of the b cytochrome component of the human neutrophil
microbiocidal oxidase: Translocation during activation" J. Cell
Biol. 97:52-61 (1983). The cavitate was collected into EGTA (2 mM
final concentration) and nuclei and undisrupted cells were pelleted
at 500 g for 10 min at 4.degree. C. The cavitate was fractionated
as described by Eklund and Gabig, "Purification and
characterization of a lipid thiobis ester from human neutrophil
cytosol that reversibly deactivates the O.sup.2-generating NADPH
oxidase" J. Biol. Chem. 265:8426-8430 (1990). Briefly, it was
layered over .sup.40% sucrose in relaxation buffer containing 2 mM
EGTA, 20 .mu.M leupeptin, 30 .mu.M antipain, and 1 mM benzamidine,
and centrifuged at 104,000 g (at r.sub.av) for 45 min at 4.degree.
C. in a rotor (model SW28; Beckman Instruments, Inc., Palo Alto,
Calif.). The top layer (FX.sub.1), the 40% sucrose layer
(FX.sub.2), and the granule pellet (FX.sub.3) were collected and
assayed for lactate dehydrogenase as a cytoplasmic marker, alkaline
phosphatase as a plasma membrane marker, and myeloperoxidase as a
marker for azurophilic granules as described by Borregaard et al.,
(1983); Geng et al., (1990).
[0024] Table I shows the distribution of marker enzymes in the
various fractions. FX.sub.2, enriched for alkaline phosphatase, was
diluted with four volumes of 0.1 M NaCl, 10 mM MOPS, pH 7.5, and
centrifuged at 111,000 g (at r.sub.av) for 60 min at 4.degree. C.
in a rotor (model 50.2 Ti; Beckman Instruments, Inc.). The
supernatant was collected and the membrane pellet was extracted
with 1% Lubrol.TM. PX, 0.1 M NaCl, 10 mM MOPS, pH 7.5, 0.02% sodium
azide, 20 .mu.M leupeptin, 30 .mu.M antipain, 1 mM benzamidine, and
stored at 4.degree. C.
[0025] HL-60 cells, maintained in suspension culture in RPMI-1640
supplemented with 10% FCS, 100 IU/ml penicillin, and 100 .mu.g/ml
streptomycin, were washed in HBSS, 10 mM MOPS, pH 7.5, and
membranes were isolated exactly as described for neutrophils.
[0026] Partial Purification of P-selectin Ligand
[0027] Neutrophil or HL-60 cell membrane extracts were applied to a
wheat germ agglutinin (WGA) affinity column (0.9.times.20 cm. 7.6
mg lectin/ml resin) equilibrated at room temperature with 0.5 M
NaCl, 10 mM MOPS, pH 7.5, 0.02% sodium azide, 0.1% Lubrol.TM. PX.
The column was washed with five column volumes of equilibration
buffer, followed by two column volumes of 0.1 M NaCl, 10 mM MOPS,
pH 7.5, 5 mM EDTA, 0.02% sodium azide, 0.01% Lubrol.TM. PX. The
column was then eluted with the above buffer containing 100 mM
N-acetylglucosamine. Protein-containing fractions were pooled and
extensively dialyzed against 0.1 M NaCl, 10 mM MOPS, pH 7.5, 0.0.2%
sodium azide, 0.01% Lubrol.TM. PX at 4.degree. C. The dialyzed WGA
column eluate was made 1 mM in CaCl.sub.2 and MgCl.sub.2 and
applied to a human serum albumin Affigel.TM.15 precolumn
(0.9.times.11 cm, 25 mg protein/ml resin) hooked in series to a
P-selectin-Affigel.TM.-15 column (0.6.times.13 cm, 2 mg protein/ml
resin). The columns were equilibrated with 0.1 M NaCl, 10 mM MOPS,
pH 7.5, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.02% sodium azide, 0.01%
Lubrol.TM. PX. After the samples were applied the columns were
washed with 100 column volumes of equilibration buffer, and eluted
with equilibration buffer containing 5 mM EDTA. Yields were
estimated by protein assays with the Micro BCA protein assay kit
using BSA as a standard.
1TABLE I Distribution of Marker Enzymes from Subcellular Fractions
of Nitrogen-cavitated Human Neutrophils. Lactate Alkaline
dehydrogenase Myeloperoxidase phosphatase FX.sub.1 (cytosol) 95.6
.+-. 0.5 0 29.0 .+-. 2.7 FX.sub.2 (membrane) 4.1 .+-. 0.5 2.6 .+-.
1.0 56.8 .+-. 8.7 FX.sub.3 (granule) 0 97.4 .+-. 1.0 14.1 .+-. 5.5
Results are expressed as the percentage of the total enzyme
activity in the cavitate (mean .+-. SD, n = 3).
[0028] P-selectin Blotting Assay
[0029] Samples were electrophoresed on 7.5% SDS polyacrylamide gels
and proteins electrophoretically tranferred to Immobilon-P.TM.
membranes (Millipore Corp., Bedford, Mass.) for 4-5 h at 0.5 A. The
positions of the molecular weight standards were marked with a pen
after staining the membranes with Ponceau S. The membranes were
blocked overnight at 4.degree. C. in 0.1 M NaCl, 10 mM MOPS, pH
7.5, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.02% sodium azide, 10%
(wt/vol) Carnation.TM. nonfat dry milk, and then washed with the
same buffer containing 0.1% Tween-20 without milk. The membranes
were incubated with [.sup.125I]P-selectin (0.5-1.0 nM), iodinated
as described by Moore et al., 1991 using standard techniques, in
0.1 M NaCl, 10 mM MOPS, pH 7.5, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2,
0.05% Lubrol.TM. PX, 1% human serum albumin for 1 h at room
temperature. After extensive washing the membrane was dried and
exposed to Kodak X-OMAT AR film (Eastman Kodak Company, Rochester,
N.Y.) for 6 18 at -70.degree. C. All the [.sup.125I]P-selectin
blots shown are autoradiograms of the entire blot, corresponding to
the area from the stacking gel interface to beyond the dye front on
the original gel.
[0030] Metabolic Radiolabeling of HL-60 Cells and Isolation of
[.sup.3H]Glucosamine-Labeled P-Selectin Ligand
[0031] HL-60 cells (1-2.times.10.sup.6 cells/ml) in 100-mm tissue
culture dishes were labeled for 48 h with 50 .mu.Ci/ml
[6-.sup.3H]glucosamine at 37.degree. C. in RPMI-1640 containing 10%
FCS, 2 mM glutamine, 100 IU/ml penicillin, and 100 .mu.g/ml
streptomycin. At the end of the labeling periods the cells were
washed three times by centrifugation and resuspension in ice-cold
PBS. The cell pellet was solubilized with 0.1 M NaCl, 10 mM MOPS,
pH 7.5, 4 mM CaCl.sub.2, 4 mM MgCl.sub.21, 1% Triton X-100, 20
.mu.g/ml aprotinin, 20 pg/ml leupeptin, 8 .mu.g/ml pepstatin, 2 mM
PMSF, 10 mM benzamidine, and 0.5 mM dichloroisocoumarin. The
solubilized cells were allowed to sit on ice for 1-2 h and then
sonicated for 20 min at 4.degree. C. in a water bath sonicator. The
cell extract was centrifuged for 5 min at 16,000 g and the
supernatant was applied to a P-selectin-Affigel-.TM.15 column
(0.25.times.13 cm, 2 mg protein/ml resin) equilibrated with 0.1 M
NaCl, 10 mM MOPS, pH 7.5, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, 0.1%
Triton X 100. The column was washed with 10-20 column volumes of
equilibration buffer and bound material was eluted with
equilibration buffer containing 10 mM EDTA. Fractions (1 ml) were
collected and monitored for radioactivity by liquid scintillation
counting.
