Regulation of leukocyte L-selectin expression
by Aiyappa Muthanna Palecanda
A thesis submitted am partial fulfillment off the requirements for the degree of Doctor off Philosophy
in Veterinary Science
Montana State University
© Copyright by Aiyappa Muthanna Palecanda (1993)
Abstract:
Neutrophil emigration into sites of inflammation and the nonrandom recirculation of lymphocytes are
essential immunological phenomena controlled, in part, by specific receptor interactions between the
leukocyte and vascular endothelium. L-selectin is an adhesion molecule expressed on all leukocytes
and is required for the initial interaction of these cells with the endothelium prior to extravasation.
Activation of leukocytes results in rapid shedding of L-selectin from the cell surface, which has been
proposed to be a mechanism of release from the endothelium prior to emigration into sites of
inflammation. Here a novel pathway leading to the shedding of L-selectin is described. I show that
chemical crosslinking agents induce a rapid activation-independent shedding of leukocyte L-selectin.
Specific crosslinking of L-selectin with monoclonal antibodies or treatment of leukocytes with the
polysaccharide fucoidin also induce downregulation of L-selectin, which cannot be explained by
activation alone. Importantly, L-selectin ligand interactions in vivo induce L-selectin downregulation.
An irreversible inhibitor of serine proteases completely blocks both crosslinking- and
activation-induced downregulation of L-selectin. Therefore, a similar serine protease is likely involved
in both pathways leading to L-selectin shedding. Based on these observations, a new model for the
regulation of leukocyte/endothelial interactions is presented.
R EG U LA T IO N OF LEUKOCYTE L-SELECTIN EX PRESSIO N
by
A iyappa MelBsamma Palecamda
A thesis sebm itted am p artial fulfillimemt
off the reqeirem em ts ffor th e degree
off
D octor off Philosophy
Em
V eterinary Science
M ONTANA STA TE UNIVERSITY
Bozemtam, 'M ontana
D ecem ber 1993
I
REGULATION OF LEUKOCYTE L-SELECTIN EXPRESSION
Aiyappa Muthanria PaIecanda
Advisor: M ark A. India, Ph.D.
Montana State University
1993
Abstract
Neutrophil emigration into sites o f inflammation and the nonrandom recirculation o f
lym phocytes are essential immunological phenom ena controlled, in part, by specific
receptor interactions between the leukocyte and vascular endothelium. L selectin is an
adhesion molecule expressed on all leukocytes and is required for the initial interaction o f
these cells with the endothelium prior to extravasation. Activation o f leukocytes results in
rapid shedding o f L-selectin from the cell surface, which has been proposed to be a
mechanism o f release from the endothelium prior to emigration into sites o f inflammation.
H ere a novel pathway leading to the shedding o f L-selectin is described. I show that
chemical crosslinking agents induce a rapid activation-independent shedding o f leukocyte
L-selectin. Specific crosslinking of L-selectin with monoclonal antibodies or treatment of
leukocytes w ith the polysaccharide fucoidin also induce downregulation of L-selectin,
which cannot be explained by activation alone. Importantly, L-selectin ligand interactions
in vivo induce L-selectin downregulation. A n irreversible inhibitor o f serine proteases
completely blocks both crosslinldng- and activation-induced dowmegulation o f L-selectin.
Therefore, a similar serine protease is likely involved in both pathways leading to L-selectin
shedding. B ased on these observations, a new m odel fo r the regulation o f
leukocyte/endothelial interactions is presented.
f
ii
APPRO V A L
of a thesis submitted by
Aiyappa Muthanna Palecanda
This thesis has been read by each member of the thesis committee and has been found
to be satisfactory regarding content, English usage, format, citations, bibliographic style,
and consistency, and is ready for submission to the College of Graduate Studies.
Date
ChairpersoK Graduate Committee
Approved for the Major Department
Date
Head, Major Department
Approved for the College o f Graduate Studies
Date
Graduate Dean
Mi
STA TEM EN T O F PE R M ISSIO N TO USE
In presenting this thesis in partial fulfillment o f the requirements for a doctoral degree at
M ontana State University, I agree that the Library shall make it available to borrowers
under rules o f the Library. I further agree that copying o f this thesis is allowable only for
scholarly purposes, consistent with "fair use" as prescribed in the U.S. Copyright Law.
Requests for extensive copying or reproduction o f this thesis should be referred to
University Microfilms International, 300 North Zeeb Road, Ann Arbor, Michigan 48106,
to whom I have granted "the exclusive right to reproduce and distribute my dissertation for
sale in and from microform or electronic format, along with the right to reproduce and
distribute my abstract in any format in whole or in part".
Signature
Date.
Sv
I dedicate this thesis to my parents, the late Mrs. Gangamma Muthanna and Mr. Palecanda
Poovaiah Muthanna, and to my brother Palecanda Shyam.
V
' A CK N O W LED G M EN TS
I would like to thank the members o f my doctoral committee. Dr. Brace L. Granger,
Dr. A l Jesaitis, Dr. Mark A. Jutila, Dr. Norman D. Reed, and Dr. C. A. Speer for their
helpful suggestions and recommendations. I particularly want to thank my advisor and
committee chairman Dr. Mark A. Jutila, who provided constructive criticism, guidance,
and encouragement on a continual basis. I also wish to express my sincere gratitude to Dr.
Norman D. Reed, for his faith in me during the earliest part of my doctoral program and for
his continued help and guidance throughout my doctoral program.
Special thanks to Davin Jutila, Kathy Jutila, Gayle W atts, Sandy Kurk, and Ginger
Perry, for their friendship and excellent technical assistance. I would also like to thank two
of my colleagues. Brace W alcheck and Rob Bar gat ze, for a pleasant and congenial work
atmosphere. My thanks to Dana Hoover for editing and correction o f manuscripts and the
dissertation.
A special appreciation to the departmental secretaries, Joan, Linda and Bert for their
help in getting through all the necessary paperwork. I would like to thank Gayle Callis and
Andy Blixt for their assistance in tissue sectioning and photography.
I wish to acknowledge the special support and encouragement I received from my
brothers, Shyam and Aran, and my sisters, Gita, Jyothi, Bollacca, Mallika and Swaroop.
A very special thanks to my wife, Lakshmi, for her understanding and encouragement
vi
TA BLE O F CO NTEN TS
PA G E
L IS T
OF
T A B L E S...... ........ ................................................. ........... x
L IS T
OF
F IG U R E S.......................................................................... »
A B S T R A C T .............................................................
1.
xiii
I N T R O D U C T I O N .......... ........................................................................ I
Id en tificatio n o f L -se le ctin ...................................................................... 2
S tru ctu re o f L -se le c tin ............................................................................... 5
F u n ctio n
o f L -se le c tin ............................... ..............................................7
R egulation o f L -selectin .......................................................................... 11
My
H y p o th e sis.............................................................................................. 13
R e f e r e n c e s .........................................................................................................14
2 . A C T IV A T IO N IN D EPEN D EN T SH ED D IN G O F
L E U K O C Y T E L -S E L E C T IN IS IN D U C IB LE BY
C RO SSLIN K IN G
A G E N T S .........................................
24
I n t r o d u c t i o n ................................................................................................. •••24
M a te r ia ls
and
M e th o d s .............................................................................. 26
A n tib o d ie s .........................................................................................................26
Preparation of leukocyte suspensions and flow cytometric analysis.........26
C rosslinking o f leukocyte L -selectin.......................................................27
A ntibody crosslinking o f L -selectin ...................................................... 28
W estern blot SD S-PA G E analysis.......................................................... 28
E L IS A
a n a ly s is ........................................
29
R e s u l t s .................................................................................................................. 29
Activation-independent down-regulation o f leukocyte L-selectin
can be induced by chemical crosslinking agents.....................................29
Treatment o f human leukocytes with BS^ causes shedding
of
L -s e le c tin ...................................................................................................32
Crosslinking of L-selectin with specific monoclonal antibodies
causes loss o f surface expression o f L-selectin...................................... 34
Activation-independent shedding o f L-selectin occurs in vivo............... 37
D i s c u s s i o n .......................................................................................................... 39.
R e f e r e n c e s ................................................
3.
42
L IG A N D IN T E R A C T IO N S IN VIVO AND
A N T IB O D Y C R O S S L IN K IN G O R FU C O ID IN T R E A T M E N T
IN VITRO LEA D S T O D O W N R E G U L A T IO N O F
L-SELEC TIN
E X P R E S S I O N ..............................................................46
I n t r o d u c t i o n ..................................................................................................... 46
M a te r ia ls
and
M e th o d s .............................................................................. 49
A ntibodies and p olysaccharid es............................................................... 49
P reparation o f cell suspension......................
Flow
49
cytom etric a n aly sis.......................................................................... 49
C h em ical
c ro s slin k in g ................................................................................ 50
A ntibody crosslinking o f L -selectin ........................................................50
Im m unofluorescence m icrosco p y.............................................................51
Crosslinking o f L-selectin with fucoidin and other polysaccharides.........51
In vivo trafficking o f L-selectin transfected L l/2 cells......................... 52
viM
M e s u E t s .................................................. ............... ..........................
52
Crosslinking o f primary anti-L-selectin mAbs is required to
induce dow nregulation o f neutrophil L-selectin....................................52
Treatment o f neutrophils or lymphocytes with the sulfated
polysaccharide fucoidin can also induce downregulation o f L-selectin......58
Crosslinker-induced shedding is a property o f L- but not E-selectin........61
In vivo trafficking of L l/2 cells results in a loss in the surface
ex pression o f L -se le c tin ........................................................................... 62
D a s c u s s i o r a ................. ............ .............................. ............................................ 67
R e f e r e n c e s ................. ........................................................................................71
4
R O L E OF A D IIS O P R O P Y L F L U O R O P H O S P H A T E (D FP)
IN H E R IT A B L E , A C T IV A T IO N -D E P E N D E N T ,
MEMBRANE-ASSOCIATED, P U T A T IV E P R O T E A S E
IN THE SHEDDING O F LEUKOCYTE L -S E L E C T IN .......... 78
I r a t r o d u c t i o m ...................................................................... ............................. 78
M aterials
and
M e th o d s ............................................................................. 80
A n t i b o d i e s ......................................................................... ;............................ 80
Preparation o f leukocyte suspensions and flow cytometric analysis........ 80
L e u k o c y te
a c tiv a tio n .................................................................................. 81
C h em ical
c ro s slin k in g ............................................................................... 81
P rotease in h ib ito r treatm en t.....................................................................81
Labeling of activation induced [^H] DiisopropylFluorophosphate (^H -D FP) binding proteins........................................ 82
Sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) and autoradiography........................................83
R e s u l t s .................... ........................................................................................... 83
Activation-induced L-selectin shedding is completely inhibited by DFP.... 83
Crosslinking-induced L-selectin shedding is also completely
inhibited
by
D F P ........................................................................................85
Upregulation of surface markers for cellular activation are not
a ffected
by
D F P ................................................................................. ........87
DFP inhibits a protease which is exposed/accessible only upon
cellular activation o r chem ical crosslinking....... .............................. 88
Cellular activation exposes previously unexpressed or hidden DFP
binding m em brane-associated p ro tein s...................................................91
D Ss cM Ss iom .............................. ...... ....................................... ............................ 93
R e f e r e n c e s ............................................. .............................. ............................96
5 . - C O N C L U S I O N S ...................... ........ .......................... ........................100
M o d e l . . . ......................................................................... ......... .......................... 104
L IST O F TABLES
TABLE
PAGE
1. Chemical crosslinking o f mouse and human leukocyte surface
proteins causes loss o f L-selectin expression..................................... 31
2. Crosslinked lysine residues must be greater than 6 Angstroms
apart to induce L-selectin dow n-regulation..........................................34
3. Cross-linking with specific mAh at 37°C causes loss of surface
expression o f leukocyte homing receptor L-selectin............................. 36
4. Effect o f primary antibodies crosslinked with whole molecule
second stage antibody bn the surface expression o f L-selectin
an d M a c -1........ ............ ................................... ................. ........................ 55
5. Exposure of neutrophils and lymphocytes to soluble fucoidin
induces L-selectin down regulation at 3 7 °C ............ ........................... 59
6 . Polysaccharides that do not affect surface expression o f L-selectin........60
7. Treatment o f the L l/2 L-selectin transfectants with anti-L-selectin
antibodies plus second stage or fucoidin induces down-regulation
at 3 7 ° C........ '................................................................................................. 64
LIST OF FIG U R ES
FIGURE
PAGE
1. Chemical cross-linking o f mouse and human leukocytes causes
L -se le ctin
d o w n -reg u latio n ......................................................................33
2. Chemical crosslinking of human leukocytes caused L-selectin
shedding from the cell surface.............................................................35
3 . The human leukocyte homing receptor L-selectin is downregulated
upon specific cross-linking at 3 7 °C ................................................... 37
4. Shed L-selectin can be detected in the plasma o f healthy adults............ 38
5 . Primary and-L-selectin antibodies alone do not induce L-selectin
d o w n -re g u la tio n ........................ ............................................................. 54
6 . Primary and-L-selectin antibodies plus second stage induce
dow nregulation o f L-selectin at 3 7 °C .................................................. 56
7. Primary and-L-selectin antibodies, followed by whole molecule,
but not Fab’, second stage, induces L-selectin down-regulation
at 3 7 ° C ...........................................................................................-.............. 57
8 . The human leukocyte homing receptor L-selectin aggregates to
form patches upon specific antibody crosslinking............................... 58
9. Immobilized fucoidin induces downregulation o f L-selectin expression.. 61
10. Chemical crosslinking causes L-selectin, but not E-selectin,
d o w n r e g u l a t i o n .........................................................................................63
11. L-selectin L l/2 transfectants, injected i.v. into mice and allowed
to recirculate, rapidly downregulate L-selectin expression................... 65
12. Anti-L-selectin antibodies block downregulation of L-selectin
on L-selectin transfectants allowed to recirculate in vivo.................... 66
13. DFP, but not other protease inhibitors, block L-selectin shedding
in response to PMA or FMLP treatment o f human leukocytes...............84
14. DFP, but not other serine protease inhibitors, block L-selectin
shedding in response to BS^ crosslinking........................................... 86
15. DFP has no effect on the activation-induced upregulation o f
certain surface markers for cellular activation..................................... 88
kM
16. Presence o f DFP is required during crosslinking or activation
to block L-selectin shedding on human neutrophils............................. 89
17 . Presence o f DFP is required during activation or crosslinking
to block L-selectin shedding on mouse bone-marrow neutrophils......... 90
18. Activation exposes membrane associated DFP binding proteins............ 92
19. Activation exposes membrane-associated 90 MD DFP binding
proteins on both lymphocytes and neutrophils......................... ........... 93
ABSTRACT
Neutrophil emigration into sites o f inflammation and the nonrandom recirculation o f
lymphocytes are essential immunological phenom ena controlled, in part, by specific
receptor interactions between the leukocyte and vascular endothelium. L-selecdn is an
adhesion molecule expressed on all leukocytes and is required for the initial interaction o f
these cells with the endothelium prior to extravasation. Activation o f leukocytes results in
rapid shedding o f L-selectin from the cell surface, which has been proposed to be a
mechanism of release from the endothelium prior to emigration into sites o f inflammation.
Here a novel pathway leading to the shedding o f L-selectin is described. I show that
chemical crosslinking agents induce a rapid activation-independent shedding o f leukocyte
L-selectin. Specific crosslinking o f L-selectin with monoclonal antibodies or treatment o f
leukocytes with the polysaccharide fucoidin also induce downregulation o f L-selectin,
which cannot be explained by activation alone: Importantly, L-selectin ligand interactions
in vivo induce L-selectin downregulation. An irreversible inhibitor o f serine proteases
completely blocks both crosslinking- and activation-induced downregulation of L-selectin.
Therefore, a similar serine protease is likely involved in both pathways leading to L-selectin
shedding. B ased on these observations, a new m odel fo r the regulation o f
leukocyte/endothelial interactions is presented.
I
C H A PTE R I
IN TRO D U C TIO N
Lymphocyte extravasation into secondary lymphoid tissues and neutrophil recruitment
into sites o f inflammation are essential immunological phenomena exemplifying immune
surveillance and protective immune responses to injury. The first step in leukocyte
extravasation is adherence to the vascular endothelium. This critical step is mediated by Lselectin on the leukocyte and its ligands, either constitutively expressed on the peripherallym ph-node high endothelial venules (PLN-HEV) or on venules in response to
inflammatory stimuli. The expression of L-selecdn and its ligand needs to be stringently
regulated to initiate the leukocyte/endothelial interaction only at sites requiring leukocyte
extravasation, such as sites o f inflammation and secondary lymphoid organs. L-selecdn is
constitutively expressed on all leukocytes and is rapidly shed from the leukocyte surface
upon activation with inflammatory chemotactic agents, such C5a, which attract cells within
the vascular bed to the underlying inflamed tissue. Activation-induced L-selectin shedding
has been suggested as a requirement for the release o f leukocytes from the endothelium in
order for these cells to continue with migration. My hypothesis is that if L-selectin
shedding is required for leukocyte extravasation, then an activation-independent mechanism
o f L-selectin shedding may be occurring at sites o f leukocyte extravasation in the absence
o f inflammation, such as the normal migration of lymphocytes into secondary lymphoid
2
organs in the periphery. My dissertation has focused on the effects of receptor crosslinking
and in vivo trafficking on L-selectin expression, and the study o f mechanisms involved in
the activation and activation-independent downregulation o f leukocyte L-selectin. Each
chapter in my dissertation deals with experiments performed to decipher the various aspects
o f L-selectin regulation, beginning with an introduction pertaining to the data presented in
that chapter and closing with a discussion and a list o f cited references. The final chapter
will assimilate all key findings. This introduction gives an overview o f L-selectin and ends
with the presentation of my hypothesis.