[0032] Analysis of [.sup.3H]Glucosamine-Labeled P-Selectin
Ligand
[0033] Metabolically labeled proteins eluted from the P-selectin
column were precipitated in the presence of 0.1 mg/ml BSA by
addition of cold TCA (10% final concentration). The resulting
pellets were washed with 1 ml acidified acetone (0.2%), solubilized
in 0.1 M NaOH and electrophoresed under reducing and nonreducing
conditions on 10% SDS-polyacrylamide gels. The gels were stained
with Coomassis blue and then processed for fluorography with
EN.sup.3HANCE (Dupont/New England Nuclear, Boston, Mass.) according
to the manufacturer's instructions. The dried gels were then
exposed to Kodak X-OMAT AR film at -80.degree. C.
[0034] Enzyme Digestion
[0035] In certain experiments, samples analyzed by P-selectin
blotting were pretreated with exo- or endo-glycosidases before
SDS-PAGE. For sialidase and endo-.beta.-galactosidase digestions of
P-selectin ligand, samples were dialyzed against 0.15 M NaCl, 50 mM
acetate, pH 6.0, 9 mM CaCl.sub.2, 0.02% azide, 0.0.sup.1%
Lubrol.TM. PX, and incubated for various times at 37.degree. C. in
the presence or absence of 200 mU/ml of enzyme. For PNGaseF and
endo-.alpha.-N-acetygalactosaminidase digestions, samples were
first reduced and denatured by boiling in 0.5% SDS, 0.5%
.beta.-mercaptoethanol for 5 min, and then a 7.5-fold molar excess
of NP-40 was added. The samples were incubated for 16 h at
37.degree. C. with either PNGaseF (20 U/ml at pH 8.6) or
endo-.alpha.-N acetylagalactosaminidase (70 mU/ml at pH 6.5) in the
presence of 5 mM PMSF and 5 mM 1, 10 phenanthroline.
[0036] Affinity purified [.sup.3H]glucosamine-labeled P-selectin
ligand was incubated for 24 h in 25 mM sodium acetate, pH 5.5 at
37.degree. C. under a toluene atmosphere in the presence or absence
of 1 U/ml of A. ureafaciens sialidase for 18 h. For PNGaseF
digestion of metabolically labeled ligand, samples were denatured
by boiling in 0.25% SDS 25 mM .beta.-mercaptoethanol for 5 min, and
NP-40 was added in eight-fold excess (wt/wt) over SDS. The samples
were incubated for 24 h with PNGaseF (3.3 U/ml) in a toluene
atmosphere. The samples were then precipitated with TCA and
subjected to SDS-PAGE and fluorography as described above.
[0037] Flow Cytometry
[0038] Human neutrophils, isolated as described by Hamburger and
McEver, (1990), were suspended (10.sup.6/ml) in HBSS containing 1%
FCS and 0.1% sodium azide (HBSS/FCS/Az). 1 ml of neutrophil
suspension was underlaid with 100 .mu.l FCS and centrifuged at 500
g for 5 min. The neutrophil pellet was resuspended in 50 .mu.l of
purified P selectin (10 .mu.l/ml, in HBSS/FCS/Az), and then
incubated sequentially with 50 .mu.l of biotin-conjugated S12 (10
.mu.g/ml, in HBSS/FCS/Az) and 20 .mu.l of
phycoerythrin-streptavidin (neat). In certain experiments, the
neutrophils were preincubated for 10-15 min with antisera or
antibodies before the addition of P-selectin. Between each step the
cells were diluted with one ml of HBSS/FCS/Az, underlaid with 100
.mu.l FCS, and centrifuged at 500 g for 5 min. All steps were
performed at 4.degree. C. After the last wash, the cells were fixed
with 1 ml of 1% paraformaldehyde in HBSS and analyzed in a FACScan
flow cytometer (FACScan is a registered trademark of Becton
Dickinson & Co., Mountain View, Calif.) formatted for two color
analysis as described by Moore, et al., (1991). Binding of
P-selectin to intact neutrophils as assessed by this assay was
Ca.sup.2+-dependent, was blocked by G1, and was abolished by
pretreatment of the cells with trypsin or sialidase.
[0039] Immunoprecipitations
[0040] WGA eluate was incubated with 10 .mu.g of anti-leukosialin
(Leu22) or an isotype matched control monoclonal antibody for 1 h
at 37.degree. C. The mixture was then incubated with protein
A-Sepharose CL4B beads saturated with rabbit anti-mouse IgG for 1 h
at 37.degree. C. The beads were pelleted, washed four times with 1
ml of 0.1 M NaCl, 20 mM Tris, pH 7.5, 1% Triton X-100, and bound
material eluted by boiling 5 min in 2% SDS, 60 mM Tris, pH 6.8, and
5% .beta.-mercaptoethanol. Immunoprecipitates and
immunosupernatants were then analyzed by P-selectin blotting and by
Western blotting using Leu22 as a probe.
[0041] Assay of Sialidase Activity in Commercial Enzyme
Preparations
[0042] The sialidase activity in O-glycanase (endo-.alpha.
N-acetylgalactosaminidase) or A. ureafaciens sialidase was assayed
by incubation of dilutions of the enzymes with 50 nmol
4-methyl-umbelliferyl-.alpha.-N-acetylneuraminic acid in 50 .mu.l
of sodium cacodylate, pH 6.5, 10 mM calcium acetate, for various
time periods. Incubations were quenched by addition of 0.95 ml 0.1
M sodium bicarbonate, pH 9.3, and assayed for released
4-methylumbellierone by fluorescence (excitation=365 nM,
emission=450 nM).
RESULTS
[0043] Identification of a P-selectin Ligand
[0044] To identify proteins from myeloid cells which bind
P-selectin, neutrophil and HL-60 cell membrane extracts were
electrophoresed on 7.5% SDS-polyacrylamide gels, transferred to
Immobilon membranes, and probed with [.sup.1251]P-selectin. When
samples were analyzed without reduction, P-selectin bound
preferentially to a glycoprotein species with an approximately
250,000 M.sub.r from both neutrophil and HL-60 cell membranes as
determined by SDS-PAGE. Cell membrane extracts (80 .mu.g
protein/lane) were electrophoresed on 7.5% SDS-polyacrylamide gels
under nonreducing or reducing conditions, transferred to Immobilon
membranes, and probed with [.sup.125I]P-selectin. Under nonreducing
conditions P-selectin also bound to proteins at the stacking gel
interface and to aminor species with an approximately 160,000
M.sub.r. When samples were analyzed after reduction, P-selectin
preferentially bound to a glycoprotein with an approximately
120,000 M.sub.r. Minor bands were observed at approximately 250,000
and approximately 90,000 M.sub.r. Under both reducing and
nonreducing conditions P-selectin also bound to the blots at the
dye front. P-selectin binding proteins were not detected when an
equivalent amount of erythrocyte membrane protein was analyzed in
parallel. The total proteins in the neutrophil cavitate were also
solubilized with SDS and analyzed for their ability to interact
with P-selectin with the blotting assay. P-selectin bound only to
proteins with apparent molecular weights of 120,000 and 90,000
under reducing conditions. Although the sensitivity of this
analysis was limited by the amount of protein that could be run on
the gel, the results indicate that we did not exclude major ligands
that were either not enriched in the membrane fraction (FX.sub.2)
or not effectively solubilized by nonionic detergent.
[0045] To further assess the specificity of the blotting assay,
neutrophil membrane extracts electrophoresed under reducing
conditions were probed with [125I] P-selectin in the presence or
absence of EDTA or anti-P-selectin mAbs. Neutrophil membrane
extracts (200 pg protein/lane) were electrophoresed on 7.5%
SDS-polyacrylamide gels under reducing conditions, transferred to
Immobilon membranes, and probed with [.sup.125I]P-selectin alone,
in the presence of 10 mM EDTA, or in the presence of 20 .mu.g/ml of
the anti-P selectin mAbs G1 or S12. [.sup.125I]P-selectin binding
to the major 120-kD and the minor 250-kD species was
Ca.sup.2+-dependent, a characteristic of all selectin-dependent
cellular interactions. Binding to both species was also blocked by
G1, a mAb to P-selectin that inhibits adhesion of myeloid cells to
P-selectin, but not by S12, a mAb to P-selectin that does not block
adhesion. Binding of [.sup.1251]P-selectin was also inhibited by a
100-fold excess of unlabeled P-selectin. The binding of
[.sup.125I]P-selectin to the dye front and to the 90,000 D protein
was not blocked by EDTA or G1, suggestion that these interactions
were nonspecific or used a specific Ca.sup.2+-independent
recognition mechanism.
[0046] Purification of P-selectin Ligand from Neutrophils
[0047] Neutrophils were disrupted and the membrane fraction
(FX.sub.2) isolated by fractionation of the cavitate as described
in Materials and Methods. The membrane fraction constituted
approximately 5-7% (n>10) of the protein in the cavitate. This
fractionation depleted both cytosolic proteins and azurophilic
granules (Table I). Proteins binding P-selectin were not detected
in the cytosolic fraction (FX.sub.1) with the blotting assay. The
final membrane pellet was solubilized with nonionic detergent and
applied to a WGA column which bound 4-5% of the protein in the
membrane extract. P-selectin blotting assays of reduced proteins
demonstrated that both the major 120,000 D and the minor 250,000 D
ligands bound quantitatively to WGA. However, the 90,000 D band and
the band at the dye front observed in the membrane extract were not
bound by WGA. After extensive dialysis, the WGA eluate was applied
to an Affigel-15.TM. precolumn in series with a P-selectin affinity
column. Approximately 2% of the protein in the WGA eluate bound to
the P-selectin column and could be eluted with EDTA. Both the
250,000 D and the 120,000 D ligands bound quantitatively to the
P-selectin column. Quantitative analysis of the protein recovered
from the P-selectin eluate indicated that the ligand(s) formed less
than 0.01% of the total protein in the neutrophil cavitate. Elution
of bound proteins from the P-selectin column with EDTA demonstrated
that the interaction of nondenatured neutrophil ligands with
P-selectin was also Ca.sup.2+-dependent. Neither species was eluted
from the Affigel-15 precolumn with EDTA.
[0048] A silver-stained SDS-polyacrylamide gel of proteins from the
various stages in the partial purification procedure was run under
reducing conditions. Samples from the indicated steps of the
isolation procedure were electrophoresed on 7.5% SDS-polyacrylamide
gels under reducing conditions, transferred to Immobilon membranes,
and probed with [.sup.125I]P-selectin. The amounts of protein
loaded onto the lanes were as follows: membrane extract and WGA
flow through, 200 .mu.g; WGA eluate and P-selecting flow through,
50 .mu.g; P-selectin eluate, 2 .mu.g. The same samples (10 .mu.g
protein/lane) were also analyzed by SDS-PAGE under the reducing
conditions followed by silver staining. The major silver-stained
band in the P-selectin eluate had an approximately 150,000 M.sub.r
which is similar to that of P-selectin itself. To determine whether
this protein represented P-selectin that had leached off the
P-selectin column, the P-selectin eluate was analyzed by SDS-PAGE
under both reducing and nonreducing conditions, followed by silver
staining, Western blotting with goat anti-P-selectin IgG, and
P-selectin blotting. The major silver-stained protein in the
P-selectin eluate was indeed P-selectin. Purified P-selectin
migrates with an approximately 120,000 M.sub.r under nonreducing
conditions; a minor component migrates with an approximately
250,000 M.sub.r. After reduction the protein migrates more slowly
with an approximately 150,000 M.sub.r. The two nonreduced bands and
the one reduced band detected by silver staining of the P-selectin
eluate co-migrated with purified P-selectin and were recognized by
anti-P-selectin IgG. The P-selectin ligand identified in the
blotting assay was not detected by silver staining and migrated
differently than P-selectin under both reducing and nonreducing
conditions. When the P-selectin eluate was electrophoresed without
reduction, P-selectin did not bind to proteins at the stacking gel
interface. Therefore, the P-selectin binding proteins at the
stacking gel interface, observed in extracts of neutrophil
membranes, were probably an artifact due to the relatively high
amount of protein loaded on the gel.
[0049] Characterization of the P-selectin Ligand
[0050] The ligand(s) on intact target cells requires sialic acids
to interact with P-selectin. To determine whether the ligand
detected by blotting of neutrophil membranes contained sialic acids
that were essential for recognition by P-selectin, neutrophil
membrane glycoproteins which bound to WGA were treated with
sialidase (200 mU/ml) for varying times before SDS-PAGE under
reducing conditions and then analyzed for their ability to bind
P-selectin. Neutrophil WGA eluate (50 .mu.g) was either
sham-treated or digested with 200 mU/ml of sialidase or with 20
U/ml of PNGaseF for 16 h, then electrophoresed on 7.5% SDS
polyacrylamide gels under reducing conditions, transferred to
Immobilon membranes, and probed with [.sup.125I]P-selectin.