Identification of L-selectin
The immune system has to keep constant surveillance of the various microenvironments
in the body to detect antigen insult. Lymphocytes which are capable of detecting and
responding to diverse antigens Were shown by in vivo experiments to recirculate from
blood to secondary lymphoid organs and back to blood via the thoracic duct (1,2). The
extravasation o f lymphocytes from blood is nonrandom and occurs at specialized high
endothelial venules (HEV) in secondary lymphoid organs (reviewed in 3-9). There are
subsets o f lymphocytes that only bind HEV in certain tissues, and this specificity is
maintained across species barriers ( 10, 11).
There are at least four different lymphocyte HEV specificities: one for peripheral
lymphoid tissues, a second for Peyer's patches, a third for lung-associated lymphoid
tissues, and a fourth at sites of chronic inflammation, such as the inflamed synovium or
skin (10,12-15). The attachment of lymphocytes to tissue-specific endothelium is mediated
by receptor/counter-receptor pairs (10). The in vivo interaction of lymphocyte subsets with
3
different HEV is reflected in an elegant in vitro model for lymphocyte/endothelial-cell
interaction developed by Stamper and W oodruff in 1976 (16). This unique specificity of
lymphocyte/HEV binding gave rise to the term "homing” and was predicted to be mediated
by receptor/counter-receptor pairs expressed by both cell types. The lymphocyte molecules
responsible for this specificity were termed "homing receptors." Thus, the receptor
involved in lymphocyte homing to peripheral lymph nodes was called the peripherallymph-node homing receptor (17).
Based on the observation that certain mouse lymphoid cell lines bind with absolute
specificity to peripheral-lymph-node HEV while others bind to mucosal HEV, attempts
were made to generate monoclonal antibodies against these receptors on lymphocytes ( 10).
Immunization of rats with a mouse B-cell lymphoma that binds to peripheral lymph node
HEV but not to Beyer's patch (mucosal) HEV was used to generate the monoclonal
antibody MEL-14 (18). M EL-14 reacted with a cell-surface determinant present on all
lymphocytes that bound peripheral HEV. Also, M EL-14 blocked adherence of lymphomas
and normal cells to peripheral-lymph-node HEV in vitro and also blocked lymphocyte
migration into the peripheral lymph nodes in vivo (17-19).
M EL-14 binds to a 90 kD glycoprotein from both peripheral HEV binding lymphomas
and normal lymphocytes (18). This 90 kD glycoprotein is called the peripheral-lymph-node
homing receptor (PLNHR) or gp9()MEL-14 Expression of the M EL-14 antigen is not
restricted to lymphocytes, since the mAb M EL-14 stains granulocytes and monocytes and
im m unoprecipitates a IOO kD glycoprotein from the neutrophil surface (20). The
gp90M E L -14 on neutrophils was also shown to mediate adhesion o f neutrophils to
inflamed endothelium (20-22). Independently, the human hom ologue o f the mouse
gp90M EL-14 antigen was identified as the pan leukocyte markers Leu- 8, T Q -1, and
DREG, found on most circulating human lymphocytes, neutrophils, and monocytes (2328).
Based on the partial amino-acid sequence o f g p 9 (# E L -1 4 an oligonucleotide probe
was derived and used to isolate the cDNA clone encoding the core polypeptide o f
gp90MEL-14 (29,30). The human homologue o f gp9()MEL-14 was independently isolated
by different laboratories through the hybridization selection screening o f human
lymphocyte cDNA library with mouse gp9QMEL-14 CDNA (31,32), immunoscreening o f
transfected cDNA clones (27), and by differential hybridization (33). The molecular
cloning o f mouse gp9()MEL-14 revealed a glycoprotein with tandem interaction domains
containing, from the NH 2-terminal, a separate carbohydrate binding (lectin) domain, an
epiderm al grow th-factor-like (EGF) dom ain, and duplicated repeats homologous to
complementary regulatory proteins (CRP) (27,29-33).
The characteristic protein mosaic architecture o f gp9()MEL-14
aiso found in two
other independently studied cell-surface glycoproteins, identifying a novel family o f
receptors recently named selectins (34). There are currently three members in the selectin
family: I) L-selectin, which is the peripheral-lymph-node homing receptor or gp90MEL14, has been called Leu- 8, LAM-1, TQ -1, LECAM -1, LEC -C A M -I, and DREG (in this
dissertation it will be referred to as L-selectin), 2) E-selectin, which is expressed on
cytokine-activated endothelial cells and is thought to mediate their binding to leukocytes at
sites o f inflam m ation (35,36), and 3) P-selectin (also known as CD62, GMP140, or
PADGEM), which is stored in Weibel-Palade bodies of endothelial cells and alpha-granules
of platelets, is rapidly mobilized to the cell surface after activation and promotes binding of
these cells to monocytes and neutrophils (37,38). All three m em bers play critically
important roles in mediating cell/cell interaction in the vasculature (29,30,35,39).
5
Structure o f L-selectin
The deduced transmembrane protein o f L-selectin is 334 amino acids long with an
unusually long hydrophobic leader sequence o f 38 am ino acids, a hydrophobic
transm em brane region follow ed by a cluster o f positively-charged residues, and a
hydrophilic cytoplasmic tail o f 18 amino acids (29,30). Hydropathy plot o f the protein
predict regions o f hydrophilicity concentrated in the NH 2-terminal 150 amino acids and a
membrane proximal 20 amino acids. The intervening extracytoplasmic portion consists o f a
relatively uncharged neutral stretch, which includes the EGF and CRP domains (29,30).
The mature protein begins with a tryptophan, which is unusual as an amino terminal
residue (29,30). It has ten potential asparagine-linked glycosyladon sites and no sites for
O-Iinked glycosyladon in the deduced sequence (29,30). This is consistent with the
antibody affinity purification and biochemical analysis o f L-selectin from the murine Tlymphoma cell line EL-4 which indicated that the core protein with a maximum size of 46.5
kD is modified by N-Iinked carbohydrates accounting for 45% o f L-selectin mass (30). The
mature protein contains 22 cysteine residues accounting for 6.6% o f the total amino acids
(29,30).
The NH 2-terminal lectin domain o f mouse L-selectin is homologous to domains found
in a diverse series o f calcium-dependent animal lectins (40,41). The lectin domain has a
large number (sixteen) o f lysine residues (29,30). The EGF-Iike domain o f L-selectin
consists of a single copy homologue of EGF-Iike sequences found in various proteins such
as growth factors, developmental gene products, extracellular matrix proteins, cell-surface
receptors, blood-clotting factors, and plasm inogen activators (29,30). The EGF-Iike
domain contains all six consensus cysteines and the glycine residues characteristic o f this
structure. The complement binding m otif consists o f two 62-amino-acid repeats (29,30).
6
This m otif is found in a number o f complementary regulatory proteins that bind C3 and C4
and other proteins, such as the JL-2 receptor. The presence o f the three distinct motifs in Lselectin suggests that the gene for L se lec d n may have evolved through exon shuffling
(29,30).
The human homologue o f mouse L selecd n shows an identical domain organization: an
NH 2-terminal lectin domain, followed by an EGF-Iike domain, two complement binding
repeats, a transmembrane domain, and a cytoplasmic tail (31-33). The overall sequence
identity between the mouse and the human L selectin is 86% in the lectin domain, 82% in
the EGF-Iike domain, and 74% and 60% in the first and second CRP domains respectively.
Also, almost identical amino-acid sequences are found in the transmembrane (95% identity)
and surrounding regions o f both human and mouse L selectin cDNA sequences (31-33).
The human L-selectin molecule immunoprecipitated from lymphocytes has a molecular
mass o f 74 kD, whereas the molecule on neutrophils is 90 kB (42). The neutrophil and
lymphocyte mRNA transcripts have the same size by northern blot analysis; therefore, the
difference in molecular mass is thought to be due to post-translational modification (42).
Recently in our laboratory, Bruce W alcheck et al. (1992) cloned the lectin domain of
the bovine homologue o f the human L-selectin (84). The nucleotide sequence o f the bovine
L-selectin lectin domain revealed a nucleotide identity of 84.2% and 80.4% with the human
and mouse homologues, respectively. The predicted amino-acid sequence o f the bovine Lselectin lectin domain has an 81.6% and 76.3% identity with the human and mouse lectin
domains, respectively. The only difference in the bovine lectin domain was the presence o f
a third N-Iinked glycosylation site. The remaining two N-Iinked glycosylation sites were
conserved between cow, mouse, and human (84).
7
Function of L-selectin
As mentioned above, two important adhesive functions are mediated by L-selectin: I)
lym phocyte adhesion to peripheral lym phoid tissues, and 2 ) neutrophil/endothelial
interactions. And-L-selecdn antibodies inhibit lymphocyte migration into peripheral lymph
nodes in vivo and the adherence o f lymphocytes to PLN-HEV in in vitro adherence
assays. Soluble, affinity-purified, lymphocyte L-selectin or a recombinant protein can bind
PLN-HEV and block lymphocyte legalization (4,18,43). Anti-L-selectin antibodies also
inhibit neutrophil localization to sites o f acute inflammation, including the dermis and
peritoneum o f mice, and block neutrophil adhesion to cytokine-stimulated endothelial cells
in vitro (44-48). Recently, a soluble L selectin-IgG chimeric molecule was also shown to
alm ost com pletely block the neutrophil influx into thioglycollate-inflam ed mouse
peritoneum in vivo (49). Early studies showed that certain monosaccharides, like Dmannose- 6-phosphate and D -fructose-1-phosphate, inhibit lymphocyte binding to PLNH EV in rat, m ouse, and hum ans (50-52). A lso, the m annose- 6-phosphate-rich
phosphomannan monoester (PPME) and the fucose-rich polysaccharide fucoidin inhibited
Iymphocyte/PLN-HEV binding (53,54). Using PPM E-derivatized microbeads, it was
shown that a calcium-dependent, lectin-like receptor on the lymphocyte surface was
required for lymphocyte binding to PLN-HEV (53,54). The monoclonal antibody M EL-14
blocked PPME binding to lymphocytes, and it was predicted that the lectin-like molecule
was the same as L-selectin (53).
A direct ELISA-based assay has confirmed the binding of the purified or recombinant
L-selectin to the mannose- 6 -phosphate-rich polysaccharide PPME in a calcium-dependent,
m annose- 6-phosphate- and fructose- 6-phosphate-inhibitable m anner (55). The ELISA
assay also showed that fucoidin, a potent inhibitor o f lymphocyte binding to HEV,
8
competes for PPME binding o f L-selectin (55). As discussed above, molecular cloning of
L-selecdn cDNA confirmed the presence of a C-type lectin domain in the NH2-terminal of
L-selectin (55); thus, L-selecdn is a mammalian lectin.
The lectin domain o f L-selectin contains a large number o f lysine residues: sixteen in
the mouse and twelve in the human, indicating a high concentration o f positive charge (3133). This is in agreement with the requirement for sialic acid on the endothelial ligand and
the fact that all the known sugars that bind L-selectin are anionic (56,57). The human and
mouse L-selectin share an overall similarity in deduced primary sequence (77% at the
protein level and 79% at the nucleotide level) and exhibit very similar carbohydrate binding
activity (58).
L-selectin has also been dem onstrated to mediate leukocyte rolling along the
endothelium (59,60). This process has been proposed as a means o f slowing the leukocyte
before it comes to a complete stop at sites o f inflammation. Neutrophil rolling in vivo can
be completely abrogated by the infusion o f anti-L-selectin antibodies, a L-selectiri-IgG
chim era, and various sulfated carbohydrates, such as dextran sulfate, fucoidin, and
sulfatides (a sulfated glycolipid)—all inhibitors o f L-selectin (59,61-64). Thus, L-selectin
appears to mediate a high avidity interaction that occurs under significant shear.
L-selectin-mediated rolling o f L-selectin cDNA transfected mouse pre-B cell line
300.19 in exteriorized rat mesenteric venules has been shown by Kansas et al. (1993) to be
dependent on the presence o f the cytoplasmic tail o f L-selectin (84). Transfectants lacking
eleven amino acids at the carboxy terminal o f L-selectin failed to bind PLN-HEV in vitro
and also failed to roll on endothelium in vivo. The rolling was also abrogated if cells were
pretreated with cytochalasin B, which disrupts actin microfilaments (84). However, the
transfectants lacking the eleven COQH-terminal amino acids did bind PPME (84). These
data suggest that the cytoplasmic domain o f L-selectin may be involved in an interaction
9
with the cytoskeleton to mediate leukocyte rolling that is independent o f ligand recognition.
The proposed ligands for L-selectin are the antigens identified by the antibody MECA79, and glycoproteins immunoisolated by a L-selectin-IgG chimeric molecule, one o f
which has been recently cloned and named G lyCAM -I (65-67). O f the two, the best
characterized ligands for L-selectin are the glycoproteins identified by the monoclonal
antibody MECA-79, which recognizes a peripheral lymphnode-specific HEV antigen found
in both mouse and man (65). MECA-79 also inhibits lymphocyte binding to PLN-HEV,
but not substantially to HEV in Peyer's patches. The antigens identified by MECA-79 are
called the peripheral-lymph-node addressin or PNad (65).
Immunoisolated PNad coated onto glass slides selectively binds lymphocytes and
lymphoid cell lines that can bind PLN-HEV (65). Lymphocyte binding to purified PNad is
calcium-dependent and abrogated by neuraminidase treatment (65). Binding o f mouse and
human lymphocytes to purified PNad is inhibitable with anti-L-selectin antibodies or
MECA-79. Also, a mouse pre-B cell line transfected with human L-selectin cDNA bound
purified PNad, whereas the untransfected parent line could not. All these data confirm that
L-selectin and PNad are receptor/ligand pairs. Using MECA-79 as an immunoadsorbent, a
number of glycoproteins o f distinct molecular weight (5 0 ,9 0 ,1 0 5 ,115,170, and 200 kD)
were isolated. It remains to be seen whether one or all of these glycoproteins are the ligand
for L-selectin (65).
Other putative ligands for L-selectin include Sgp50 and Sgp90 (66), which are sulfated,
fucosylated, and sialylated glycoproteins isolated by affinity columns using L-selectin-IgG
chimeric molecule from PLN-HEV. These components (Sgp50 and Sgp90) are not detected
in other lymphoid organs, such as spleen, thymus, or Peyer's patches. The binding o f the
L-selectin-IgG chimera to Sgp50 and Sgp90 is calcium dependent and inhibited by MEL14, PPME, and treatment o f the Sgps with sialidase (66).These Sgps are also precipitated
by the anti-peripheral-lymph-node-addressin (PNad) antibody MECA-79. N-glycanase
10
treatment o f SgpSO and Sgp90 does not diminish their molecular weight, indicating the
absence of N-Iinked carbohydrate chains (66).
N-terminal amino-acid microsequencing o f purified SgpSO was used to clone a cDNA
encoding the protein proposed to be the ligand for L-selecdn (67). The cDNA encodes a
novel, serin e/th reo n in e-rich , m ucin-like glycoprotein now called G lyC A M 1/(glycosylation-dependent cell-adhesion molecule) (67). G lyCAM -I is HEV-associated,
and contains predominantly O-Iinked carbohydrate chains with a requirement for sulfation
to bind L-selectih (68).
R ecent
reports have established that E-, P-, and L -selectin all recognize the
fucosy lated and sialylated tetrasaccharide called sialyl Lewis X and related carbohydrates
(69-74). Therefore, all selectins are suspected to bind ligands that are distinctive
modifications of the core carbohydrate, such as sialyl Lewis X.
Recently, using a recombinant L-selectin as an immunohistochemical probe, potential
ligands for L-selectin have been demonstrated on myelinated regions o f the central but not
the peripheral nervous system (75). Based on antibody blocking studies, L-selectin has
been shown to mediate binding of lymphocytes to myelinated regions o f mouse central
nervous system. These studies raise the possibility that a L-selectin-dependent mechanism
may be a factor in the pathogenesis o f certain central-nervous-system demyelinating
diseases (75). It is interesting to note that treatment of rats with fucoidin or mannose- 6phosphate—two carbohydrate-based inhibitors o f L-selectin, has been reported to prevent or
delay the induction of experimental autoimmune encephalomyelitis in rats (76,77).
I
11
Regulation o f L-selectin
L selecdn is rapidly shed from the cell surface upon activation o f mouse neutrophils
with various inflammatory mediators, and the shed form is 5-10 kD smaller in size than the
membrane-associated form. (78). Mouse lymphocytes activated in vitro by exposure to
phorbol esters shed a soluble L-selectin that is 12 kD smaller than the intact receptor (79).
Similarly, L-selectin is also released upon stimulation o f human lymphocytes (23,28). The
loss o f L-selectin expression has been proposed to be important for the release o f the
leukocyte from the vascular endothelium in order to enter the underlying tissues (78). This
model is consistent with leukocyte entry into sites o f inflammation since neutrophils that are
found in inflamed lesions are L-selectin negative (78). However, the concentration o f
inflammatory mediators used in the in vitro assays to cause L-selectin downregulation is
high and are unlikely to be attained in vivo. Also, activation o f leukocytes in vitro leads to
a total loss o f cell-surface expression o f L-selectin; whereas, lymphocytes which enter
peripheral lym ph nodes via L-selectin-dependent adhesion pathw ays do n o / show
appreciable loss o f L-selectin expression (85). Therefore, an alternate mechanism for the
downregulation of L-selectin expression may exist.
Upon activation, neutrophils upregulate other adhesion receptors o f the integrin family,
particularly C D l lb/CD18 (expression o f which is obligatory for neutrophil localization at
sites o f inflammation) (78). H iis intricately controlled and inverse regulation o f L-selectin
and C D l lb/CD18 is proposed necessary for the neutrophils to detach from the endothelium
and begin the process o f transendothelial migration into inflammatory sites (78). The
downregulation o f L-selectin has been hypothesized to be mediated by a surface protease
that becomes functional upon activation o f the neutrophil. Recent reports have shown that
low-dose chymotrypsin treatment o f leukocytes can cause shedding o f L-selectin.m vitro
12
(45). The presence o f lysine and tyrosine residues near the plasm a membrane on the
extracytpplasmic portion o f the L-selectin molecule lend support to the model that activation
o f the neutrophil may indeed "turn on" a serine protease-like enzyme to cleave and release
L-selectin.
It has been suggested that a Pi-linked form o f L-selectin may be expressed, and,
therefore, a phospholipase may be involved in the regulation o f L-selectin expression (80).
This hypothesis was based on isolation o f two cDNA clones o f different lengths from Tcell libraries which encode either a longer transmembrane protein or a shorter Pi-linked
form o f L-selectin. PI-PLC treatment o f COS cells transfected with the shorter clone
diminished reactivity to anti-L-selectih antibodies by FACs analysis, whereas PI-PLC
treatm ent o f COS cells transfected with the longer clone had no effect on L-selectin
expression (80). However, the presence o f Pi-linked L-selectin on the leukocyte surface
and its functional significance is controversial. Recently, Ord et al. found no splice site in
the genomic DNA that would yield a mRNA encoding a Pi-linked form o f L-Selectin (81).
Also, leukocytes, isolated from patients with paroxysmal nocturnal hemoglobinuria (PNH)
who have defective expression o f Pi-linked proteins, had normal levels of L-selectin
expression (81). Therefore, the significance o f a Pi-linked form o f L-selectin and its
regulation remains to be determined.
R ecently, Tedder et al. showed that activation o f lym phocytes with anti-CD3
monoclonal antibodies causes a transient increase in the affinity o f L-selectin to bind PPME
(82). Sim ilarly, neutrophil activation with cytokines, such as TN F, also transiently
increases PPME binding by L-selectin. In both o f these cases, the cell-surface expression
o f L-selectin did not increase, suggesting a conformational change in L-selectin that
enhances receptor affinity. These authors propose that leukocyte lineage-specific activation
in vivo may be the regulatory mechanism that induces enhanced ligand binding by Lselectin prior to being shed from the cell surface (82).
Ill
[I
13
Mv Hypothesis
The prevailing hypothesis is that shedding o f L-selectin is required for the release o f the
bound leukocyte from the endothelium and extravasation into the underlying tissues. If
gross activation of the leukocyte is required for total shedding of L-selectin and this in turn
is required for extravasation, then this hypothesis fails to address the following issues: (I)
lymphocytes require L-selectin to bind HEV, but do not become L-selectin negative upon
entering uninflamed lymph nodes (79); (2) neutrophils bind uninflamed HEV via L-selectin
in ex vivo binding assays, but do not enter uninflam ed lym phoid tissues in vivo,
suggesting that neutrophils must release from the HEV and re-enter circulation; (3) our
recent finding that L-selectin can be detected in the plasma o f individuals who do not show
any overt signs o f acute inflammation indicates that L-selectin may be shed in the absence
o f inflammatory mediators in vivo. Therefore, a mechanism o f L-selectin downregulation
that does not rely on overt activation o f the cell may exist.
I propose that crosslinking o f L-selectin to its endothelial ligand causes or enhances
j
downregulation. This hypothesis is consistent with shedding being involved in the
circumstance listed above. For example, the entry o f lymphocytes into normal lymphoid
tissue would result in crosslinking of L-selectin to its ligand on the PLN-HEV. This in turn
would cause shedding o f L-selectin and releasing o f the lymphocyte from the endothelium
in order to proceed with the process of extravasation. The presence o f L-selectin-positive
■
'
lymphocytes in lymphoid tissue could be explained by shedding occurring only at the sites
of leukocyte/endothelial-cell contact and not over the entire cell surface as seen in overtly
activated cells. Similarly, the L-selectin-mediated neutrophil-rolling phenomenon observed
I
in vivo would result in crosslinking of L-selectin to its ligand on the endothelium, which
: i|
14
causes shedding at sites o f neutrophil/endothelium contact enabling the neutrophil to
continue rolling. If inflammatory mediators are present, then upregulation o f C D l lb/CB18
occurs which would mediate firm attachment and emigration. The lack o f neutrophil
extravasation into uninflamed lymphoid tissue, even though neutrophil L-selectin binds
HEV, could also be explained by our hypothesis. The binding o f neutrophils to HEV
would result in crosslinking of L-selectin and subsequent releasing o f neutrophils from the
HEV. The presence o f L-selectin in the plasma o f individuals showing no signs o f overt
inflammation may be due to the shedding o f L-selectin induced by constant crosslinking
occurring under physiological conditions.
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67. Lasky, L A., M.S. Singer, D. Dowbenko, Y. Imai, W .J. H enzel, C. Grimley,
C. Fennie, N. Gillett, S.R. W atson, and S.D. Rosen. 1992. A n endothelial ligand
for L-selectin is a novel mucin-like molecule. Cell 69:927-934.
68 . Im ai, Y ., L.A . L asky, and S.D. Rosen. 1993. Sulfation requirem ent for
GlyCAM -1, an endothelial ligand for L-selectin. Nature (London) 361:555-557.
69. Low e, J.B ., L.M . Stoolm an, R.P. N air, R.D. Larsen, T.L. B erhend, and
R.M. M arks. 1990. ELA M -I dependent cell adhesion to vascular endothelium
determined by a transfected human fucosyltransferase cDNA. Cell 63:475-484.
70. W alz, G ., A. A ruffo, W. K olanus, M. B evilacqua, and B. Seed. 1990.
Recognition by ELAM -I o f the sialyl-Lex determinant on myeloid and tumor cells.
Science (Wash. D.C.) 250:1132-1135.
7 1. Tiem eyer, M., S I. Swiedler, M. Ishihara, M. M oreland, H. Schweingruber, P.
Hirtzer, and B K. Brandley. 1991. Carbohydrate ligands for endothelial-leukocyte
adhesion molecule-1. Proc. Natl. Acad. Sci. USA 88:1138-1142.
72. Policy, M .J., M .L. P hillips, E. W ayner, E. N udlem an, A .K . Singhal, S.
Hakomori, and J.C. Paulson. 1991. CD62 and endothelial cell-adhesion molecule I
(ELA M -I) recognize the same carbohydrate ligand, sialyl-Lew is x. Proc. Natl.
Acad. Sci. USA 88:6224-6228.
73. Berg. E.L., I. M agnani, A.R. W am ock, M.K. Robinson, and E.C. Butcher.
1992. Comparison of L-selectin and E-selectin ligand specificities: The L-selectin
can bind the E-selectin ligands sialyl Lex and sialyl Lea. Biochem. Biophys. Res.
Commun. 184:1048-1055.
74. Foxall, C., S.R. W atson, D. Dowbenko, C. Fennie, L A . Lasky, M. Kiso, A.
Hasigawa, D. Asa, and B.K. Brandley. 1992. The three members o f the selectin
receptor family recognize a common carbohydrate epitope, the sialyl Lewis x
oligosaccharide. J. Cell Biol. 117:895-902.
75. H uang, K ., J.S . G e o ffre y , M .S. S in g er, and S.D . R osen. 1991. A
lym phocyte hom ing receptor (L-selectin) mediates the in vitro attachment o f
lym phocytes to m yelinated tracts o f the central nervous system . J. Clin.
Invest. 88:1778-1783.
76. W illenborg, D O., and C R. Parish. 1988. Inhibition o f allergic encephalomyelitis
in rats by treatment with sulfated polysaccharides. J. Immunol. 140:3401-3405.
77. W illenborg, D O., C R. Parish, and W .B. Cowden. 1989. Phosphosugars are
potent inhibitors o f central nervous system inflammation. FASEB I. 3:1968-1971.
78. Kishim oto, T.K ., M.A. Jutila, E.L. Berg, and E.C. Butcher. 1989. Neutrophil
M ac-1 and M el-14 adhesion proteins inversely regulated by chemotactic factors.
Science (Wash. D.C.) 245:1238-1241.
79. Jung, T.M ., and M.O. Dailey. 1990. Rapid m odulation o f hom ing receptors
(gp90MEL-14) induced by activators o f protein kinase C. Receptor shedding due to
accelerated proteolytic cleavage at the cell surface. J. Immunol. 144:3130-3136.
80. C am erini, D ., S.P. Jam es, I. Stam enkovic, and B. Seed. 1989. L eu8/TQ1 is the hum an equivalen t o f the M el-14 lym ph node hom ing
receptor. Nature 342:78.
S l.O rd , D C., T.J. Ernst, L.J. Zhou, A. Ram baldi, O. Spertini, J. Griffin, and T.F.
Tedder. 1990. Structure o f the gene encoding the human leukocyte adhesion
m o le c u le-1 (TQ 1, L e u - 8) o f lym phocytes and n eu tro p h ils. J. Immunol.
265:7760.
82. Spertini, O., G.S. K ansas, J.M. M unro, J.D. Griffin, and T.F. Tedder. 1991.
Regulation of leukocyte migration by activation of the leukocyte adhesion molecule1 (LAM -I) selectin. Nature (London) 349:691-694.
23
83. K ansas, G.S., K. Ley, J.M . M unro, and T.F. Tedder. 1993. Regulation o f
leukocyte rolling and adhesion to endothelial venules through the cytoplasmic
domain o f L-selectin. J. Exp. Med. 177:833-838.
84. W alcheck, B., M. W hite, S. K urk, T.K. K ishim oto, and M .A. Jutila. 1992.
Characterization o f the bovine lymph node homing receptor: a lectin cell adhesion
molecule (LECAM). Eur. J. Immunol. 22:469-476.
85. P ilarski, L.M ., E.L. Turley, A .R .E. Shaw , W .M . G allatin, M .P. Ladefoute,
R. G illitzer, I.G.R. Beckm an, and H. Zola. 1991. FM C46, a cell protrusion
associated leukocyte adhesion m olecule -1 epitope on human lymphocytes and
thymocytes. J. Immunol. 47: 136-143.
24
CHAPTER 2
A CTIV A TIO N -IN D EPEN D EN T SH EDDING O F L E U K O C Y TE L=
SE L E C T IN IS IN D U CIBLE BY C RO SSLIN K IN G AGENTS
Introduction
The entry o f peripheral blood leukocytes into extravascular tissues is controlled by
highly specific and coordinated receptor-ligand interactions between the circulating
leukocyte and the vascular endothelium (1,2,3). L-selectin (also termed Leu- 8, T Q -1,
gp 90 M EL-M l a m - 1, peripheral lymph node homing receptor, and LECAM -1) represents
one of several types of leukocyte adhesion proteins involved in these events. L-selectin was
originally defined as a tissue-specific homing receptor, which controls lymphocyte
adhesion to high endothelial venules in peripheral lymphoid tissues (3, 4). Recently, in
vitro and in vivo monoclonal antibody (mAb) blocking studies have suggested that Lselectin also contributes to neutrophil and monocyte adhesion to endothelial cells at sites o f
inflammation (5-11). L-selectin appears to be involved in the initial contact o f the peripheral
blood leukocyte with the vascular endothelium, including the process o f leukocyte"rolling"
along the vessel wall ( 12, 13).
Expression o f L-selectin is inversely regulated on the surface o f leukocytes in
comparison to other adhesion proteins. Within 5-15 minutes o f stimulation in vitro with
25
chemotactic factors, or other activating agents, L selecdn is shed from the neutrophil and
monocyte cell surface (4, 9, 15). In contrast, C D l 1/CD 18 molecules increase in surface
expression and function. Lymphocyte L-selectin can also be downregulated by activation
with phorbol esters, but shedding of the lymphocyte molecule takes longer (15-30 minutes)
(16, 17). Leukocytes also rapidly downregulate L-selectin expression in vivo during
migration in response to inflammation (4, 6). It is thought that the release o f L-selectin is
due to a unique protease, but the specific molecular events regulating shedding are difficult
to dissect because o f the array of cellular processes induced by gross cell activation.
It has been proposed that the loss o f L-selectin after activation is an important signal
allowing the release of the leukocyte from the vascular endothelium and entry into the
underlying tissue (9). This model is consistent with leukocyte entry into sites o f
inflammation where down-regulation o f L-selectin expression can clearly be shown on the
cells migrating into the inflammatory site. No such correlation has been seen with the entry
o f leukocytes into uninflamed tissues, however. For example, lymphocytes that enter
peripheral lymph nodes via L-selectin adhesion pathways do not show appreciable loss of
L-Selectin (18). However, L-selectin modulation on trafficking lymphocytes might still be
involved in their entry into lymph nodes if the event is more subtle than that on grossly
activated cells. For example, shedding may only take place at the site o f lymphocyte contact
with the endothelium. For this to take place another mechanism o f down-regulation
independent of activation-induced down-regulation must exist.
In this chapter I have tested whether crosslinking L-Selectin, which may be a result of
the interaction o f the molecule with endothelial cells, triggers shedding independent o f
activation. To detect such a change, I used a chemical method to crosslink L-selectin over
the entire cell surface at 4 °C . Treatm ent w ith the chem ical crosslinker, [bis
(sulfosuccinimidyl) suberate (ES3)], caused a rapid shedding o f L-selectin from the cell
26
surface. To mimic in vivo ligand binding I used specific anti-L-selectin monoclonal
antibodies to crosslink L-selectin. A combination o f anti-L-selectin monoclonal antibodies
crosslinked and induced loss o f surface expression o f L-selectin. I also demonstrated in
vivo that activation-independent shedding o f L-selectin occurs. Based upon the results
described here I propose a novel mechanism o f L-selectin regulation. Also, my results
provide the basis for a workable system to examine the molecular events required for Lselectin shedding.
Materials and Methods
Antibodies
DREG 152, DREG 200, DREG 56, DREG 110, DREG 55, EL246 and Leu -8 (Becton
Dickinson, Mountain View, CA) are mouse mAb which recognize human L-selectin (4,
19). The DREG mAb are IgG Is, whereas, Leu -8 is a IgG2a. M EL-14 (rat IgG2a) (20) and
poly-anti-M EL-14 antigen (rabbit polyclonal anti-sera, gift o f S. Rosen, University o f
California, San Francisco) recognize mouse L-selectin. MJ64 (rat IgG2a (21)) and IM7 (rat
IgG2a (21)) recognize mouse Pgp-I (mouse CD44) and H erm es-3 (mouse Ig G l)
recognizes human CD44 (22). FD445.1 (rat IgG2b (23)) and M l/7 0 (rat IgG2b (24))
recognize mouse LFA -I and M ac-1, respectively, and 30G12 (rat IgG2a) recognizes
mouse T200. Finally, anti-M ac-1 (mouse IgG2a) was purchased from Becton Dickinson.
Preparation of leukocyte suspensions and flow cytometric analysis
Human peripheral blood and mouse bone marrow leukocytes were prepared as
previously described with no modifications (4, 6). The mouse m yeloid cell line W EHI
78/24, which expresses high levels o f L-selectin (5), was also used for the regulation
27
studies described below. All cell suspensions were washed in HBSS prior to treatment
with the different crosslinking agents (see below).
Immunofluorescence staining of cells was carried out in 4ml tubes. Briefly, IxlO^ cells
were initially incubated in 5% rabbit serum for 10 minutes on ice to block Fe receptors. The
cells were washed and then incubated with primaiy antibody at 50-100 (ig/ml for 20 min on
ice. A fter washing, bound antibodies were revealed by incubation with FITC conjugated
goat anti-mouse Ig (second stage) (Sigma) at a 1:100 dilution in 5% FBS in DMEM. Flow
cytometric analysis was performed on a FACScan (Becton and Dickinson) as described (7,
8). In some experiments direct stains with phycoerythrin-labeled Leu -8 or FTlC labeled
DREG 56 and DREG 200 were done. Background fluorescence was established by
sttuning with conjugated second stage alone. Data were collected from 10,000 cells and are
presented as either histograms or mode fluorescence.
Crosslinking o f leukocyte L-selectin
For chemical crosslinking o f cell surface proteins, both human and mouse leukocytes
w ere treated with eith er B S ^ [bis
(su lfo su ccin im id y l)
su b erate]
or S-D ST
(disulfosuccinimidyl tartate). BS 3 is a bifunctional and membrane-impermeable chemical
crosslinker, which covalently crosslinks proteins 11.4 Angstroms apart via lysine residues.
S-DST has the same specificity as BS3 , but crosslinks proteins 6.4 Angstroms apart.
Conditions used for crosslinking were similar to those used by others (5x1
cells/ml in 5
mM B S 3 or S-DST for 15-25 minutes on ice) (25). The crosslinking agents were
quenched by washing the cells in PBS plus 5% rabbit serum (Sigma) or a Tris-glycine
buffer. Controls included cells incubated on ice in HBSS alone. A fter treatment, the cells
were always kept on ice to prevent activation and membrane turnover and stained with the
antibodies listed above for flow cytometric analysis.
28
Antibody crosslinking of L-selectin
Human leukocytes were treated with either a combination o f anti-L-selectin monoclonal
antibodies (DREG 152 and EL246) or anti CD45 antibody L3B12 at a final concentration o f
50ug/ml on ice for 20 min, washed, and a FITC conjugated anti-mouse antibody was used
to crosslink the primary antibodies and thereby L-selectin or CD45. After washing away
excess second stage the cells were incubated at either 37°C or on ice for 15 min, washed
and blocked with 6 % mouse serum in the presence o f 2mM azide, and stained with
phycoerythrin (PE) labelled Leu -8 as an additional means o f detecting surface L-selectin
expression. Similarly treated cells were also stained with PE-M ac-I instead o f Leu -8 to
monitor upregulation of C D l lb/GD18 due to cell activation. Flow cytometric analysis was
performed on a FACScan (Becton-Dickinson).
Western blot SDS-PAGE analysis
Blood was collected from healthy adults into heparinized tubes and centrifuged at 200g
to collect the red and white blood cell pellet and the plasma layer. The white blood cells
were further purified by dextran sedimentation and used in flow cytometric analysis. The
plasma was clarified by centrifugation at 20,(K)Og for 10 min, mixed with an equal volume
o f 2x nonreducing SDS-solubilization buffer, run on a 7% SDS-PAGE gel, and transferred
to nitrocellulose with a BioRad transblot apparatus per manufacturer's directions. Filters
were incubated with 50% horse serum in TBST (10 mM Tris-HCl, pH 7.4,150 mM NaCl,
and 0.05% Tween-20) for one hour. Using a 25 lane mini-blot apparatus (Immunonetics,
Cambridge, MA), the filters were then incubated for 30 min with either DREG 56, DREG
152 or an isotype control antibody, H erm es-3, at 50 [ig/ml concentrations. The
nitrocellulose filters were then washed in TEST, incubated with goat anti-mouse Ig-alkaline
29
phosphatase conjugate (Sigma, A-9654) diluted 1:200, and then washed again. The blots
were developed by addition o f substrate solution from Promega Biotech.