[0051] Sialidase digestion for 30 min increased the apparent
molecular weight of the major 120,000 D ligand, a shift
characteristic of heavily sialylated glycoproteins. Longer
sialidase digestion did not further alter the electrophoretic
mobility of the ligand but did abolish its ability to bind
[.sup.125I]P-selectin. Sialidase treatment had a similar effect on
the minor 250 kD ligand.
[0052] These results demonstrate that the ligand(s) contains sialic
acid residues that are critical for recognition by P-selectin, but
suggest that only a portion of the sialic acid residues are
required for binding.
[0053] To examine whether the ligand contained N-linked glycans,
neutrophil membrane glycoproteins which bound to WGA were digested
with PNGaseF. This treatment did not affect [.sup.125I]P-selectin
binding but did decrease the apparent molecular weight of the
ligand, consistent with the enzymatic removal of one or two
N-linked glycan chains. This demonstrates that the ligand contains
at least one N-linked oligosaccharide chain that is not required
for P-selectin binding. Although one could not directly assess
whether N-linked glycans were quantitatively removed from the
ligand, conditions that normally cleave such glycans from most
proteins were used.
[0054] Prolonged treatment of neutrophil membrane extracts with
endo-.alpha.-N-acetylgalactosaminidase (O-glycanase) abolished
binding of [125I]P-selectin in the blotting assay, whereas sham
digestion was without effect. This was a surprising result, since
only nonsialylated Gal.beta.1-3GalNAc disaccharides O-linked to
serine or threonine residues are known substrates for the enzyme.
Assays using a synthetic sialidase substrate confirmed the presence
of a small amount of sialidase (0.01 mU/mU O-glycanase)
contaminating the O-glycanase. Although the level of activity was
small, it was stable to prolonged incubations under the conditions
recommended by the manufacturer for use of the O-glycanase
preparation. To prove that the contaminating sialidase was
responsible for the loss of P-selectin binding, the digestions were
repeated in the presence of a competitive sialidase inhibitor,
Neu2en5Ac. Under these conditions
endo-.alpha.-N-acetylgalactosaminidase digestion had no effect on
[125I] P-selectin binding to the ligand or the apparent molecular
weight of the ligand. Because the ligand requires sialic acid to
interact with P-selectin, the blotting assay could not be used to
assess the role of O-linked glycans in recognition by
P-selectin.
[0055] Isolation of a P-selectin Ligand from Metabolically Labeled
HL-60 Cells
[0056] P-selectin blotting of denatured membrane proteins from
myeloid cells may not detect molecules whose ability to bind
P-selectin is dependent on secondary and/or tertiary structure. As
an independent approach to identify ligands for P-selectin, HL-60
cells were metabolically labeled with [.sup.3H]glucosamine,
solubilized with nonionic detergent, and applied to a P-selectin
affinity column. After extensive washing, bound material was eluted
with EDTA and analyzed by SDS-PAGE followed by fluorography.
Samples were electrophoresed on 10%/o SDS polyacrylamide gels under
both nonreducing and reducing conditions and analyzed by
fluorography. Other samples were either sham treated or digested
with 1 U/ml of sialidase for 24 h or with 3.3. U/ml of PNGaseF for
24 h, and then electrophoresed on 10% SDS polyacrylamide gels under
reducing conditions and analyzed by fluorography.
[0057] A single metabolically labeled species was eluted, which
co-migrated under both nonreducing and reducing conditions with the
major species detected in neutrophil and HL-60 cell membranes by
blotting with [.sup.125I]P-selectin. Only 0.15-0.5 of the total
[.sup.3H]glucosamine-la- beled HL-60 glycoproteins bound to the
P-selectin column, indicating that the ligand is not abundant.
Sialidase treatment of the [.sup.3H]glucosamine-labeled P-selectin
ligand from HL-60 cells produced the same increase in apparent
molecular weight that was observed for the major neutrophil ligand
identified by the P-selectin blotting assay. In addition, PNGaseF
treatment caused the same decrease in the apparent molecular weight
of the HL-60 cell ligand that was observed for the neutrophil
ligand.
[0058] Comparison of the P-selectin Ligand with Known Neutrophil
Membrane Proteins
[0059] The properties of the major 120,000 D P-selectin ligand were
compared with those of three well-characterized neutrophil membrane
proteins with similar apparent molecular weight. The first two
molecules, lamp-1 and lamp-2, are abundant neutrophil proteins that
are predominantly localized in lysosomal membranes but are also
expressed in small amounts on the cell surface. These proteins have
a large number of complex N-linked glycan chains, many of which
carry the sialyl Le.sup.x tetrasaccharide. Polyclonal antisera (1:5
dilution) and mAbs (40 .mu.g/ml) to lamp-1 (CD3) and lamp-2 (BB6)
had no effect on binding of P-selectin to neutrophils as assessed
by flow cytometry.
[0060] Membrane extracts (200 .mu.g protein/lane) were
electrophoresed on 7.5% SDS-polyacrylamide gels under nonreducing
or reducing conditions, transferred to Immobilon membranes, and
probed with [.sup.125I] P-selectin or murine monoclonal antibodies
directed against human lamp-1 (CR3), human lamp-2 (BB6), human
L-selectin (DREG-200), or human leukosialin (Leu22). Western blot
analysis of neutrophil membranes with mAbs to lamp-1 and lamp-2
showed that the electrophoretic mobilities of these proteins under
nonreducing conditions were distinct from that of the P-selectin
ligand. In contrast to the P-selectin ligand, the electrophoretic
mobilities of lamp-1 and lamp-2 are not affected by sialidase
treatment. Although lamp-1 and lamp-2 from myeloid cells are rich
in lactosaminoglycans sensitive to endo-.alpha.-galactosidase,
treatment of intact neutrophils with the enzyme did not affect
binding of [.sup.1251]P-selectin. Pretreatment of crude neutrophil
membrane extracts or WGA column eluate with endo
.beta.-galactosidase (200 mU/ml, 1-2 h, 37.degree. C.) also did not
affect the apparent molecular weight of the ligand or its ability
to bind [.sup.125I]P-selectin. These data argue that lamp-1 and
lamp-2 are not ligands for P-selectin even though they carry many
sialyl Le.sup.x structures.
[0061] The third molecule whose apparent molecular weight is
similar to the 120,000 D P-selectin ligand is CD43 (leukosialin,
sialophorin), a heavily sialylated membrane protein present on
platelets and all leukocytes. It carries numerous O-linked sugar
chains and is differentially glycosylated by cells of various
hematopoietic lineages. Like the P-selectin ligand, treatment of
leukosialin with sialidase increases its apparent molecular weight.
However, in contrast to the P-selectin ligand, the electrophoretic
mobility of leukosialin was unaffected by reduction. Monospecific
polyclonal anti-human leukosialin antisera (1:5 dilution) did not
inhibit P-selectin binding to neutrophils as assessed by flow
cytometry. Furthermore, immunodepletion of leukosialin from
neutrophil membrane extracts did not deplete P-selectin ligand as
assessed by the blotting assay. Finally, leukosialin purified from
HL-60 cells did not bind P-selectin. Neutrophil WGA eluate (50
.mu.g) and leukosialin purified from HL-60 cells (0.5 .mu.g) were
electrophoresed under reducing conditions on 7.5%
SDS-polyacrylamide gels, transferred to Immobilon, and probed with
[.sup.125I]P-selectin. The same membrane was then probed with the
monoclonal anti-human leukosialin antibody Leu22.