ELISA analysis
Human peripheral blood leukocytes (SxlOfyml) were treated with 5 mM BS^ or buffer
alone for 15 min, centrifuged at 300g, and the supernatant fluid collected. The supernatants
were diluted 1:2 in coating buffer (0.1M bicarbonate buffer, pH 9.6) and 2-fold serial
dilutions incubated in the wells of a 96-well Immulon type-1 plate (Dynateck-Fisher) for
two hours at room temperature in a humidified chamber. The plates were washed, blocked
in 0.5% BSA overnight, and probed with DREG 152 and DREG 200 mAbs. Negative
controls included second stage alone and an isotype m atched (Ig G l) mAb which
specifically recognizes a sheep leukocyte antigen (SH43). The primary mAbs were revealed
by alkaline phosphatase conjugated anti-mouse Ig (Sigma) followed by development in
Sigma 104 phosphate substrate in 0.1M diethanolanine buffer, pH 9.8. The absorbance,
measured at 405 nM, o f each well was read on a Biorad multi-well ELISA reader. Values
reflect optical density (o.d.) readings o f the DREG mAbs minus the background given by
the isotype negative control, which was automatically calculated by the ELISA reader. Each
data point reflects the mean+SEM from 6 separate experim ents. Controls included
supernatant fluid from cells treated with buffer alone and medium containing ju st the
crosslinking agent, but no cells.
Results
Activation-independent down-regulation of leukocyte L-selectin
can be induced by chemical crosslinking agents
W e employed a system whereby L-selectin was crosslinked at 4 °C using chemical
30
crosslinking agents. BS^ crosslinks surface proteins within 11.4 Angstroms o f each other
by form ing covalent bonds between adjacent lysine residues (25, Pierce product
information). The human and mouse L-selectin lectin domains, which are thought to be
important in adhesion o f L-Selectin to its ligand (26^28), are rich in lysine. Recently, Lsclectin has been shown to be located in clusters on the ends o f the microvilli and ruffles o f
cells (18, 29). We hypothesized that adjacent L-selectin molecules could be crosslinked by
these agents, and thus mimic crosslinking that may occur during the interaction o f Lselectin and a multivalent ligand. Importantly, conditions could be met which precluded the
contributions o f activation (the experiments could be done at 4°C and for short periods of
time).
Both human and mouse leukocytes were treated with BS^ on ice for 15 min, as
described in the Materials and Methods. The crosslinking procedures did not grossly alter
I ,
the cells. They exhibited normal cell size, granularity, and auto-fluorescence as measured
by flow cytometry. The crosslinked cells had normal morphology determined by light
microscopy, as well. Cell death (propidium iodide uptake) and lysis did not occur (data not
shown). Surface expression, including distribution, of a number o f control proteins were
unaltered (see below).
The effect o f BS^ crosslinking on L-selectin expression was then examined. Mouse
leukocytes, which expressed high levels o f L-selectin, showed greatly reduced staining
with an anti-murine L-selectin mAb (MEL-14) after treatment (Table I, Figure I). t h e
effect was not epitope specific, since BS3 treatment also reduced staining with a polyclonal
anti-murine L-selectin (Table-1). The effect o f BS3 on the staining o f six other murine
leukocyte specific mAbs, which included anti-Pgp-1, T200, SK105, LFA -1, and Mac-1,
was tested (Table I) and only the staining o f one, anti-Pgp-1 mAb (M j64), was
significantly reduced. Since a second anti-Pgp-1 mAb, (IM7), did not show a reduction in
31
staining, the loss o f M J64 staining was likely due to BS^ altering the M J64 epitope. The
unaltered expression o f SK105 and Mac-1 showed that BS^ did not activate the cells, since
a rapid increase in the surface expression o f these antigens has previously been shown to
be a sensitive marker o f activation (6-9).
T able I. Chemical crosslinking o f mouse and human leukocyte surface proteins causes
loss of L -selectin expression.
PercemftSEe off
AmftiEemexroressioiiL&l
L-selectin (MEL-14)
Mouse leukocvtes
L-selectin (Poly-MEL-14)
5± I
20± 10.2
T200
100+7.6
PgP-I (MJ64)
PgP-I (IM7)
53±11.4
94+3.4
LFA-I
121+15.3
. Mac-1
Human neutrophils
'
Human monocvtes
Human Ivmphocvtes
87±15
L-selectin (DREG55)
6+ 2.6
L-selectin (BREG56)
16+1.5
L-selectin (DREG 110)
5+3.8
L-selectin (DREG 152)
15+2.8
L-selectin (DREG2G0)
28±3.3
L-selectin (Leu-8)
10+1.4
M ac-1
46+5.8
CD44
96±9.2
L-selectin (DREG 55)
18±8.0
Mac-1
43+8.8
CD44
91±4.0
L-selectin (DREG 55)
9+3.6
95+4.6
CD44
a) Mouse leukocytes included bone marrow cells and the W EHI 78/24 cell line, which
behaved identically. Human cells were isolated from peripheral blood by 1% dextran
sedimentation, as described (16). Cells were treated with 5 mM BS^ for 15 minutes on ice,
washed, and the effect on staining with the indicated antibodies determined by flow
32
cytometry. Human neutrophils, monocytes, and lymphocytes were identified by their
distinctive side and forw ard light scatter profiles. Mode fluorescence values were
determined for BS3-Ueated and untreated cells. The data are presented as the percentage o f
control untreated cell expression o f each o f the antigens (mode fluorescence after
BS3Zmode fluorescence o f control x 100), where values close to 100 reflect little effect o f
the crosslinking agent. Background fluorescence did not change after treatment with BS3.
Values reflect mean±SEM of at least 3 different experiments.
In the human, the staining o f six different anti-human L-selectin mAbs was greatly
reduced after treating human leukocytes with BS 3 (Table I, Figure I). BS 3 had equal
effects on human neutrophils, monocytes, and lymphocytes (Table I). Kinetic analysis
showed that B S 3 caused >50% reduction in L-selectin expression within 5 min on all
leukocyte types (data not shown). No significant changes were noted in the expression o f
CD44 on human cells after treatment with BS 3 (Table I and Figure I), however, anti-Mac1 staining of human neutrophils and monocytes was greatly reduced (Table I).
We next compared the effects o f BS 3 with another chemical crosslinker, S-DST. SDST, which has the same specificity for lysines as BS3, but only crosslinks lysines within
6 Angstroms of each other (Pierce), had no effect on human L-Selectin expression (Figure
I and Table 2). However, S-DST treatment still caused the loss o f anti-M ac-1 staining on
human neutrophils and monocytes (Table 2). This demonstrated a unique specificity to
BS3, which was related to the distance between crosslinked lysine residues that induces
shedding. It also showed that the activity o f BS 3 was not simply due to non-specific effects
o f the crosslinking procedure.
Treatment o f human leukocytes with BS2 causes shedding of L-selectin.
Even though we demonstrated reduced L-selectin staining with six different mAbs and
one polyclonal anti-L-Selecdn, it was still possible that BS 3 just altered multiple antigenic
epitopes, as was seen for M J64 and anti-M ac-1 staining. To address this issue, and
determine if BS 3 induced L-selectin down-regulation was sim ilar to that induced by
33
activation, we tested for shed L-selectin in the supernatant o f treated cells. Serial dilutions
I
T . ; : 11."Y
*
I!
CD
U
CD
>
ro
CD
CD
JD
E
ZD
C
cr
■*>
Log Fluorescence
F igure I. Chemical cross-linking of mouse and human leukocytes causes L-selectin
down-regulation. Mouse bone marrow and human peripheral blood leukocytes were
prepared as described in materials and methods and treated with BS^ for 15 minutes on ice.
(A) shows anti-L-selectin (MEL-14) and anti-T200 (30G12) staining of control (MEL-14,
solid l in e _____ and 30G12, dashed lin e _______ ) and BS^ treated (M EL-14, dotted
line.......and 30G12, spaced dotted line..................) mouse leukocytes; (B) shows anti-Lselectin (DREG-55) and anti-CD44 (Hermes 3) staining of control (DREG-55, solid line
_____ and Hermes 3, dashed lin e _______) and BS^ treated (DREG55, dotted line.........
and Hermes3, spaced dotted line.............) human leukocytes; and panel (C) shows anti-Lselectin (DREG55) and anti-CD44 (Hermes 3) of control (DREG55, solid lin e_____ and
H erm esi, dashed l i n e _______) and S-DST treated (DREG55, dotted line...........and
Hermes3, spaced dotted line............. ) human leukocytes.
34
T able 2. Crosslinked lysine residues must be greater than 6 Angstroms apart to induce Lselecdn down-regulation.
Antigeim
C e llu la r an tig en
exroresgiom a s p e rc en t
S-DST
L-selectin (Leu-8)
Mac-1
CD44
8
108
15
29
93
101
a) Human cells were isolated from peripheral blood by 1% dextran sedimentation, as
described (16). Cells were treated with 5 mM BS^ or S-DST, or HBSS for 15 minutes on
ice, washed, and the effect on staining with the indicated antibodies determined by flow
cytometry. M ode fluorescence values were determined for BS^ and S-DST-treated, and
untreated cells. The data are presented as the percentage of control untreated cell expression
of each o f the antigens (mode fluorescence after BS^ or S-D ST treatm ent/m ode
fluorescence o f control x 100), where values close to 100 reflect little effect o f the
crosslinking agents. Data are the means o f two separate experiments.
of supernatant fluid collected from treated leukocytes were coated onto Immulon plates for
ELISA analysis. DREG 152 and DREG 200 were used to test for the presence of shed Lselectin, since these antibodies were specifically raised against the shed form of L-selectin
(4). Both mAb showed reactivity with a protein in the supernatant fluid, which could be
diluted 1:16 and still exhibit significant activity (Figure 2). An irrelevant isotype negative
control mAb or second stage alone did not show any reactivity with the crosslinkedinduced shed L-selectin, nor was there significant reactivity o f DREG 152 and DREG 200
with supernatant fluids from control untreated cells incubated under similar conditions
(Figure 2). Finally, the crosslinking agent itself did not account for the positive reactivity in
the ELISA, since medium plus BS^ alone was negative (data not shown).
Crosslinking o f L-selectin with specific monoclonal antibodies causes loss of surface
expression o f L-selectin.
To mimic a more physiologically relevant ligand we used a combination of anti-L-
35
0 . 0 8
—T
■D-CREG2C0 Crcsslinkec supernatant
- 4 - OREGl52 Crossiinkea supernatant
DREG200 Control supernatant
0.C6 _
-O-OREGl 52 Control supernatant
0.02 -I
1 /i
1 /8
1/1 6 1 / 3 2
1 / 6 4 1/1 2 8 1 / 2 5 4 I / 5 0 8
Dilution
F igure 2. Chemical crosslinking of human leukocytes caused L-selectin shedding from
the leukocyte cell surface. Human peripheral blood leukocytes were isolated from
heparinized blood by dextran sedimentation (4), washed, treated with BS^ for 15 minutes
and the supernatant fluid collected. Serial dilutions of the fluid were tested for the presence
o f L-selectin by ELISA analysis using DREG 152 and DREG200 as described in the
M aterials and Methods. Values represent absorbance, measured at 405 nM, minus
background versus the dilution of the supernatant fluid. Each data point reflects the
mean+SEM from 6 separate experiments. A comparison of the reactivity o f the DREG
mAbs with supernatant fluid from BS 3-treated and control cells is provided.
selectin antibodies to crosslink leukocyte L-selectin. We treated cells with a combination of
mouse anti-human L-selectin antibodies and a second stage anti-mouse antibody
to
crosslink the primary and-L-selectin antibody (as described in the materials and methods).
Crosslinking of L-selectin with specific monoclonal antibodies at 37°C caused loss o f
surface expression of L-selectin (Figure 3 and Table 3). The crosslinking was done at
37°C to allow for more efficient crosslinking by patching at physiological temperature.
That the loss in surface expression of L-selectin is due to specific crosslinking and not a
36
T ab le 3. Cross-linking with specific mAb at 37°C causes loss o f surface expression o f
the leukocyte homing receptor L-selectin
Recem tor
M ode flMoreseem.ee c ro s s -lin k e d
Cross-flimking^secoMlarv mAb
4fi£_
L-selectin
57 -
CD45
66
4d
None
Lflia^Ek
Mas=l£
3Z.QCL
49
42£L
27
2Z.SC.
8
APC.
82
2Z.eC
348
79
46
37
73
323
3d
36
37
53
301
a) Human peripheral blood neutrophils were treated with and CD45 mAb (L3B12) or a
combination o f anti-L-selectin antibodies (DREG 152 and BL246) on ice for 20 min,
washed, and the primary mAb was cross-linked with a HTC-Iabeled secondary mAb
(FITC-goat anti-mouse IgG). After washing away excess second-stage reagent, the
cells were either incubated at 37°C or on ice for 15 min, and the expression o f the
cross-linked antigens was determined by flow cytometry. Neutrophils were identified
by their characteristic forward and side scatter profiles and the data are presented as
mode fluorescence, for a representative experiment among three performed.
b) As an additional means o f detecting L-selectin expression, cells treated as in “a” were
blocked with 6% mouse serum in the presence of 2mM sodium azide for 15 min on ice
and stained with PE-conjugated Leu -8 (mouse anti-human L-selectin, which binds a
distinct epitope from that recognized by DREG-152 and EL246 mAb).
c) Cells treated as in “b” but stained with PE-anti M ac-1 instead of Leu- 8.
d) Cells stained with second-stage reagent alone.
result of activation is demonstrated by the fact that even though incubation of cells alone or
crosslinking o f a different surface protein caused similar levels o f activation (upregulation
o f C D l lb/CD18), only L-selectin specific crosslinking induced a loss in surface expression
of L-selectin (Table 3). As an additional means o f detecting L-selectin expression we used
PE-Leu -8 (which binds a distinct epitope from DREG152 and EL246, M.A. Jutila,
personal com m unication) to stain cells crosslinked with anti-L-selectin monoclonal
antibodies or control cells crosslinked with anti-CD45 antibody. As shown in Figure 3 and
Table 3, loss in surface expression of L-selectin was observed only on anti-L-selectin mAb
crosslinking of L-selectin.
37
L-Sclectln
CD45
TS *
T5T
Tsy
Leu-8 Staining
Figure 3. The human leukocyte homing receptor L-selectin is down-regulated upon
specific antibody cross-linking at 370 C. Human peripheral blood cells were treated as
described in the legend for Table 3 with either mAb against CD45 (L3B12, left panel) or a
combination o f antibodies against L-selectin (DREG-152 and EL246), right panel) and then
cross-linked with an anti-mouse second-stage reagent on ice. After cross-linking, the cells
were incubated at either 37°C or on ice for 15 min, blocked with 6% mouse serum in the
presence o f 2 mM sodium azide and then stained with PE-conjugated Leu -8 to determine
the surface expression o f L-selectin.
Activation-independent shedding of L-selectin occurs in vivo.
To determine if activation-independent shedding of leukocyte L-selectin occurs in vivo,
we harvested plasm a from normal individuals [those not undergoing any noticeable
inflammatory events or infection, and whose peripheral blood leukocytes showed no
obvious signs o f activation (normal CDl 1/18 and L-selectin levels by flow cytometry)] and
38
1
2
3
-7
- r t*
i .I I
4
5
6
7
r
Figure 4. Shed L selecdn can be detected in the plasma of healthy adults. Plasma from an
individual, who showed no obvious signs of disease or ongoing inflammatory events, was
separated on a 8% SDS-PAGE under non-reducing conditions. The separated proteins
were transferred to nitrocellulose and probed with DREG 152 and DREG 56 anti-L-selectin
mAbs. Lanes 1,2, and 3 were probed with 10^g/m l, SOpg/ml, or 100 Hg/ml DREG 56
mAb respectively. Lanes 5 and 6 were probed with lOOpg/ml and 50pg/ml DREG152,
respectively. Lane 4 was probed with an isotype negative control mAb and lane 7 was
probed with second stage alone. The distance of migration of molecular weight markers
were as indicated and are in kD.
tested for the presence of circulating L-selectin. To increase our chances of detecting shed
L-selectin in human plasma, SDS-PAGE/Westem blot analysis was done. This allowed
separation of any soluble L-selectin molecule from other serum proteins, such as
39
immunoglobulin, which would lead to high background with our anti-Ig detection systems.
A large smear was detected in all lanes which was greater than I(X) kD and most likely
represented different immunoglobulin isotypes (Figure 4). The DREG naAbs reacted with
two discrete bands in the plasma, which ran at molecular weights o f approximately 65-70
kD (Figure 3). The different molecular weight bands may have reflected different leukocyte
forms o f L-selectin (neutrophil vs lymphocyte, for example), which have previously been
shown to be o f different size (7, 10). No reactivity was seen with an irrelevant isotype
control mAb and second stage control (Figure 3). The same pattern was repeated with
seven different plasma samples.
Discussion
W e demonstrate in this chapter that a novel activation-independent mechanism o f
shedding o f leukocyte L-selectin can occur in vitro and perhaps in vivo as well. W e
propose that L-selectin-dependent leukocyte-endothelial cell interactions result in
crosslinking and shedding o f the molecule. Importantly, this hypothesis is consistent with
the presence o f L-selectin in blood, since L-selectin-endothelial cell interactions are
constantly occurring in vivo. For example, lymphocytes constitutively recirculate through
peripheral lymphoid tissues o f the body via L-selectin mediated adhesion pathways (3).