[0062] Based on studies in which an antibody to L-selectin
(DREG-56) partially inhibited neutrophil adhesion to
P-selectin-transfected cells, it was suggested that L-selectin is
an important glycoprotein ligand on myeloid cells for P-selectin by
Picker et al., "The neutrophil selectin LECAM-1 presents
carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140"
Cell 66:921-933 (1991). Although L-selectin is present in membrane
extracts and WGA eluates of neutrophil membranes, as detected by
Western blotting, [125I] P-selectin did not bind to L-selectin in
the blotting assay. In addition, the anti-L-selectin mAb DREG-56
(100 .mu.g/ml) had no effect on the binding of purified P-selectin
to quiescent neutrophils as assessed by flow cytometry. Neutrophils
were preincubated for 15 min with buffer alone, 100 pg/ml of the
anti-L-selectin monoclonal antibody DREG-56, or 100 .mu.g/ml of the
anti-P-selectin mAb Gl before addition of buffer or P-selectin.
P-selectin binding was then detected by sequential incubation of
the cells with biotinylated S12 (a noninhibitory monoclonal
antibody to P-selectin) and phycoerythrin-streptavidin as described
in Materials and Methods.
[0063] Parallel control assays showed that the neutrophils
expressed high levels of L-selectin detactable by DREG-56. Binding
of the anti-L-selectin mAb DREG-56 to the neutrophils was assessed
by indirect immunofluorescence using a phycoerythrin-conjugated
anti-murine IgG.sub.1 antibody. Identical results were obtained
with the anti-L-selectin mAbs DREG-55 and DREG-200. Thus,
interactions with L-selectin do not appear to contribute to the
binding of fluid-phase P-selectin to intact neutrophils or to
immobilized proteins from neutrophil membrane extracts.
[0064] The following additional observations have been made
relating to the ligand.
[0065] Treatment of the .sup.3H-glucosamine-labeled P-selectin
ligand from HL-60 cells with neuraminidase releases approximately
30% of the radioactivity as sialic acid.
[0066] Strong acid hydrolysis of the .sup.3H-glucosamine-labeled
P-selectin ligand from HL-60 cells releases both
.sup.3H-glucosamine and .sup.3H-galactosamine in the approximate
ratio of 2:1, indicating that the ligand contains both
N-acetylglucosamine and N-acetylgalactosamine. In most
glycoproteins structurally defined to date, the glucosamine and
galactosamine occur as the acetylated derivatives.
[0067] The presence of N-acetylgalactosamine is indicative of the
presence of O-linked oligosaccharides (or Ser/Thr-linked
oligosaccharides) in which GalNAc is commonly found in O-glycosidic
.alpha.-linkage directly to amino acid. The indicated presence of
O-linked oligosaccharide is confirmed by the further observation
that the ligand binds quantitatively to the Jacalin-Sepharose, an
immobilized plant lectin that binds to the core disaccharide
sequence Ga1.beta.1-3GalNAc.alpha.-Ser/Thr in glycoproteins.
Jacalin-Sepharose can bind to O-linked oligosaccharides that have
modifications of this simple core. Thus, these results are not in
conflict with the lack of sensitivity of the ligand to O-glycanase.
When the .sup.3H-glucosamine-labeled P-selectin ligand from HL-60
cells is treated with mild base in the presence of sodium
borohydride (50 mM NaOH, 1 M NaBH.sub.4, 16 hr at 45.degree. C.) to
cause a beta-elimination reaction, approximately two-thirds of the
radioactivity is released as a moderately sized oligosaccharide
with more than four sugar residues, as defined by chromatography on
a column of BioGel P-10 in bicarbonate buffer. It is well known
from previous published studies that oligosaccharides in
O-glycosidic linkage, but not in N-glycosidic linkage, are
susceptible to release from peptide by this treatment. These
results further support the observation that the ligand contains a
considerable amount of O-linked oligosaccharides.
[0068] Treatment of the .sup.3H-glucosamine-labeled P-selectin
ligand from HL-60 cells with O-glycanase and neuraminidase does not
demonstrably affect the apparent mobility of the ligand any more
than neuraminidase alone. This indicates that the ligand does not
contain large amounts of the simple oligosaccharides in O-linkage
to Ser/Thr residues. O-glycanase is an endoglycosidase which
cleaves the sequence Gal.beta.1-3GalNAc.alpha- .-Ser/Thr in
glycoproteins to release the disaccharide Gal.beta.1-3GalNAc.
Treatment of the .sup.3H-glucosamine-labeled P-selectin ligand from
HL-60 cells with neuraminidase causes a decrease in the
electrophoretic mobility of the ligand.
[0069] The presence of numerous O-linked oligosaccharides on the
ligand is confirmed by the sensitivity of the
.sup.3H-glucosamine-labeled P-selectin ligand from HL-60 cells to
an enzyme termed O-glycoprotease. This enzyme is a protease which
recognizes and cleaves peptide bonds within glycoproteins that
contain numerous sialic acid-containing O-linked
oligosaccharides.
[0070] The ligand contains the sialyl Lewis x (SLe.sup.x) antigen
(NeuAca2-3Gal.beta.1-4 [Fuc.alpha.1-3]GlcNAc.beta.-R). When the
.sup.3H-glucosamine-labeled P-selectin ligand from HL-60 cells was
reapplied to a column of P-selectin-Affigel.TM.-15 it rebound. When
this chromatography was done in the presence of antibody to the
SLe.sup.x antigen (CSLEX1 monoclonal antibody (Fukushima, et al.,
Cancer Res. 44:5279-5285, 1984)), purchased from Dr. Paul Teraski,
University of California at Los Angeles, binding was more than 90%
reduced. In contrast, when a control experiment was done in which
the rechromatography occurred in the presence of antibody to the
Le.sup.x antigen (which lacks sialic acid), there was little if any
effect. The CSLEX1 anti-SLe.sup.x antibody bound to the ligand as
assessed by Western blotting.
[0071] The ligand contains poly-N-acetyllactosamine sequences of
the type [3Gal.beta.1-4GlcNAc .beta.].sub.n The
.sup.3H-glucosamine-labeled P-selectin ligand from HL-60 cells
quantitatively binds to a column of immobilized tomato lectin, a
plant lectin which has been shown to bind to
poly-N-acetyllactosamine sequences within glycoproteins.
[0072] The ligand from HL-60 cells contains fucose. When HL-60
cells are incubated with 6.sup.-3H-fucose, the P-selectin ligand is
radioactively labeled. All incorporated radioactivity is in fucose.
Furthermore, when the .sup.3H-fucose-labeled ligand is treated with
mild base and sodium borohydride to effect beta-elimination,
3H-fucose-labeled oligosaccharides are released that are both high
molecular weight and moderate molecular weight, as estimated by
chromatography on a column of BioGel P-10.
[0073] The differential mobility of the major ligand during
SDS-PAGE in the presence and absence of reducing agents suggests
that the native ligand is a disulfide-linked homodimer or that a
120,000 D subunit is disulfide linked to a distinct subunit that is
not directly involved in P-selectin binding. Since only a 120,000 D
band was detected after electrophoresis of reduced P-selectin
eluate from metabolically labeled HL-60 cells, a heterodimer would
have to consist of nonidentical subunits with the same apparent
molecular weight and which undergo the same change in
electrophoretic mobility after sialidase and PNGaseF digestion.
Alternatively, the 120-kD-labeled subunit would have to be
disulfide-linked to a subunit of similar apparent molecular weight
that is not labeled with [.sup.3H]glucosamine. A homodimeric ligand
with two equivalent binding sites might enhance the avidity of the
interaction with P-selectin. The ability of [.sup.125I]P-selectin
to bind to the ligand after reduction and denaturation with SDS
suggests that higher order structural features of the protein are
not critical for recognition.
[0074] The blotting assay also detected two minor ligands. The
first has an approximately 250,000 M.sub.r under reducing
conditions. Because its mobility is identical to that of the major
ligand under nonreducing conditions, it may represent a
subpopulation of the major ligand that is resistant to reduction.
The second has an approximately 160,000 M.sub.r under nonreducing
conditions. Binding of P-selectin to both minor ligands was
Ca.sup.2+ dependent and blocked by the mAb GI.
[0075] The isolation of a single glycoprotein from metabolically
labeled HL-60 cells suggests that P-selectin has a marked
preference for a particular ligand structure. L-selectin, which is
expressed on leukocytes and binds to sialylated structures on
endothelial cells, interacts preferentially with 50,000 D and
90,000 D sulfated, fucosylated glycoproteins from murine peripheral
lymph nodes (Imai, et al., J. Cell Biol. 113:1213-1222, 1991).
Thus, both P-selectin and L-selectin appear to interact with a
small subset of glycoprotein ligands.
[0076] It has been demonstrated that L-selectin on neutrophils
carries the sialyl Le.sup.x epitope and that a mAb to L-selectin
partially blocks neutrophil adhesion to cells transfected with
P-selectin cDNA (Picker, it al., Cell 66:921-933, 1991). Based on
these observations, it was proposed that L-selectin on neutrophils
is a predominant ligand for P-selectin. However, no direct
interaction of L-selectin with P-selectin was demonstrated. In the
present study, binding of P-selectin to L-selectin in neutrophil
membrane extracts was not detectable. Furthermore, the binding of
P-selectin to intact neutrophils is unaltered by antibodies to
L-selectin or by neutrophil activation that causes shedding of
L-selectin from the cell surface. Although it is conceivable that
L-selectin has weak affinity for P-selectin, the significance of
this potential interaction remains to be established.
[0077] A recombinant P-selectin IgG chimera was shown to bind to
myeloid cells and to a sulfatide, Gal(3-SO.sub.4) .beta.1-Ceramide
by Aruffo et al., "CD62/P-selectin recognition of myeloid and tumor
cell sulfatides" Cell 67:35-44 (1991). Sulfatide also inhibited
interaction of the chimera with monocytoid U937 cells, as reported
by Aruffo et al., (1991). It was not demonstrated whether binding
of the P-selectin chimera to the cells or to sulfatide was
Ca.sup.2+ dependent, a fundamental characteristic of
selectin-dependent cellular interactions. Protease digestion of
intact cells should increase the accessibility of P-selectin to
potential glycolipid ligands such as sulfatides. However, protease
treatment abolishes binding of P-selectin to neutrophils and HL-60
cells as well as adhesion of neutrophils to immobilized P-selectin.
In addition, although erythrocytes and platelets express
sulfatides, they do not specifically interact with P-selectin.
Thus, it seems unlikely that sulfatides are the principal mediators
of adhesion of myeloid cells to P-selectin. It remains to be
determined whether sulfatides inhibit binding of P-selectin to
myeloid cells by specific competition with a glycoprotein ligand or
by indirect effects. It is possible that the P-selectin ligand
described herein is sulfated or contains other structural features
that are mimicked by sulfatides.
[0078] Previous studies by Zhou et al., (1991); and Polley et al.,
(1991) have shown that P-selectin interacts with .alpha.(2-3)
sialylated, .alpha.(1-3) fucosylated lactosaminoglycans, of which
one is the sialyl Le.sup.x tetrasaccharide. However, several
observations suggest that the sialyl Le.sup.x tetrasaccharide per
se does not bind with high affinity to P-selectin. First, some
investigators (Moore et al., 1991; Aruffo et al., 1991; Polley, et
al.), but not all, have found that sialyl Le.sup.x inhibits
interactions of myeloid cells with P-selectin. Second, CHO cells
transfected with a fucosyltransferase express sialyl Le.sup.x yet
bind P-selectin with significantly lower affinity than do myeloid
cells (Zhou et al., 1991). Third, HT-29 cells, which also express
sialyl Le.sup.x, do not interact at all with P-selectin (Zhou et
al., 1991). Finally, several neutrophil membrane proteins known to
carry the sialyl Le.sup.x structure, are distinct from the major
glycoprotein ligand identified herein and do not bind P-selectin in
the assays described here. These observations suggest that the
ligand contains structural features in addition to the sialyl
Le.sup.x tetrasaccharide that enhance the affinity and/or
specificity of its interaction with P-selectin.
[0079] A blotting assay of neutrophil and HL-60 cell membrane
extracts was used to search for ligands for P-selectin. As
described previously in U.S. Ser. No. 07/650,484,
[.sup.125I]P-selectin bound preferentially to a glycoprotein of
M.sub.r 120,000 as assessed by SDS-PAGE under reducing conditions.
Under nonreducing conditions, the ligand for P-selectin had an
apparent Mr of 250,000, suggesting that it is a disulfide-linked
homodimer. In initial studies, the ligand was partially purified by
serial affinity chromatography on wheat germ agglutinin (WGA) and
P-selectin affinity columns. Proteins bound to the P-selectin
column were eluted with EDTA. The glycoprotein ligand was greatly
enriched in the EDTA eluate from the P-selectin column, as assessed
by the intensity of the band identified by [.sup.1251]P-selectin
blotting. As noted in U.S. Ser. No. 07/650,484, however, the ligand
stained poorly with silver, consistent with its being an unusually
heavily glycosylated protein. In the initial purifications, the
only contaminating protein present noted by silver staining of the
gel was a small amount of P-selectin itself which had been leached
from the affinity column. Using a new P-selectin affinity column
and more extensive washing procedures documented in the methods,
the ligand has now been isolated free from contaminants. This
conclusion is based on observation that there are no silver
staining bands present but the ligand is clearly identified by its
ability to interact with [.sup.125I]P-selectin in the blotting
assay.