Recently, others have shown that leukocyte rolling along the vascular endothelium in vivo,
which occurs in the absence o f overt inflammation, involves L-selectin (12,13). Therefore,
crosslink induced shedding o f leukocyte L-selectin within the vascular lumen could be a
common event.
It has been previously proposed that loss o f L-Selectin allows the cell which is tightly
bound to the vascular wall to release and migrate into the underlying tissues (4). However,
40
activation was required and the model was primarily directed toward leukocyte entry into
sites o f inflammation. The results in this chapter provide the basis for a mechanism
whereby L-selectin shedding could be important in the entry of leukocytes into uninflamed
tissues. In this model crosslinking o f L-selectin via its endothelial ligand would be all that
is necessary for L-selectin shedding. Shedding would take place only at the site on the
leukocyte cell surface which makes contact with the vascular endothelium. Thus, the cells
entering uninflamed tissues would still express significant levels o f L-selectin, which is
consistent with observations that lymphocytes which have recently em igrated into
uninflamed peripheral lymph nodes are L-selectin positive (18). The apparent total loss o f
L-selectin on leukocytes in some sites o f inflammation, such as the 4 hour thioglycollateinflamed peritoneum, would be due to a combination o f crosslinking and activation via
inflammatory factors (6). Finally, the model presented here provides a rapid and specific
regulation for L-selectin expression on all leukocyte types, since the effects of BS^ were
the same for neutrophils, monocytes, and lymphocytes. This is in contrast to the effects o f
activation, which is rapid for neutrophils (9), but rather slow for lymphocytes (16,17).
To test our hypothesis directly, a natural, multi valent ligand for L-selectin that binds
leukocytes in suspension and does not cause activation must be identified. In the absence o f
this we relied on other means to crosslink L-selectin and show that activation is not
required for release o f the cell surface protein. A t the very least, the chemical means o f
inducing L-selectin shedding described here should be a more useful tool than gross cell
activation to study the molecular events involved in how L-selectin is released. Though
BS^ is not specific for L-selectin and does alter antigenic epitopes on many different
surface antigens, it is possible that BS^ mimics endothelial cell crosslinking of adjacent Lselectin molecules. BS^ reacts with lysines in surface proteins, and the binding domain of
L-selectin is rich in this amino acid (27,30). That crosslinking, and not the chemical
41
crosslinker or activation o f cells, caused loss o f surface expression o f L-selectin was
demonstrated by the use o f anti-L-selectin specific monoclonal antibodies to crosslink and
induce loss o f L-selectin expression. W e demonstrate that the crosslinking agent may have
to link lysines that are greater than 6 Angstrom apart in order to cause shedding o f Lselectin, which could imply the crosslinking o f separate and adjacent proteins versus within
the same molecule. It is also possible that the adjacent proteins are both L-selectin, since it
has been recently shown that L-selectin molecules are preferentially clustered at the ends o f
the microvilli o f cells (18, 29). Further, if L-selectin gets crosslinked to an unknown
surface protein the unknown molecule must also be susceptible to shedding, since the
covalently attached complex clearly comes off o f the cell surface. There are few examples
of surface proteins which show this type of regulation (4).
It has been previously proposed that the release o f L-selectin is due to an unique
activation-dependent proteolytic process (8,9 ). W e thought that either the protease had to
be translocated from intracellular pools to the cell surface or it was present on the cell
surface and simply needed to be "turned on" by activating signals: the point of regulation
would be at the level o f the protease. The results presented here suggest that neither o f
these events may be required. A conform ational change in L-selectin induced by
crosslinking, which exposes a protease sensitive region, may be the critical event
controlling shedding. Gross activation may indirectly cause the same conformational
change, but unlike crosslinking, it occurs over the entire cell surface. Therefore, the
regulation o f L-selectin shedding may be controlled by the conformation o f the molecule
and not the activation o f the protease. This would provide an extremely rapid process for
regulation of L-selectin.
In conclusion, in this chapter, we have shown that crosslinking L-selectin on the cell
surface o f mouse and human leukocytes results in shedding o f the molecule, which is
42
independent o f leukocyte activation. These results suggest a new and highly specific
m echanism o f adhesion protein regulation, which is uniquely dependent upon the
interaction o f the adhesion receptor with its specific ligand. Further, chemical crosslinking
o f L-selectin may provide a better system to study the events involved in shedding o f the
molecule.
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12. Ley, K., P. G aehtgens, C. Fennie, M .S. Singer, L A . Lasky, and S.D. Rosen.
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13. von A ndrian, U. H., J.D. Chambers, L. McEvoy, R.F. Bargatze, K.E. Arfors,
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609-618.
16. Huang, K., I. Suhn-Young, W.E. Samlowski, and R A . Daynes. 1989. M olecular
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17. Jung, T.M . and M O. D ailey. 1990. Rapid m odulation o f hom ing receptors
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18. P ilarsk i, L.M ., E.L. Turley, A .R.E. Shaw, W .M. G allatin, M .P. Laderoute,
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19. Camerini, D., S.P. James, I. Stamenkovic, and B. Seed. 1989. L eu- 8/T Q -l is the
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25. S taros, I. V. 1982. N -H ydroxysulfosuccinim ide activ e esters.: B is (N hydroxysulfosuccinim ide) esters o f tw o dicarboxylic acids are hydrophilic,
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H. R odriguez, T. N guyen, S. Stachel, and S.D. Rosen. 1989. Cloning o f a
lymphocyte homing receptor reveals a lectin domain. Cell. 56:1045-1055.
28. Yednock, T A ., E.C. Butcher, L.M. Stoolman, and S.D. Rosen. 1987. Receptors
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29. Picker, L. J., R A . W am ock, A R. B um s, C.M. D oerschuk, E.L. Berg, and
E.C. Butcher. 1991. The neutrophil selectin LEC A M -I presents carbohydrate
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30. Siegelman, M .H., M. van de Rijn, and I.L. Weissman. 1989. M ouse lymph node
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46
C H A PTE R 3
LIG AN D IN TER A C TIO N S IN VIVO AND ANTIBODY C RO SSLIN K IN G
O R FU C O ID IN TR E A T M E N T IN VITRO LEADS TO
D O W N R EG U LA TIO N O F LEU K O C Y TE L-SE L E C T IN
Introduction
The nonrandom recirculation of lymphocytes and the recruitment of neutrophils to sites
of inflammation are essential immunological phenomena mediated by highly specific,
leukocyte-endothelial cell binding events (1-7). Two types o f leukocyte-endothelial cell
interactions have been defined in vivo: I) an initial interaction represented by rolling o f the
leukocyte along the endothelium, and 2) a subsequent tight, static adhesion prior to
extravasation (8-10). Two classes o f leukocyte adhesion molecules, selecdns and integrins,
regulate these binding events (5,6,9-11). L-selectin is a member o f the selectin family,
which also includes E- and P-selectin (1,4,5,11-17). Selectins are characterized at the
m olecular level by a N-terminal, mammalian, C-type lectin domain, followed by an
epidermal growth factor (EGF)-Jike domain and a series o f consensus repeats similar to
domains found in complement binding proteins (1,5,15,18-20). L-selectin is expressed by
leukocytes and is involved in regulating the initial rolling interaction with the vascular
endothelium (8-13) by specific recognition, via its lectin dom ain, o f sialylated
carbohydrates expressed by endothelial cells (21-27). A nti-L-selectin m onoclonal
47
antibodies (mAbs) and certain sulfated polysaccharides, like fucoidin, block leukocyteendothelial cell adhesion in vitro and leukocyte rolling in vivo (11,28-33). L-selectin
mediated rolling is a prerequisite to inflammation, since reagents that block rolling also
block inflammation in vivo (1,5). Tight adhesion o f leukocytes to the endothelium and
subsequent transendothelial migration are mediated by the leukocyte integrins, which bind
endothelial cell ligands M onging to the immunoglobulin superfamily (34-37). L-selectin
mediated rolling is necessary for neutrophil function, since L-selectin mediated rolling is
required for the tight adhesion o f neutrophils mediated by 62-integrins at physiological
shear rates in vivo (38).
Leukocyte activation results in an inverse regulation o f L-selectin and integrin
expression (39). In response to activating signals, L-selectin is rapidly shed from the cell
surface, whereas the leukocyte integrins are upregulated in expression and/or function (29,
35-39). Kishimoto et al. (1989) (39) proposed that the loss o f L-selectin allows the bound
leukocyte to release from the endothelium and emigrate into underlying inflamed tissues
(28,30,39). These pathways are described in detail in many recent papers and reviews
(1,5,8,9,28-30,39,40). Importantly, the regulation o f L-selectin expression and function in
these models is dependent on large concentrations o f activating signals delivered by
chemotactic agents or cytokines released by inflamed tissues or the endothelium itself.
In the last chapter, we showed that treatment o f neutrophils with chemical crosslinkers
or a combination o f two anti-L-selectin antibodies induces downregulation of L-selectin
expression that cannot be accounted for by activation (defined as an inverse increase in the
beta-2 integrin M ac-1 expression) alone (41). From these results, w e proposed that
activation-independent shedding may occur during the interaction o f L-selectin with its
ligand on endothelial cells (41). This hypothesis suggests the existence of a unique
m echanism o f L-selectin regulation that could occur during leukocyte-endothelial cell
48
binding events taking place in the absence o f inflammatory or activating signals, such as
leukocyte margination and lymphocyte homing or recirculation. These two types o f
interactions are constantly occurring in vivo and involve L-selectin (1,2-6,39,40). Our
hypothesis predicts that shedding o f leukocyte L-selectin into the blood is continuous
during these events and is supported by our observations that high levels o f shed L-selectin
are detectable in the plasma o f healthy adults (41), which has recently been confirmed by
Schleiffenbaum et al. (42).
To further test our hypothesis, it was imperative that we demonstrate a similar event via
more physiological means o f crosslinking L-selectin than those used in our earlier report.
W e used human selectin cDNA-transfected, mouse lymphoma cell lines to determine
whether crosslinked-induced downregulation was unique to L-selectin or was a property o f
other selecdns as well. We extended our earlier analysis by testing the effect of crosslinking
L-selectin with function blocking anti-L-selectin mAbs whose epitopes map to the lectin
dom ain, polysaccharides that bind L-selectin, and, importantly, L-selectin-dependent
interactions in vivo on the regulation of L-selectin expression. W e found that L-selectin
transfectants rapidly downregulate L-selectin following crosslinking, whereas E selecdn on
transfected cells was unaffected. We also found that downregulation o f L-selectin could be
induced by any o f the m eans o f potentially cross-linking L-selectin listed above.
Im portantly, L-selectin-specific ligand interactions in vivo also induced L-selectin
downregulation. Our results provide physiological evidence for a novel mechanism o f Lselectin regulation that acts independently or synergistically with activation in regulating
leukocyte-endothelial cell interactions and is unique to L-selectin.
49
Materials and Methods
Antibodies and polysaccharides
D REG -55, D REG -56, D REG-110, D REG-152, D REG-200, and Leu-8 (Becton
Dickinson, M ountain View, CA) are mouse mAbs that recognize human L-selectin [12].
The DREG mAbs are all Ig G l, whereas Leu-8 is an IgG2a. EL-246 is a mouse IgG l that
recognizes both human leukocyte L-selectin and endothelial-cell E-selectin [48]. L3B12
(mouse IgG2b) recognizes CD45 on human leukocytes [41], and Hermes-3 (mouse Ig G l)
recognizes human C D 44 [47]. FITC-Iabelled goat anti-m ouse (second stage) was
purchased from Sigma (Sigma Chemical Co., St. Louis, MO). Fab' goat anti-mouse IgG
was a gift from Jackson Laboratories (Jackson ImmunoResearch Lab. Inc., W est Grove,
PA) and was FITC-conjugated according to standard procedures. Finally, anti-M ac-1
(mouse IgG2a) was purchased from Becton Dickinson. The polysaccharides, fucoidin,
mannan, and chondroitin sulfate were purchased from Sigma. Dextran and dextran sulfate
were obtained from Pharmacia (Pharmacia LKB Biotechnology Inc., Piscataway, NI).
Preparation o f cell suspension
Human peripheral-blood leukocytes were prepared, as previously described [12,28].
The murine Abelson-virus-transformed C57L pre-B cell lines, L I -2, stably transfected with
either E- or L-selectin cDNA, were a gift from E. C. Butcher, Stanford University [46].
M ouse bone-m arrow cells were harvested, as previously described, and used as an
additional source o f neutrophils [12,28] .
Flow cytometric analysis
Staining o f cells was carried out in 4-ml tubes. Briefly, I x IO6 cells were initially
50
incubated in 5% rabbit serum for 10 min on ice to block Fe receptors. The cells were
washed and then incubated with primary antibody at 50 jig/ml for 20 m in on ice. A fter
washing, bound antibodies were revealed by incubation with FFTC-conjugated goat anti
mouse Ig (second stage) (Sigma) at a 1:100 dilution in 5% FBS in BBSS. Flow cytometric
analysis was perform ed on a FACScan (Becton Dickinson), as described [29]. In some
experiments, direct stains with PE-labelled Leu-8 or M ac-1 were done. Background
fluorescence was established by staining with conjugated, second-stage reagent alone. Data
are presented as contour plots or mode fluorescence.
Chemical crosslinking
For chemical crosslinking o f cell surface proteins, both human leukocytes and L- or Eselecdn transfected L l/2 cells were treated with BS^ [bis (sulfosuccinimidyl) suberate].
B S^ is a homobifunctional and membrane-impermeable chem ical crosslinker, which
covalently crosslinks proteins 11.4 A apart via lysine residues [41]. Conditions used for
crosslinking were as previously described (5x1
cells/ml in 5 mM DSA, 15 - 25 min on
ice) [41]. The crosslinking agent was quenched by washing the cells in PBS plus 5% rabbit
serum or a Tris-glycine buffer. Controls included cells incubated on ice in BBSS alone.
After treatment, the cells were kept on ice to prevent activation and membrane turnover, and
then they were stained with the antibodies listed above for flow cytometric analysis.
Antibody crosslinking of L-selectin
Human leukocytes or L-selectin-transfected L l/2 cells were treated with anti-L-selectin
antibodies (DREG-55, DREG-56, DREG-110, DREG-152 o r DREG-200), and-CD45
antibody L3B12, or anti-CD44 antibody Hermes-3 at a final concentration o f 50 (ig/ml in
HBSS on ice for 20 min and washed in HESS. The primary antibody was either cross-
X
SI
linked with FITC-conjugated goat anti-mouse IgG or detected with FTTC-conjugated Fabi
goat anti-mouse IgG on ice for 20 min. A fter washing away excess second-stage reagent,
cells were incubated at 37°C or on ice for 15 min, washed, and the fluorescence intensity
o f the second stage was determined by flow cytometry. After mAb crosslinking (on ice or
at 37°C), cells were blocked with 10% mouse serum in the presence o f 2 mM azide and
stained with PE-Leu-8 as an additional means o f detecting L-selectin expression. Similarly
treated cells were stained with PE-M ac-I instead o f Leu-8 to m onitor upreguladon o f
C D llb /C B 1 8 due to cell activation. Flow cytometric analysis was perform ed on a
FACScan.
Immunofluorescence microscopy
To visualize patch of cap formation, human leukocytes, treated with either anti-Lselectin or anti-CD45 antibodies on ice for 20 min, were washed, primary antibodies
crosslinked with a PE-conjugated, goat anti-mouse second stage for 20 min, washed again,
and then incubated at 37°C or oh ice for 15 min. These cells were then fixed in 1%
paraformaldehyde and observed with a fluorescence microscope.
ry^giinkinp o f L-selectin with fucoidin and other W vsacchaddes
Hum an leukocytes or L-selectin transfected L l/2 cells were treated with fucoidin,
mannan, chondroitin sulfate (all from Sigma), or dextran T500 (Pharmacia) at 500-2000
|ig/m l HBSS on ice for 20 min and then either incubated at 37°C or kept on ice for an
additional 15 min. A fter washing away excess sugars, the level o f L-selectin, CD45, o r
M ac-1 expression was determined by flow cytometric analysis, as described above. None
of the sugars used blocked anti-L-selectin mAb staining. Results are presented as percent of
control, where percent of control = mode fluorescence of control cells incubated with or
'
' -
''I
!1I
52
without fucoidin at 4°C , divided by mode fluorescence o f cells treated with fucoidin and
incubated at 37°C, and the resulting value multiplied by 100.
In vivn trafficking of L-selectin-transfected L l/2 cells
L 1/2 L-selectin transfectants were labeled w ith FITC according to established
procedures with m inor modifications [43,44]. Briefly, 5 x IO^ cells in Ca & M g-free
HBSS were incubated with FTiC (30 pg/ml) at room temperature for 20 min. Excess FlTC
was removed by washing the cells three times in BBSS. Labeled cells were resuspended to
a concentration o f 8 x I O^ cells per ml, and Im l was injected into G D I or BALB/c mice
intravenously via the tail vein. Similarly labeled cells, incubated in the dark at 37°C in the
presence o f 20% mouse serum for the duration o f the experiment, served as controls.
Fifteen m in after in vivo trafficking, peripheral blood was collected by retra-orbital
bleeding and RBCs were lysed by hypotonic lysis. The in vivo trafficked and control cells
were stained with anti-human, L-selectin antibodies and the prim ary antibodies revealed
with a PE-conjugated, goat anti-mousej second-stage antibody. The expression o f Lselectin was determined by two-color flow cytometric analysis on a FACscan. To block Lselectin-m ediated interactions in vivo, FITC-Iabeled L l/2 cells were incubated with
blocking anti-L-selectin mAbs (DREG-110 and DREG-200) at 50 (J.g/ml in HBSS for 10
min on ice, washed, injected into mice and allowed to traffic, as described above, and then
the expression of L-selectin was determined by flow cytometry.