[0080] As described in U.S. Ser. No. 07/650,484, partial removal of
sialic acids with sialidase slowed the mobility of the ligand, a
feature characteristic of heavily sialylated glycoproteins.
Extensive sialidase digestion abolished recognition of the ligand
by P-selectin. It has now been demonstrated that the ligand contain
both N- and O-linked oligosaccharides.
[0081] A form of the ligand in which the carbohydrate components
are radiolabeled has also been purified by P-selectin affinity
chromatography, as described above. SDS-PAGE analysis of the
P-selectin column eluate, followed by fluorography, indicates that
the only labeled protein has an Mr of 250,000 under nonreducing
conditions and 120,000 under reducing conditions. The radiolabeled
ligand has the same shifts in electrophoretic mobility following
treatment with sialidase or PNGase F. Thus, all the features of the
radiolabeled ligand correspond to those of the ligand identified by
the P-selectin blotting assay. Because only a single radiolabeled
species is isolated from the P-selectin affinity column, the
carbohydrate structures of the ligand can be analyzed in detail by
procedures that have been developed, for example, as reported by R.
D. Cummings and S. Kornfeld, J. Biol. Chem 257:11235-11240 (1982)
and R. D. Cummings, et al., J. Biol. Chem. 258: 15261-15273
(1983).
[0082] In summary, the glycoprotein ligand for P-selectin from
myeloid cells has the characteristics of a disulfide-linked
homodimer with each subunit having an apparent Mr of 120,000 as
assessed by SDS-PAGE. The protein has some N-linked carbohydrate
but its most striking feature is the presence of a large number of
clustered O-linked glycans, most of which appear to be larger than
the usual simple O-linked chains cleaved by O-glycanase. Although
the ligand contains the sLe.sup.x structure, the data indicate that
additional structural features in the ligand are required to confer
high affinity binding to P-selectin. These features may include,
but are not limited to, carbohydrate structures of more complexity
than sLe.sup.x itself, clustering of many glycan chains to increase
avidity, and specific orientations of the glycans relative to the
protein backbone.
[0083] Preparation of Diagnostic and Therapeutic Agents Derived
from the Protein or Carbohydrate Components of the Glycoprotein
Ligand for P-selectin.
[0084] The glycoprotein ligand for P-selectin described above has a
variety of applications as diagnostic reagents and, potentially, in
the treatment of numerous inflammatory and thrombotic
disorders.
[0085] Diagnostic Reagents.
[0086] Antibodies to the ligand can be used for the detection of
human disorders in which P-selectin ligands might be defective.
Such disorders would most likely be seen in patients with increased
susceptibility to infections in which leukocytes might not be able
to bind to activated platelets or endothelium. Cells to be tested,
usually leukocytes, are collected by standard medically approved
techniques and screened. Detection systems include ELISA
procedures, binding of radiolabeled antibody to immobilized
activated cells, flow cytometry, immunoperoxidase or immunogold
analysis, or other methods known to those skilled in the arts.
[0087] Antibodies directed specifically to protein or carbohydrate
components of the ligand can be use to distinguish defects in
expression of the core protein or in glycosyltransferases and/or
modifying enzymes that construct the proper oligosaccharide chains
on the protein. The antibodies can also be used to screen cells and
tissues other than leukocytes for expression of the protein or
carbohydrate components of the ligand for P-selectin.
[0088] Complementary DNA clones encoding the protein component of
the ligand can be isolated and sequenced. These probes can be used
as diagnostic reagents to examine expression of RNA transcripts for
the ligand in leukocytes and other tissues by standard procedures
such as Northern blotting of RNA isolated from cells and in situ
hybridization of tissue sections.
[0089] A similar approach can be used to determine qualitative or
quantitative disorders of P-selectin itself. The glycoprotein
ligand, carbohydrates, or appropriate derivatives thereof, is
labeled and tested for its ability to bind to P-selectin on
activated platelets from patients with disorders in which
P-selectin might be defective.
[0090] The ligand, or components thereof, and also be used in
assays of P-selectin binding to screen for compounds that block
interactions of P-selectin with the ligand.
[0091] Clinical Applications.
[0092] Since P-selectin has several functions related to leukocyte
adherence, inflammation, tumor metastases, and coagulation,
clinically, compounds which interfere with binding of P-selectin
and/or the other selecting, including E-selectin and L-selectin,
such as the carbohydrates, can be used to modulate these responses.
These compounds include the P-selectin ligand, antibodies to the
ligand, and fragments thereof. For example, the glycoprotein
ligand, or components thereof, particularly the carbohydrate
moieties, can be use to inhibit leukocyte adhesion by competitively
binding to P-selectin expressed on the surface of activated
platelets or endothelial cells. Similarly, antibodies to the ligand
can be used to block cell adhesion mediated by P-selectin by
competively binding to the P-selectin ligand on leukocytes or other
cells. These therapies are useful in acute situations where
effective, but transient, inhibition of leukocyte-mediated
inflammation is desirable. In addition, treatment of chronic
disorders may be attained by sustained administration of agents,
for example, by subcutaneous or oral administration.
[0093] An inflammatory response may cause damage to the host if
unchecked, because leukocytes release many toxic molecules that can
damage normal tissues. These molecules include proteolytic enzymes
and free radicals. Examples of pathological situations in which
leukocytes can cause tissue damage include injury from ischemia and
reperfusion, bacterial sepsis and disseminated intravascular
coagulation, adult respiratory distress syndrome, tumor metastasis,
rheumatoid arthritis and atherosclerosis.
[0094] Reperfusion injury is a major problem in clinical
cardiology. Therapeutic agents that reduce leukocyte adherence in
ischemic myocardium can significantly enhance the therapeutic
efficacy of thrombolytic agents. Thrombolytic therapy with agents
such as tissue plasminogen activator or streptokinase can relieve
coronary artery obstruction in many patients with severe myocardial
ischemia prior to irreversible myocardial cell death. However, many
such patients still suffer myocardial neurosis despite restoration
of blood flow. This "reperfusion injury" is known to be associated
with adherence of leukocytes to vascular endothelium in the
ischemic zone, presumably in part because of activation of
platelets and endothelium by thrombin and cytokines that makes them
adhesive for leukocytes (Romson et al., Circulation 67: 1016-1023,
1983). These adherent leukocytes can migrate through the
endothelium and destroy ischemic myocardium just as it is being
rescued by restoration of blood flow.
[0095] There are a number of other common clinical disorders in
which ischemia and reperfusion results in organ injury mediated by
adherence of leukocytes to vascular surfaces, including strokes;
mesenteric and peripheral vascular disease; organ transplantation;
and circulatory shock (in this case many organs might be damaged
following restoration of blood flow).
[0096] Bacterial sepsis and disseminated intravascular coagulation
often exist concurrently in critically ill patients. They are
associated with generation of thrombin, cytokines, and other
inflammatory mediators, activation of platelets and endothelium,
and adherence of leukocytes and aggregation of platelets throughout
the vascular system. Leukocyte-dependent organ damage is an
important feature of these conditions.
[0097] Adult respiratory distress syndrome is a devastating
pulmonary disorder occurring in patients with sepsis or following
trauma, which is associated with widespread adherence and
aggregation of leukocytes in the pulmonary circulation. This leads
to extravasation of large amounts of plasma into the lungs and
destruction of lung tissue, both mediated in large part by
leukocyte products.
[0098] Two related pulmonary disorders that are often fatal are in
immunosuppressed patients undergoing allogeneic bone marrow
transplantation and in cancer patients suffering from complications
that arise from generalized vascular leakage resulting from
treatment with interleukin-2 treated LAK cells
(lymphokine-activated lymphocytes). LAK cells are known to adhere
to vascular walls and release products that are presumably toxic to
endothelium. Although the mechanism by which LAK cells adhere to
endothelium is not known, such cells could potentially release
molecules that activate endothelium and then bind to endothelium by
mechanisms similar to those operative in neutrophils.
[0099] Tumor cells from many malignancies (including carcinomas,
lymphomas, and sarcomas) can metastasize to distant sites through
the vasculature. The mechanisms for adhesion of tumor cells to
endothelium and their subsequent migration are not well understood,
but may be similar to those of leukocytes in at least some cases.
Specifically, certain carcinoma cells have been demonstrated to
bind to both E-selectin, as reported by Rice and Bevilacqua.
Science 246:1303-1306 (1991), and P-selectin, as reported by
Aruffo, et al., Proc. Natl. Acad. Sci. USA 89:2292-2296 (1992). The
association of platelets with metastasizing tumor cells has been
well described, suggestion a role for platelets in the spread of
some cancers. Since P-selectin is expressed on activated platelets,
it is believed to be involved in association of platelets with at
least some malignant tumors.
[0100] Platelet-leukocyte interactions are believed to be important
in atherosclerosis. Platelets might have a role in recruitment of
monocytes into atherosclerotic plaques; the accumulation of
monocytes is known to be one of the earliest detectable events
during atherogenesis. Rupture of a fully developed plaque may not
only lead to platelet deposition and activation and the promotion
of thrombus formation, but also the early recruitment of
neutrophils to an area of ischemia.
[0101] Another area of potential application is in the treatment of
rheumatoid arthritis.
[0102] In these clinical applications, the glycoprotein ligand, or
fragments thereof, can be administered to block selectin-dependent
interactions by binding competitively to P-selectin expressed on
activated cells. In particular, carbohydrate components of the
ligand, which play a key role in recognition by P-selectin, can be
administered. Similarly, natural or synthetic analogs of the ligand
or its fragments which bind to P-selectin can also be administered.
In addition, antibodies to the protein and/or carbohydrate
components of the ligand, or fragments thereof, can be
administered. The antibodies are preferably of human origin or
modified to delete those portions most likely to cause an
immunogenic reaction. Carbohydrate components of the ligand or the
antibodies, in an appropriate pharmaceutical carrier, are
preferably administered intravenously where immediate relief is
required. The carbohydrate(s) can also be administered
intramuscularly, intraperitoneally, subcutaneously, orally, as the
carbohydrate, conjugated to a carrier molecule, or in a drug
delivery device. The carbohydrate can be modified chemically to
increase its in vivo half-life.
[0103] The carbohydrate can be isolated from cells expressing the
carbohydrate, either naturally or as a result of genetic
engineering as described in the transfected COS cell examples, or,
preferably, by synthetic means. These methods are known to those
skilled in the art. In addition, a large number of
glycosyltransferases have been cloned (J. C. Paulson and K. J.
Colley, J. Biol. Chem. 264:17615-17618, 1989). Accordingly, workers
skilled in the art can use a combination of synthetic chemistry and
enzymatic synthesis to make pharmaceuticals or diagnostic
reagents.
[0104] Protein fragments of the ligand can also be administered as
a pharmaceutically acceptable acid- or base- addition salt, formed
by reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamies.
[0105] Carbohydrates that are biologically active are those which
inhibit binding of leukocytes to P-selectin. Suitable
pharmaceutical vehicles for administration to a patient are known
to those skilled in the art. For parenteral administration, the
carbohydrate will usually be dissolved or suspended in sterile
water or saline. For enteral administration, the carbohydrate will
be incorporated into an inert carrier in tablet, liquid, or
capsular form. Suitable carriers may be starches or sugars and
include lubricants, flavorings, binders, and other materials of the
same nature. The carbohydrate can also be administered locally at a
wound or inflammatory site by topical application of a solution or
cream.
[0106] Alternatively, the carbohydrate may be administered in, on
or as part of, liposomes or microspheres (or microparticles).
Methods for preparing liposomes and microspheres for administration
to a patient are known to those skilled in the art. U.S. Pat. No.
4,789,734 describe methods for encapsulating biological materials
in liposomes. Essentially, the material is dissolved in an aqueous
solution, the appropriate phospholipids and lipids added, along
with surfactants if required, and the material dialyzed or
sonicated, as necessary. A good review of known methods is by G.
Gregoriadis, Chapter 14. "Liposomes", Drug Carriers in Biology and
Medicine pp. 287-341 (Academic Press, 1979). Microspheres formed of
polymers or proteins are well known to those skilled in the art,
and can be tailored for passage through the gastrointestinal tract
directly into the bloodstream. Alternatively, the carbohydrate can
be incorporated and the microspheres, or composite of microspheres,
implanted for slow release over a period of time, ranging from days
to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673,
and 3,625,214.
[0107] The carbohydrates should be active when administered
parenterally in amounts above about 1 .mu.g/kg of body weight. For
treatment of most inflammatory disorders, the dosage range will be
between 0.1 to 30 mg/kg of body weight. A dosage of 70 mg/kg may be
required for some of the carbohydrates characterized in the
examples.
[0108] The criteria for assessing response to therapeutic
modalities employing antibodies or carbohydrate is dictated by the
specific condition and will generally follow standard medical
practices. For example, the criteria for the effective dosage to
prevent extension of myocardial infarction would be determined by
one skilled in the art by looking at marker enzymes of myocardial
necrosis in the plasma, by monitoring the electrocardiogram, vital
signs, and clinical response. For treatment of acute respiratory
distress syndrome, one would examine improvements in arterial
oxygen, resolution of pulmonary infiltrates, and clinical
improvement as measured by lessened dyspnea and tachypnea. For
treatment of patients in shock (low blood pressure), the effective
dosage would be based on the clinical response and specific
measurements of function of vital organs such as the liver and
kidney following restoration of blood pressure. Neurologic function
would be monitored in patients with stroke. Specific tests are used
to monitor the functioning of transplanted organs; for example,
serum creatinine, urine flow, and serum electrolytes in patients
undergoing kidney transplantation.
[0109] Modifications and variations of the present invention,
methods for modulating binding reactions involving P-selectin using
carbohydrate derived from or forming a portion of the P-selectin
ligand, or antibodies to the ligand, will be obvious to those
skilled in the art from the foregoing detailed description. Such
modifications and variations are intended to come within the scope
of the appended claims.
* * * * *