Results'
Cmsslinlrihp of primary anti-L-selectin mAbs is required to
induce downregulation of neutrophil L-selectin
In chapter 2 , 1 showed that a combination o f two anti-L-selectin mAbs and whole-
53
molecule second stage induced a loss in L-selectin expression without a significant increase
in M ac-1 expression (41). It was important to know whether receptor perturbation or
specific crosslinking was involved in the downregulation seen in our earlier study. Also,
the antibodies used in our earlier study have been mapped to two different sites on Lselectin, one to the short consensus complement binding repeats (mAb EL246) and the
other (DREG-152) to the lectin domain (ref. 48 and M.A. India, unpublished). It is known
that the lectin domain of L-selectin is required for function and agents which bind the lectin
domain, such as mAbs and the polysaccharide fucoidin, inhibit function (11, 28-33, 38).
W e have extended our observations by testing whether crosslinking o f single anti-Lselectin mAbs which bind the lectin domain o f L-selectin (all the DREG mAbs, ref. 12, and
T.K. Kishimoto, personal communication), induces downregulation. Binding o f L-selectin
w ith three different anti-L-selectin antibodies o r control antibodies alone and then
incubating at 37°C did not alter the level o f L-selectin expression (Figure 5). In contrast,
treating leukocytes with any o f four anti-L-selectin prim ary antibodies (DREG-152,
DREG-200, DREG-56 or D R EG -110), followed by whole-molecule second stage, and
then incubating at 37°C caused a significant loss in the surface expression o f L-selectin
(reduction ranged from 50-75%) (Figure 6 and Table 4). As controls, cells treated to
crosslink CD44 or CD45 under similar conditions with anti-CB44 or anti-CD45 mAbs did
not alter L-selectin expression (Figure 6 and Table 4). Furthermore, the expression o f
C D llb/C D 18 was the same on cells following crosslinking CD44, CD45, or L-selectin,
though expression increased compared to cells on ice (Table 4 and Ref. 41).
To determ ine if surface antigen crosslinking was necessary to induce the effects
described above, we treated cells with anti-L-selectin mAbs followed by either Fab' or
whole-molecule second stage. Required crosslinking was dem onstrated since a similar
downregulation did not occur if Fab' was used instead o f a whole IgG molecule as the
second-stage reagent (Figure 7).
54
Antihodv
DREG-152
DREG-200
DREG-56
DREG-110
Hermes-3
0
20
40
60
80
100
Percent of control
mode fluorescence
F ig u re 5. Primary anti-L-selcctin antibodies alone do not induce L-selectin downregulation. Human peripheral-blood neutrophils were treated with anti-L-selcctin antibodies
(DREG-56, DREG-152 or DREG-200) or anti-CD45 antibody (L3B12) on ice for 20 m in ,
w ashed, and either incubated at 37°C or on ice for 15 min. Following the 15 min
incubation, the cells were washed, treated with goat anti-mouse second stage antibody for
20 min on ice in the presence of 2mM azide, and the expression o f L-selectin and CD45
was determined by flow cytometry. Cells treated as above were blocked with 10% mouse
serum in the presence o f 2mM azide for 15 min on ice and stained with PE-conjugated Leu8 (mouse anti-human L-selectin, which binds an epitope distinct from those recognized by
D REG -56, DREG-200 and DREG-152 mAb). Neutrophils were identified by their
characteristic forward and side scatter profiles, and the data are presented as percent of
control mode fluorescence (Leu-8 staining) from a representative experiment among at least
three performed, wherein control are cells treated as above, except that all incubations were
on ice. Background fluorescence values were subtracted from all positive values.
55
T ab le 4. Effect o f prim ary antibodies crosslinked with whole molecule second stage
antibody on the surface expression of L-selecdn and M ac-1.
H staged
Leu-Sfe
Mac-Ig
Antibodv
ICE
3Z>C_
ICE
3Z9C_
J£Eu
a m
DREG-110
202
61
22
10
17
209
DREG-200
195
53
34
8
21
220
DREG-152
234
85
38
12
36
215
D R E G -56'
175
74
53
29
20
209
Hermes-3
335
280
53
46
29
168
L3B12*
I(X)
92
57
46
74
195
a) Human peripheral blood neutrophils were treated with different anti-L-selectin antibcxiies
(DREG_56, D R E G -110, D R EG -152 o r DREG-200), anti-CD44 (Hermes-3), or andCD45 (L3B12) on ice for 20 min washed, and the primary mAh was cross-linked with a
FITC-Iabeled secondary mAh (FITC-goat anti-mouse IgG). A fter washing away excess
second stage reagent, the cells were either incubated at 37oC or on ice for 15 min, and the
expression o f the cross-linked L-selecdn was determined by flow cytometry. Neutrophils
were identified by their characteristic forward and side scatter profiles and the data are
presented as mode fluorescence, from a representative experiment repeated at least three
times,
b) As an additional means o f detecting L-selectin expression, cells treated as in “a” were
blocked with 10% mouse serum in the presence o f 2mM azide for 15 min on ice and
stained with PE-conjugated Leu-8 (mouse anti-L-selectin, which binds a distinct epitope
from that recognized by DREG-56, DREG-200, DREG-110 and DREG-152).
c) Cells treated as in “b” but stained with PE-anti-Mac-1 instead o f Leu-8
Background fluorescence with isotype m atched control or second stage alone was
subtracted from all positive values, ^separate experiment.
56
Antibody
DREG-152
DREG-200
DREG-56
DREG-110
Hermes-3
0
20
40
60
80
100
Percent of control
mode fluorescence
Figure 6. Primary anti-L-selectin antibodies plus second stage induce downregulation of
L-selectin at 37°C. Human peripheral-blood neutrophils were treated with different anti-Lselectin antibodies (DREG-56, DREG-110, DREG-152 or DREG-200) or anti-CD44 mAb
(Hermes-3) on ice for 20 min, washed, and the primary mAb was bound by a FITClabelled secondary mAb (FITC-goat anti-mouse IgG). After washing away excess second
stage reagent, the cells were either incubated at 37°C or on ice for 15 min, and the
expression of the crosslinked L-selectin was determined by staining with the anti-L-selectin
mAb PE-Leu-8, as described in figure I. Neutrophils were identified by their characteristic
forward and side scatter profiles, and the data are presented as percent of control mode
fluorescence as described in the legend for figure I.
We examined the cells treated (for 15 min) to crosslink either L-selectin or CD45 by
immunofluorescence microscopy. As shown in figure 8, cells treated to crosslink L-selectin
57
DREG-56
DREG-200
O
20
40
60
80
100
Percent of control
mode fluorescence
Figure 7. Primary anti-L-selectin antibodies, followed by whole molecule, but not Fab',
second stage, induces L-selectin down-regulation at 37°C . Human peripheral-blood
neutrophils were treated with anti-L-selectin antibodies (DREG-56 o r DREG-200) on ice
for 20 min, washed, and the primary mAb was either cross linked with a FTTC-conjugated
secondary mAb [(FlTC -goat anti-m ouse IgG) hatched bars] or treated with FITCconjugated Fab' [(FITC-goat anti-mouse IgG) solid bars]. A fter washing away excess
second stage reagent, the cells were either incubated at 37°C or on ice for 15 min, and the
expression of L-selectin was determined by flow cytometry. Neutrophils were identified by
their characteristic forward and side light scatter profiles, and the data are presented as
mode fluorescence from a representative experiment repeated at least three times.
Background fluorescence o f isotype-matched control or second stage antibody alone were
subtracted from all positive values.
showed distinct clustering or patching o f L-selectin. In contrast, cells treated to crosslink
CD45 showed distinct capping at the 15 min time point. This result was the same as seen in
our earlier report (41), suggesting that the observed loss in fluorescence intensity detected
by flow cytometry was likely not due to rapid (15 min) capping and internalization of Lselectin.
58
F igure 8. T h e h u m a n le u k o c y te h o m in g r e c e p to r L - s e le c tin a g g r e g a te s to f o r m p a tc h e s
u p o n s p e c if ic a n tib o d y c r o s s lin k in g . H u m a n p e r ip h e r a l b lo o d c e lls w e r e tr e a te d a s in th e
le g e n d f o r f ig u r e 2 w ith e ith e r a n d - L - s e le c d n m A b ( D R E G - 15 2 , p a n e l A a n d B ) o r a n tiC D 4 5 m A b ( L 3 B 1 2 , p a n e l C a n d D ) a n d th e n c r o s s - lin k e d w ith a P E - a n ti- m o u s e s e c o n d s ta g e r e a g e n t o n ic e . A f t e r c r o s s - l i n k i n g , th e c e l ls w e r e w a s h e d , f i x e d in 1 %
p a r a f o r m a ld e h y d e a n d p h o to g r a p h e d o n a N ik o n F lu o r e s c e n c e m ic r o s c o p e . D is tin c t
p a tc h in g , b u t little c a p p in g , o f L - s e le c tin w a s d e te c te d ( P a n e l B , s o lid b la c k a r r o w h e a d s ) ;
w h e r e a s a n tib o d y c r o s s - lin k in g o f C D 4 5 p r e d o m in a n tly le d to c a p p in g ( P a n e l D , s o lid
b la c k a r r o w h e a d s ) . C o n tr o l c e lls tr e a te d to c r o s s - lin k e ith e r L - s e le c tin ( P a n e l A ) o r C D 4 5
( P a n e l C ) a n d k e p t o n ic e d id n o t n o t e x h ib it c a p o r p a tc h fo rm a tio n .
T r e a tm e n t o f n e u tro p h ils o r ly m p h o c y te s w ith th e s u lfa te d p o ly s a c c h a r id e
f u c o id in c a n a ls o in d u c e d o w n r e g u la d o n o f L - s e le c tin
V a r io u s s u lf a te d p o ly s a c c h a r id e s , lik e f u c o id in , b in d to a n d in h ib it th e f u n c tio n o f L s e le c tin , p r e s u m a b l y b y m im ic k in g th e e n d o t h e li a l lig a n d f o r L - s e l e c ti n w h ic h is a
f u c o s y la te d , s ia ly la te d g ly c o p r o te in ( 2 1 - 2 4 ) . F u c o id in h a s a ls o b e e n s h o w n to p o te n tly
59
block leukocyte rolling in rat mesentery in vivo (33). Therefore, we tested whether
incubating leukocytes with concentrations o f fucoidin (0.5 - 2 mg/ml) used by others to
inhibit L-selectin (21-23) would affect the surface expression o f L-selectin. Table 2 shows
that treatment o f human leukocytes in suspension with fucoidin at 37°C caused a 30-40%
loss in surface expression o f L-selectin. Importantly, the level o f M ac-1 expression was
similar on cells treated with or without fucoidin (Table 5), which indicated that activation
could not solely account for these results as in our earlier report (41). Other surface
molecules, like CD45, did not change in expression following treatment with fucoidin
(Table 5). Sugars, like mannan, chondroidn sulfate, and dextran, at similar concentrations
as used with fucoidin, did not affect L-selectin expression (Table 6). Dextran sulfate
induced a loss in neutrophil L-selectin in one experiment (Table 6), but this effect was not
consistent (data not shown).
T able 5. Exposure o f neutrophils and lymphocytes to soluble fucoidin induces L-selectin
down-regulation at 37°C.
PereeM of comitrol mode ffBaoreseemceS
NeMftroiohiBs
LvmmMeMes
Without
With
Without
With
Fucoidin
Fucoidin
Fucoidin
Fucoidin
L-selectin
96 ± 2
65 ± 14*
99±5
63 ± 4*
L-selectin
93 ± 4
63 ± 4 *
95 ± 1 0
70 ± 8 *
L-selectin
90 ± 5
62 ± 9 *
95 ± 5
63 ± 4 *
Mac-1
263 ± 6 5
232 ± 6 5
8 6 ± 11
90 ± 6
CD45
179 + 20
150 + 39
111 + 11
104 + 6
Antigen
a) Human peripheral blood leukocytes were treated with fucoidin (500 p.g/ml) on ice for 20
min and then transferred to either 37°C or kept on ice for another 15 min. The cells were
then washed and the expression o f L-selectin, M ac-1 or CD45 determ ined by flow
cytometry as described in the materials and methods. The data from 3-6 experiments are
60
presented as percent o f control (cells treated with fucoidin and kept on ice prior to staining
as opposed to cells treated with fucoidin and the moved to 37°C ) mode fluorescence.
Background fluorescence with isotype m atched control or second stage alone was
subtracted from all positive fluorescence values.
* Difference significant at p = <0.005.
T able 6 Polysaccharides that do not affect surface expression ofL-selectm .
I
M ode Fluorescemce
N e u tro p h ils
L y m p h o c y te s
Sugar
ICE
IO L
3Z2£
Dextran
49
64
66
68
Dextran
106
74
57
51
113
136
210
195
91
102
210
118
sulfate
Chondroitin
sulfate
Mannan
Human peripheral blood leukocytes were treated with dextran, dextran sulfate, chondroitin
sulfate or mannan (500p.g/ml) for 20 min on ice and then transferred to either 37°C or kept
on ice for another 15 min. The cells were then washed and the expression o f L-selectin
determined by staining with and-L-selectin antibody Leu-8 and analyzed by flow cytometry
as described in the m aterials and methods. The data, which represent one o f three
perform ed, are presented as mode fluorescence o f Leu-8 staining. Background mode
fluorescence values were subtracted from all positive values.
W e tested whether fucoidin, immobilized on polystyrene plates, could induce L-selectin
downregulation. Polystyrene plates were coated overnight with fucoidin, washed, and
human leukocytes added and allowed to interact with the plate on a rotator at 37°C for 20
min. As shown in figure 9, lymphocytes lost L-selectin expression following interaction
with immobilized fucoidin. Dextran, similarly immobilized on polystyrene plates, had no
effect on L-selectin expression (Figure 9).
61
Percent of control
mode fluorescence
Control
Dextran
coated
Fucoidin
coated
F ig u re 9. Im m obilized fucoidin induces downregulation o f L-selectin expression.
Polystyrene plates were coated with either fucoidin or dextran (2mg/ml) in PBS at 4oC
overnight and excess sugar removed by three cycles of washing. Human peripheral blood
leukocytes were allowed to interact with immobilized sugars at 37°C for 15 min under
constant rotation. After the incubation, cells were washed, stained with anti-L-selectin
antibody DREG-56, and analyzed on a FACScan (Becton-Dickinson. Lymphocytes were
identified by their characteristic forward and side scatter profiles, and the data, from a
representative experiment repeated at least three times, are presented as percent o f control
mode fluorescence, wherein control is mode fluorescence o f DREG-56 staining on cells
incubated at 37°C under rotation for the duration o f the experiment. Background
fluorescence values o f second-stage antibody alone were subtracted from all positive
values.
Crosslinker-induced shedding is a property of L- but not E-selectin
We used E- and L-selectin cDNA-transfected cell lines to determine if the chemical
means of crosslinking that induced L-selectin downregulation in our previous report (41)
62
was specific for L-selectin. The chemical crosslinking agent was BS^, which crosslinks
proteins within I l A via adjacent lysine residues. The lectin domains o f L- and E-selectin
have comparable numbers o f lysines (twelve and ten respectively), so it was likely that BS^
would interact comparably with each protein. As shown in figure 10, treatment o f the Lselectin transfectants with 5mM BS^ for 15 min caused complete loss o f L-selectin
expression. The same treatm ent o f E-selectin transfectants had no effect. Further, the
crosslinker had no effect on expression o f other surface proteins, like CB45 (Figure 10).
The loss in L-selectin expression was not due to a selective loss in the antigenic epitope
recognized by the anti-L-selectin mAb, since an antibody that recognizes both L- and Eselectin (EL-246) (48) loses reactivity with L-selectin but not E -selectin-positive cells
following chem ical crosslinking (data not shown). These results show that chemical
crosslinking-induced shedding occurs with L-, but not E-selectin.
W e then tested whether the treatments that induce L-selectin downregulation on normal
cells, shown above, produce similar results with the L-selectin transfectants. As shown in
table 7, anti-L-selectin mAbs and fucoidin-induced L-selectin downregulation on the L 1/2
transfectants. This indicated that the expression o f L-selectin on the transfected cells was
regulated in a manner similar to that on normal cells.
In vivo trafficking of L l/2 cells results in a loss in the surface expression o f L-selectin
W e used the L l/2 L-selectin transfectants to test the effect o f in1vivo trafficking and
ligand interaction on L-selectin expression. In vitro studies have shown that the L-selectin
transfected L l/2 cells bind peripheral lymph node-high endothelial venules (PLN-HEV) in
the Stam per-W oodruff assays, whereas nontransfected controls do not (46). Intrayital
video microscopy has shown that L-selectin transfected L l/2 cells roll, but do not bind
tightly to lymphoid HEV and eventually release and re-enter the circulation (R. Bargatze,
personal communication). L l/2 cells express low levels o f adhesion molecules, such as
C D lla/C D 18, that are required for transendothelial migration, which may account for
V.
63
Si
Log10 Fluorescence
Figure 10. Chemical crosslinking causes L-selectin, but not E-selectin, downregulation.
L l/2 cells, transfected with either L- or E-selectin cDNA, were untreated (dashed-----line) or treated with the chemical crosslinker BS^ (dotted .......line) for 15 min on ice as
described in the materials and methods section. (A) shows and-L-selecdn (DREG-56)
staining of untreated and treated L-selectin-expressing L l/2 cells; (B) shows anti-E-selectin
(EL-246) staining of untreated and treated E-selectin-expressing L l/2 cells; and panel (C)
shows anti-CD45 (L3B12) staining of untreated and treated L-selectin-expressing L l/2
cells.
64
TaM e 7. Treatment of the L l/2 L-selectin transfectants with anti-L-selectin antibodies plus
second stage or fucoidin induces down-regulation at 37°C.
Leu-A sftaimHHifiS
SE.
Antibody treated
49
28
Fucoidin treated
27
18
a) L l/2 cells expressing L-selectin were treated with, fucoidin or anti-L-sele'ctm mAb
(DREG-152) plus second stage cross-linking antibody as described in the materials and
methods section. A fter sugar or mAb cross-linking, cells were washed and incubated on ice
or at 37oC for 15 min, washed and stained with anti-L-selectin antibody (Leu-8) and
analyzed on a FACScan (Becton-Dickinson). The data are presented as mode fluorescence
o f Leu-8 staining. Background mode fluorescence values were subtracted from all positive
values.
their inability to extravasate. The fact that these cells re-enter circulation allows for multiple
interactions with lymphoid tissue HEV, which does not occur with normal lymphocytes
that proceed with extravasation once they have bound the HEV. W e tested the effect o f in
vivo trafficking on L-selectin expression on the L l/2 transfectants. The data from three
independent experim ents, presented in figure 11, show that L-selectin transfectants,
allowed to traffic for only 15 min in vivo, downregulated L-selectin expression; whereas
cells incubated at 37°C for the same duration in the presence of 20% mouse serum did n o t
A s a com parison, E-selectin-transfected L l/2 cells, allowed to traffic under sim ilar
conditions, did not downregulate E-selectin (data not shown).
To determine if a L-selectin-ligand interaction was necessary for the observed in vivo
L-selectin dowmegulation, we treated L l/2 L-selectin transfectants with mAbs shown to
block L-selectin function (ref. 28 and M.A. Jutila, unpublished observations) prior to in
vivo trafficking. As shown in figure 12, pretreatment with anti-L-selectin antibodies
65
!
I
I
Exp #1
Control
M H =I 17
Exp. #1
In-Vivo Trafficked I
MFI=4.0
I
I
I
:
c
I
Exp. #2
Control
MFI= 159
Exp. #3
Control
MFI=Bl
Exp. #2
In-Vivo Trafficked
M FI-2.0
Exp. #3
In-Vivo Trafficked
MFI=5.0
--------------------- ---------------------►
Log Fluorescence FITC (labelled Ll-2 cells)
Figure 11. L selecdn L l/2 transfectants, injected i.v. into mice and allowed to recirculate,
rapidly downregulate L selecdn expression. L l/2 cells, transfected with L selecdn were
labeled with FTTC and injected into either BALB/c (expt. #1 and #3) or C D l (expt. #2)
mice, as described in the materials and methods section. Fifteen minutes later, peripheral
blood was collected, erythrocytes were lysed and leukocytes stained with anti-L-selectin
(DREG-56) mab, followed by PE-anti-mouse second-stage antibody. Similarly labeled
(FITC) L-selectin transfectants, incubated at 37°C in the presence o f 20% mouse serum for
the duration of the experiment, were used as controls. The FITC-Iabeled L-selectin
transfectants incubated in vitro or allowed to recirculate in vivo were analyzed by two
color flow cytometry under the same fluorescence and compensation settings. Data are
presented as contour plots wherein the X-axis shows the fluorescence intensity of the FTTC
label and Y-axis shows L-selectin expression detected by treatment with anti-L-selectin
antibody (DREG-56) followed by PE-conjugated second stage in three independent
experiments. The left panels represent transfectants incubated in vitro (control), whereas
the right panels represent transfectants allowed to recirculate in vivo (in vivo trafficked).
MFI = mean fluorescence intensity.
66
Untreated
DREG-110
DREG-200
O
20
40
60
80
100
Percent of control
mean fluorescence
F igure 12. Anti-L-selectin antibodies block downregulation o f L-selectin on L-selectin
transfectants allowed to recirculate in vivo. L l/2 L-selectin transfectants were labeled with
FTTC and then treated with anti-L-selectin antibodies (DREG-110) for 15 min on ice,
washed, and injected into BALB/c mice for in vivo trafficking, as described in the
materials and methods section. Following in vivo trafficking, cells were collected from the
blood and stained with PE-anti-mouse IgG or PE-anti-L-selectin antibody Leu-8 and
analyzed by flow cytometry to determine L-selectin expression. Data are presented as
percent of control mean fluorescence, wherein control are cells pretreated with anti-Lselectin antibodies and incubated at 37°C in the presence o f 20% mouse serum for the
duration of the experiment.
almost completely inhibited the L-selectin downregulation that occurred in vivo. Similar
67
numbers o f cells (percent FITC-positive) were recovered from m ice following in vivo
trafficking when the cells were untreated or pretreated with control (anti-CB45) or anti-Lselectin mAbs prior to injection (data not shown). Therefore, preferential sequestration o f
untreated L-selectin-positive cells in the control m ice could not account fo r our
observations, as there would have been a significant increase in the number o f FITCpositive cells recovered following anti-L-selectin mAb pretreatment. These results suggest
that L-selectin- dependent interactions in vivo lead to L-selectin downregulation.
Discussion
Selectins are thought to be important in regulating diverse leukocyte-endothelial cell
interactions (1-7). L-selectin is required for the initial adhesion and stopping o f neutrophils
on inflamed endothelium, which is the first step in the emigration o f the cell into underlying
inflam ed tissue (1,5,6, 9-11,28). In vitro activation o f neutrophils with chemotactic
agents, like C5a, results in rapid shedding o f L-selectin and an inverse upregulation o f
M ac-1 (11,12,39,40). Based on these findings, it has been hypothesized that neutrophils
initially bind inflamed endothelium through a L-selectin-mediated adhesion event and are
then activated by the local inflammatory mediators, inducing shedding o f L-selectin and an
upregulation o f M ac-1 to begin the process o f emigration (39,40). In chapter 2 , 1 showed
that treatment o f neutrophils with chemical crosslinkers causes a rapid and activationindependent loss in the surface expression o f L-selectin (41), which led us to hypothesize
that normal leukocyte trafficking and rolling may result in L-selectin cross-linking and
shedding in vivo.
Here we demonstrate that treating leukocytes with different means o f potentially crosslinking L-selectin induces a loss in the surface expression o f the antigen. Four different
68
function blocking anti-L-selectin monoclonal antibodies which map to the lectin domain
(12, and Kishimoto T.K. personal communication) were able to cause a loss in surface
expression o f L-selectin when further cross-linked with a second-stage antibody. The antiL-selectin mAbs alone or treated with a noncross-linking second stage had no effect on Lselectin expression. In addition to monoclonal antibodies, sugars, like fucoidin, which bind
L -selectin and block rolling in vivo, induced L-selectin dowmregulation. A lso,
crosslinked-induced shedding was unique to L-selectin and not another member o f the
selectin family, E-selectin. Using L l/2 cells stably transfected with L-selectin cDNA, we
showed that L-selectin-dependent interactions in vivo cause a loss in surface expression o f
L-selectin. Our observations suggest that receptor crosslinking or engagement leads to
down-regulation of L-selectin expression.
Crosslinking-induced shedding o f L-selectin triggered by antibodies or fucoidin had to
be done at room temperature or 37°C. Rapid patching o f L-selectin occurred at 37°C
following antibody crosslinking (see figure 8). The clustering o f L-selectin molecules,
which doesn't happen at 4°C , may potentiate their shedding by bringing them in close
contact with a membrane protease (see below). If this clustering is required, it may explain
the temperature dependence o f the phenomenon. The requirement o f higher temperature
may also indicate that cell signalling events occur following the crosslinking o f L-selectin
which activate a protease involved in L-selectin shedding. At this time we cannot rule out
this possibility. Subtle activation o f neutrophils does happen follow ing antibody
crosslinking o f L-selectin, as indicated by an increase in M ac-1 expression (see table I).
However, similar increases in M ac-1 expression can be seen following crosslinking o f
CD44, CD45, or simply manipulating cells at 37°C (see table 4 and Ref. 41). Thus, it is
unlikely that the level of general activation induced by crosslinking accounts for our results.
It could be that crosslinking o f L-selectin leads to specific signalling o f only L-selectin
69
shedding and not generalized cell activation, or crosslinking o f L-selectto potentiates the
shedding o f L-selectin induced by subtle activation o f the cell, such as that caused by in
vitro manipulation. In support o f the latter possibility, we have found that crosslinking o f
L-selectin potentiates the effect o f activation (FMLP) induced shedding of L-selectin, such
that far less (100 fold) activating agent is required to cause complete surface loss o f antigen
(Palecanda A., unpublished observation).
It is likely that only a fraction o f surface L-selectin is available for crosslinking in the
assays used in this study. Anti-L-selecdn antibodies or fucoidin directly caused only a 3075% drop in surface L-selectin expression. Other studies have shown that much o f Lselectin is clustered on the tips of processes and microvilli o f leukocytes (45,49,53). It may
be that Only a fraction o f these molecules are localized in such a m anner to facilitate
crosslinking.
Our data show that crosslinking or engaging L-selectin potentiates L-selectin shedding
from the leukocyte surface. From these results we propose the following. First, shedding
o f L-selectin following the initial interaction of neutrophils with inflamed endothelial cells
may contribute to rolling o f the leukocyte, which has been shown by others to be a Lselecdn-dependent event (8,9,11,38). Second, crosslinker-induced shedding may facilitate
the transendothelial cell migration o f leukocytes into inflammatory sites and/or provide a
mechanism for L-selectin positive cells which will not enter into a certain inflammatory site
to release and re-enter circulation. The same event may take place during lymphocyte
recirculation through peripheral lymph nodes (PLN). According to our model, lymphocytes
would bind PLN-HE V via L-selectin, which in turn is shed at the site o f contact and the cell
proceeds with emigration. The loss of total surface expression o f L-selectin in this setting
would be minimal (only at the site o f initial contact between the lymphocyte and the
endothelium) and likely not detectable on the cell once it has entered the tissue, as shown
by others (42).
I
70
An important step in testing our hypothesis that crosslinking of-L-selectin in vivo leads
to shedding, will be to examine the effects o f native ligand. Proposed ligands for L-selectin
include the peripheral-lymph-node addressin (PNAd) and E-selectin (45,46). Recently, a
novel, mucin-like ligand (Gly CAM-1) for L-selectin expressed in lymph nodes has been
described (24,52). W e are presently attem pting to use PNAd, im m une-isolated and
adsorbed onto glass surfaces, to mimic immobilized endothelial ligand for L-selectin.
Preliminary results suggest that PNAd can support rolling and subsequently induce an
activation-independent loss in the surface expression o f L-selectin on both L-selectin
transfectants and mouse bone-marrow neutrophils. However, we have been unable to draw
definite conclusions from these experiments because not all preparations o f PNAd induce
these effects. PNAd, isolated by the M ECA-79 mAb, is a m ixture o f many different
glycoproteins (46), which are not equally represented in different preparations. Since we
do not know which glycoprotein(s) is the predominant receptor for L-selectin, it remains to
be seen whether one or a subset o f these glycoprotein species can by itself mediate Lselectin binding and crosslinking.
The molecular mechanisms involved in the actual shedding of L-selectin are not known.
Kishimoto et al. (39) proposed that L-selectin release is due to proteolysis, and the fact
that chymotrypsin treatment causes L-selectin release supports this hypothesis (29). An
alternate phosphotidyl inositol (GPI)-Bnked form o f L-selectin has been described (50),
suggesting the involvement o f phospholipase C in L-selectin regulation; but experimental
data does not support this hypothesis, since Qrd et al. (51) found no spBce sites in the
genomic DNA sequences that would yield mRNA message encoding the Pi-linked form.
U sing specific protease inhibitors, we have implicated a unique membrane-associated
serine protease as mediating L-selectin shedding (See Chapter 4) following crosslinking or
activation. This analysis shows the molecular mechanism involved in L-selectin shedding
71
in the two processes is similar and is the first direct demonstration o f a role for proteolysis.
A s indicated above, crosslinking o f L-selectin may directly lead to activation o f this
protease. W e are currently attempting to isolate and characterize the regulation o f the
protease to begin to address this question.
In conclusion, we have shown that anti-L-selectin m A b crosslinking and the
polysaccharide fucoidin induce a loss in L-selectin expression. Activation alone cannot
account for the observed downregulation o f L-selectin, since the same level o f M ac-1
upregulation occurs on cells treated to crosslink L-selectin or other surface antigens. Also,
in vivo trafficking leads to L-selectin downregulation which can be blocked by anti-Lselectin antibodies. Based on these studies, w e propose that crosslinking or receptor
engagement contributes to the downregulation o f L-selectin in vivo.
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78
CHAPTER' 4
■' R O L E O F A D H SO PR O PY L FL U O R O PH O SPH A TE (DFP) SN H ZEiiA BLE,
A C TIV A TIO N -D EPEN D EN T, M EM BRA N E-A SSO CIA TED , PU TA TIV E
PR O T E A SE IN T H E SH EDDING O F LEUKOCYTE L-SEL EC TIN
Introduction
L-selectin (previously called peripheral-lymph node-homing receptor, LECAM -1, and
LAM -1), a member o f a new family of adhesion proteins called selectins, is expressed by
all leukocytes and is involved in neutrophil binding to inflamed endothelial cells and
lym phocyte adhesion to high-walled endothelium in peripheral lymph nodes (1-5).
M embers o f the Selectin family have been shown to mediate leukocyte rolling on the
vascular endothelium which slows the flow o f the cells within the blood, promoting their
initial transient adhesiveness (1,2,5). Direct experimental evidence shows that L-selectinm ediated rolling is prerequisite for inflammation in vivo, since reagents that block this
event also inhibit inflammation (2,6-11). Huang et al. suggest that L-selectin may also
mediate leukocyte targeting to myelin-rich regions in pathological demyelinating reactions
( 12).
The expression o f L-selectin is uniquely regulated. Unlike m ost adhesion proteins that
increase in expression and/or function, L-selectin is rapidly shed from the cell surface
following stimulation o f the leukocyte with activating agents, such as chemotactic factors
79
(13). Cross-linking of L-selectin by chemical means and anti-L-selectin antibodies leads to
rapid loss o f the surface antigen (14). Others have suggested that the shedding o f L-selectin
is im portant in the release o f the leukocyte from the vascular endothelium prior to
extravasation into the underlying tissues (4,13). W e and others have shown that shed Lselectin is present in the plasm a from healthy individuals (14,15). The mechanisms
involved in the in vivo and in vitro shedding o f L-selectin are not known.
Shed L-selectin is 5-10 kD smaller than the transmembrane form, suggesting that its
release involves proteolysis (13,16,25); however, no direct evidence for this has been
dem onstrated. Protease inhibitors, such as pepstatin, L-l-tosylam ido-2-phenylethylchloromethyl ketone (TPCK)I, phenanthroline, and aprotinin, have no effect on L-selectin
regulation (16, M.A. Jutila, unpublished observation and see below). In this report, we
present an analysis of the effects o f diisopropyl fluorophosphate (DFP) on the regulation o f
L-selectin on neutrophils and lymphocytes. Inhibition o f any protease by DFP is generally
considered diagnostic for its identification as a serine protease (17-21). A unique property
o f DFP is that it can pass through the cell membrane, which most protease inhibitors cannot
do (22). D FP treatm ent o f neutrophils has been shown to only minimally alter their
respiratory burst in response to cellular activation; and other functions, like phagocytosis or
depolarization o f membrane potential, are unaffected (22,23). W e show here that DEP is a
potent inhibitor o f L-selectin shedding. D FP does not affect the activation-induced
upregulation of certain markers o f activation, thus, it is unlikely that DFP is blocking a
common leukocyte signalling pathway, but, rather, it inhibits the protease involved in the
release o f L-selectin. The D FP inhibitable protease is somehow m asked or hidden in
unactivated leukocytes. Also, the protease seems to be membrane associated and is not
released upon activation of the leukocyte.
80
Materials and Methods
Antibodies
D REG -110 and Leu-8 (Becton Dickinson, Mountain View, CA) are mouse antibodies
that recognize human L-selectin (7,25). D REG-110 is an Ig G i, whereas Leu-8 is an
IgG 2a . M el-14 is a rat IgG2a that recognizes mouse L-selectin (25). L3B12 recognizes
human CD45 and 30G12 (rat XgG2a) recognizes mouse T200 (7,14). BM39.9 is a rat IgG2
that recognizes an uncharacterized 30kD activation antigen on mouse neutrophils (Jutila,
M.A., unpublished) and TIB218 (rat IgG2a) recognizes mouse GDIS. All the monoclonal
antibodies were purified from hybridoma supernatants by ammonium sulfate precipitation
according to standard procedures. PE conjugated Leu-8 was purchased from Becton
Dickinson. FITC-Iabelled goat anti-mouse (second stage) was purchased from Sigma
(Sigma Chemical Co., St. Louis, MO).
Preparation o f leukocyte suspensions and flow cytometric analysis
Human peripheral blood and bone-marrow leukocytes were prepared as previously
described with no modifications (7). Control and cross-linked or activated cells (see below)
were stained and analyzed for L-selectin, C D l8, or BM39.9 expression on a FACScan
(Becton Dickinson), as previously described (7,14). Briefly, I x IO^ cells were initially
incubated in 5% rabbit serum for 10 min on ice to block Fe receptors. The cells were
washed arid then incubated with primary antibody at 50 pg/m l for 20 min on ice. A fter
washing, bound antibodies were revealed by incubation with FTTC-conjugated goat anti
mouse Ig (second stage) (Sigma) at 1:100 dilution in 5% FBS in HESS. Flow cytometric
analysis was perform ed on a FACScan (Becton Dickinson), as described (25). In some
81
experiments, direct stains with PE-labelled Leu-8 were done. Background fluorescence
was established by staining with isotype matched control antibodies and second stage
reagent.'
Leukocyte activation
For leukocyte activation, isolated human peripheral-blood leukocytes or mouse bonemarrow neutrophils were treated with either 50 ng/ml PMA o r I x 10 ^ M FM LP (both
from Sigma Chemical co., St. Louis, M O) in HBSS (containing 2% FBS and IOmM
Hepes) for 20 min at 37°C in the presence or absence o f protease inhibitors (see below),
w ashed in PBS w ith 2% horse serum, stained, and L-selectin, CD 18, or BM 39.9
expression was determined by flow cytometry, as described above.
Chemical crosslinking
H um an peripheral-blood leukocytes and m ouse bone-m arrow neutrophils were
collected and treated with the crosslinking agent, BS^ (Pierce Chemical Co., Rockford,
CL), in the presence or absence o f protease inhibitors (see below), as described (14).
Briefly, isolated human leukocytes or mouse bone-marrow neutrophils at a concentration o f
I x IO ? cells/ml, in HBSS containing 2% FBS plus IOmM HEPES, were incubated for 15
min on ice with 5mM BS^. A t the end o f the incubation period, the cells were washed
twice in PBS with 2% horse serum, stained with anti-L-seleCtin mAbs, and L-selectin
expression was determined by flow cytometry, as described above.
Protease inhibitor treatment
Isolated human peripheral-blood leukocytes or mouse bone-marrow neutrophils were
treated with 2.7mM DFP, 10 pg/m l each o f leupeptin, pepstatin, chymostatin, or Im M
phenylmethylsulfonyl fluoride (PMSF) (all from Sigma).in BBSS, containing 2% FBS and
82
IOmM Hepes, for 10 min on ice. The concentrations o f the inhibitors were based on the
manufacturer's recommendations for maximal activity. The protease inhibitor solutions
were maintained at a pH o f 6-7. The cells were then either activated with PMA or EMLP or
treated with the crosslinker BS^ in the presence o f the proteases as described above. In
some experiments, the protease treated cells were washed twice with PBS prior -to the
treatment with activating or crosslinking agents. After washing away the activating agents
o r the crosslinker, cells were stained and analyzed for L-selectin expression by flow
cytometry. A s a control, the expression o f CB45 on leukocytes was monitored by flow
cytometry following all treatments.
binding proteins
Isolated human peripheral-blood leukocytes were washed in HBSS containing 2% FBS
and 10 mM HEPES and suspended in the same buffer at a concentration o f 30 x 10&
cells/ml. To I ml o f the cell suspension, either pretreated with 2.7mM cold DFP for 10 min
on ice and w ashed o r untreated, 10 p i o f ^H -D FP (10 pC i, specific activity o f 1.0
Ci/mmol, New England Nuclear, Boston, MA) was added and allowed to interact for 10
min on ice. The mixture was incubated for 20 min at 37°C in the presence or absence o f 50
ng of PM A and then centrifuged at 15,000g for 2 min at 4°C; the supernatant was removed
and saved, and the cells were suspended in I ml o f lysis buffer (2% NONBDET P-40) and
incubated for 45 min on ice. The suspension was then centrifuged at 15,000g for 20 min
and the supernatant removed and saved for SDS-PAGE and autoradiography. Cells labelled
with ^H -D FP under the same conditions on ice without the addition o f PMA were also
lysed and analyzed by SDS-PAGE and autoradiography. The specificity o f the binding o f
^H-DFP was shown in competition assays using cold DFP.
83
Sodium dodecvl sulfate (SDS Vpolvacrvlamicite eel electrophoresis (PAGE)
and autoradiography
Hum an leukocytes were reacted with ^H -D FP (in the presence o r absence o f the
activating agent PMA) and lysed as described above. The lysates were mixed with 6x
treatment buffer and analyzed by SDS-PAGE (29). Fluorographic detection, o f radiolabeled
proteins was done according to previously established procedures (31). Briefly, the gel
was washed in 30% methanol and then impregnated with sodium salicylate (Sigma) for I
hour at room temperature, dried onto filter paper and loaded into a cassette with XAR-5
film (Eastman Kodak); exposure was done at -70°C for 3-21 days.
Results
Activation-induced L-selectin shedding is completely inhibited by PER.
Protease inhibitors, such as TPCK, TLCK, aprotinin, and leupeptin, have very little or
no effect on L-selection shedding (16, M.A. Jutila, unpublished report, and see below).
W e have extended these studies to the analysis o f DFP. We first tested the effects o f DFP
on activation-induced shedding o f L-selectin. Human- leukocytes were pretreated with DFP
for 10 min prior to stimulation with PMA or the chemotactic factor FMLP. As shown in
figure 13, DFP treatment completely blocked the shedding o f L-selectin, that was induced
by the activating agents. In contrast, PM SF o r chymostatin had no effect on L-selectinregulation, and leupeptin showed only slight inhibition ( < 25% o f control, figure 13).
D ose response analysis (0.5 to 21.6 mM tested) showed that 2.7mM, DFP was m ost
effective at blocking L-selectin shedding (data not shown)^ and was the dose used
throughout the course of the study. DFP was effective on both human and mouse
Figure 13
Percent of control mode fluorescence (anti-L-selectin staining)
Protease
inhibitor
None
Activating 0
agent
None
NEUTROPHILS
40
60
80
0
20
None
None
DFP
DFP
DFP
PMSF
NA
Chymostatin FMLP
NA
Leupeptin
NA
FMLP
LYMPHOCYTES
40
60
80
85
F ig u re 13. DFP, but not other protease inhibitors, block L-selectin shedding in response
to PM A or FM LP treatment o f human leukocytes. Human peripheral-blood leukocytes,
collected as described in the methods section, were untreated or treated with protease
inhibitors (2.7 mM DFP, 10 pg/ml each chymostatin, leupeptin, pepstatin or I mM PMSF)
for 10 min on ice and then incubated with PMA (50 ng/ml) or FM LP (IOr^M) for 20 min at
37°C . A fter washing in PBS, the cells were stained with anti-L-selectin antibody Leu-8
and analyzed by flow cytometry on a FACScan (Becton Dickinson). The results are
presented as percent o f control mode fluorescence o f Leu-8 staining. Controls are untreated
cells incubated at 4°C for the duration o f the experiment. Neutrophils and lymphocytes
were detected by their distinct forward and side light scatter profiles. Background staining
o f isotype-matched controls were deducted from all positive values. All experiments were
repeated at least four times. NA = Not applicable (FMLP is a neutrophil activating
agent).course o f the study. DFP was effective on both human and mouse leukocytes, and
sim ilar effects were seen on neutrophils as well as lymphocytes (Fig. I and see Fig. 4
below). DFP was not toxic to the cells, nor did it affect other surface antigens, such as
CD45 (data not shown, and see below). These results demonstrate that specific inhibition
o f leukocyte serine proteases blocks the shedding o f L-selectin following activation o f the
cell.
leukocytes, and similar effects were seen on neutrophils as well as lymphocytes (figure 13
and figure 17 below). D FP was not toxic to the cells, nor did it affect other surface
antigens, such as CD45 (data not shown, and see below). These results demonstrate that
specific inhibition of leukocyte serine proteases blocks the shedding o f L-selectin following
activation o f the cell.
Crosslinking-indnced L-selectin shedding is alsQOompletelvJnhiMMjaLDEE
W e recently showed that chemically cross-linking surface antigens with BS^ causes Lselectin shedding (14). Subsequently, we have shown that cross-linking o f L-selectin with
antibodies, sugars that bind L-selectin, or L-selectin-dependent interactions in vivo leads
to loss o f L-selectin expression (Palecanda, A., R.F. Bargatze, R A . W amock, and M A .
Jutila. 1993. Ligand interaction in vivo and receptor crosslinking in vitro modulates Lselectin expression. Manuscript submitted). W e tested the effects o f DFP on BS3-M u c e d
L-selectin shedding. As shown in figure 14, D FP pretreatm ent o f human leukocytes
completely inhibited the loss o f L-selectin expression following treatment of the cells with
Figure 14
Percent control mode fluorescence (anti-L-selectin staining)
NEUTROPHILS
Protease
inhibitor
None
None
DFP
DFP
PMSF
Chymostatin
O
20
LYMPHOCYTES
40
60
80
100
87
F ig u re 14. DFP, but not other serine protease inhibitors, block L-selectin shedding in
response to BS^ crosslinking. Human peripheral-blood leukocytes, collected as described
in die methods section, were untreated or treated with 2.7 mM D FP for 10 min on ice and
then incubated with 5 mM BS^ for 15 min on ice. After washing in PBS, the cells were
stained with anti-L-selectin antibody Leu-8, and the expression o f L-selectin was
determined by FACS. The results are presented as percent o f control mode fluorescence as
described in the legend for figure I.
B S^, whereas PM SF and chymostadn had no e ffe ct Also, expression o f other leukocyte
surface m olecules, such as CB45, did not change under any o f the above treatment
conditions (data not shown).These results demonstrate that the release o f L-selectin induced
by crosslinking or activation likely involves the same proteolytic mechanism.
,
Upregulation o f surface markers for cellular activation are not affected bv DFP
Some protease inhibitors have been shown to block certain activation-induced activities
o f neutrophils, presumably by altering some signalling pathways (24). W e have recently
generated a monoclonal antibody (mAb BM 39.9) against a mouse neutrophil surface
antigen that is rapidly upregulated in response to cellular activation (Jutila, M .A.,
unpublished). Also, the common beta chain (CD 18) of beta-2 integrins on leukocytes has
been shown to increase in expression following activation (30). W e tested the effect o f
DFP on the expression o f CD 18 and the BM39.9 antigen following cellular activation to
determine if DFP was nonspecifically blocking proteases involved in signalling. The data
presented in figure 15 show that DFP has no effect on the activation-induced upregulation
o f CD 18 or the BM39.9 antigen. Thus, D FP is unlikely to be blocking general signal
transduction pathways. Also, doses o f D FP used in this study have been shown not to
affect the activation o f the neutrophil as measured by their oxidative burst, phagocytosis, or
depolarization o f membrane potential (22,23). Additional results also show that inhibition
o f signalling pathways is not required for the effect o f DFP, since DFP blocks BS^induced L-selectin shedding, which can be done at 4°C (Fig. 14) and in the presence o f
88
sodium azide (14). These results show that DFP likely blocks the protease that actually
cleaves L-selectin.
Mode Fluorescence
T reatm ent
Antibodv
None
None
Mel-14
TIB218
None
BM39
PMA
PMA
Mel-14
TIB218
PMA
BM39
PMA+DFP
Mel-14
PMA+DFP
TDB218
PMA+DFP
BM39
Figure 15. DFP has no effect on the activation-induced upregulation o f certain surface
markers for cellular activation. Mouse bone marrow neutrophils, collected as described in
the methods section, were treated with the protease inhibitor DFP (2.7 mM), for 10 min on
ice and then incubated with PMA (50 ng/ml) or FMLP (10'^M ) for 20 min at 37°C. After
washing in PBS, the cells were stained with anti-L-selectin antibody Mel-14, and CD18
antibody TIB218 or the antibody against a neutrophil activation m arker BM39.9, and
analyzed by flow cytometry on a FACScan (Becton Dickinson). The results are presented
as mode fluorescence staining. Controls are untreated cells incubated at 4 °C for the
duration of the experiment. Neutrophils were detected by their distinct forward and side
light scatter profiles. Background staining of isotype-matched controls were deducted from
all positive values. All experiments were repeated at least four times.
DFP inhibits a protease which is exposed/accessible only upon
cellular activation or chemical crosslinking
To gain further insight into the nature o f the protease involved in the release of Lselectin, we determined whether DFP presence is required during the activation or
89
crosslinking of the leukocytes to be effective at blocking L-sclcctin shedding. We treated
human and mouse leukocytes with DFP for 10 min and washed it from 1/2 of the cells.
We then treated both cell preparations with BS^ or FMLP for 15 min, as described above,
and then stained for L-sclcctin expression. As shown in figures 16 and 17, BS^ and FMLP
caused a loss in L-selectin expression on the cells treated with DFP and washed, but no
loss was seen on the cells treated with DFP and not washed. Similar results were seen with
both mouse and human leukocytes (Figures 16 and 17). Histograms o f one flow cytometric
Mode fluorescence of anti-L-selectin staining
Protease
Inhibitor
None
T reatm en t
None
BS3
None
FMLP
DFP
BS3
DFP
FMLP
DFP
Washed
BS3
DFP
Washed
FMLP
None
F ig u re 16. Presence of DFP is required during crosslinking or activation to block Lselectin shedding on human neutrophils. Human peripheral-blood leukocytes, collected as
described in the methods section, were treated with 2.7 mM DFP for 10 min on ice and
either washed or not washed prior to treatment with FMLP or the crosslinker BS^ ( see
legend for figures I and 2). A fter washing in PBS, the cells were stained with anti-Lselectin antibody Leu-8 and analyzed by flow cytometry on a FACScan (Becton
Dickinson). The results are presented as percent of control mode fluorescence o f Leu-8, as
described in the legend for figure I.
90
No treatment
A
No treatment
D'
E
1
Fi
I
LogioFluorescence
Anti-L-selcctin staining
F igure 17. Presence of DFP is required during activation or crosslinking to block Lselectin shedding on mouse bone-marrow neutrophils. Mouse bone-marrow leukocytes,
isolated as described in the methods section, were treated with 2.7 mM D FP for 10 min on
ice and then either washed (Panel B and E) or not washed (Panel C and F) prior to
treatment with PMA (Panel B and C) or the cross-linker BS^ (Panel E and F). Panel A and
B are untreated control cells incubated at 4°C for the duration o f the experim ent After
washing in PBS, the cells were stained with the anti-L-selectin antibody M el-14 (see
methods section), and the expression o f L-selectin was determined by flow cytometry. Data
are presented as histograms.
analysis are shown in figure 5 to demonstrate that the observed effects occurred over the
entire leukocyte population. Since it is known that DFP irreversibly binds active serine
91
proteases (17-21), these results suggest that activation o r cross-linking o f cell surface
antigens exposes or allows access to the protease involved in the shedding o f L-selectin,
making it accessible to DFP binding and inhibition. This washing step also shows that DFP
pretreatment is not toxic to the cells.
;
Cellular activation exposes previously unexpressed or hidden DFP
binding membrane-associated proteins
According to our analysis, DFP inhibits a protease that is only exposed upon cellular
activation or chemical crosslinking. In order to determine if, indeed, there are DFP binding
proteins exposed after cellular activation, we activated human peripheral-blood leukocytes
with PM A in the presence o f ^H-DFP and analyzed the cell lysate by SDS-PAGE and
autoradiography. The autoradiograph in figure 18 shows that activation leads to exposure
o f membrane-associated DFP binding proteins (60-90 kD) not present on unactivated cells.
As shown in figure 6 (lane B), preincubation with cold DFP inhibits ^H-DFP binding to
almost all the DFP binding proteins. Activation o f leukocytes with FM LP also resulted in
the emergence of membrane-associated 60-90 kD DFP binding proteins (data not shown).
To determine if there were differences in the activation-induced expression of DFP binding
proteins between lymphocytes and neutrophils, we isolated lymphocytes and neutrophils
separately and then activated them in the presence o f ^H-DFP. The autoradiograph in figure
19 shows that activation leads to the exposure o f previously hidden membrane-associated
60-90 kD DFP binding proteins both on lymphocytes and neutrophils. There are distinct
differences in the expression of activation-induced DFP proteins expressed by lymphocytes
and neutrophils. Lymphocytes have very low levels of DFP binding proteins o f the 30 kD
DFP binding proteins which is the predominant band seen on neutrophil membranes. Also,
the activation-induced 47 kD DFP binding protein is expressed only on neutrophils and the
60 kD protein is expressed only on lymphocytes, whereas the 90 kD protein is expressed
by both neutrophils and lymphocytes. Importantly, the 60 and 90 kD activation-induced
DFP binding proteins are membrane associated and not released into the supernatant.
92
Hence activation o f leukocytes does indeed lead to exposure of previously masked DFP
binding proteins. Furthermore, these proteins are likely contained in the membrane,
because they are readily solubilized by treating leukocytes with 2% NP-40.
A)
1 2
200
LANES
3
B)
4
1 2
LANES
3
4
-►
90 * ►
"*
69 - ►
30
Figure 18. A ctivation exposes membrane associated DFP binding proteins. Human
peripheral-blood leukocytes, collected as described in the methods section, were labelled
with
DFP in the presence or absence of the activating agent PMA as described in the
materials and methods section. Labelled cells were lysed and analyzed by SDS-PAGE and
autoradiography as described in the text. Panel A, shows the radiograph of the gel exposed
to radiographic film for 3 weeks and panel B, shows the same gel re-exposed for 6 weeks
Lane I, cells labeled with % DFP in the presence of PMA; Lane 2, cells pretreated with
cold DFP, washed and then labelled with ^H-DFP; Lane 3, cells labelled with 3H-DFP
without any activating factors; and Lane 4, cells pretreated with cold DFP, washed, and the
labelled with
DFP in the presence of PMA. Molecular weight markers are numbered
corresponding to their w eight
93
______ LANES________
I
2
3
4
5
6
200
90
69
30
Figure 19. Activation exposes membrane-associated 90 kD DFP binding proteins on
both lymphocytes and neutrophils. Human peripheral-blood leukocytes, collected as
described in the methods section, were passed over an Histopaque (Sigma) column to
separate lymphocytes according to the manufacturer’s directions. The polymorhonuclear
cells and erythrocyte containing pellet was resuspended in HBSS and neutrophils isolated
by sedimentation as described in the materials and methods. The mononuclear cells and
neutrophils were separately labeled with ^H-DFP in the presence or absence o f the
activating agent PMA as described in the materials and methods section. Labelled cells were
lysed and analyzed by SDS-PAGE and autoradiography as described in the text. Lane I,
lymphocyte lysate following activation with PMA in the presence o f ^H-DFP. Lane 2,
supernatant from lymphocytes activated with PMA in the presence o f ^H-DFP. Lane 3,
supernatant from neutrophils activated with PMA in the presence o f ^H-DFP. Lane 4,
Neutrophil lysate following activation with PMLA in the presence o f ^H-DFP. Lane 5,
supernatant from neutrophils labeled with ^H DFP in the absence o f activation, and Lane
6, neutrophil lysate labeled with ^H DFP in the absence of activation. Molecular weight
markers are numbered corresponding to their weight.
Discussion
In this chapter, we show for the first time that inhibition of serine proteases blocks the
release of L-selectin following both activation of the leukocyte and cross-linking o f the
94
surface antigen. These results are significant because they provide direct evidence for the
role o f proteolysis in the shedding o f L-selectin and demonstrate that the same proteolytic
mechanism is involved in both activation and crosslinked-induced processes. It is likely
that the inhibition o f L-selectin shedding defined here is due to blocking o f a protease
which acts directly on L-selectin and not blocking o f general signalling pathways. The
protease inhibitor used in these studies (DFP) does not affect neutrophil phagocytosis,
polarization, or respiratory burst following activation (22,23). W e show that DFP does not
block activation-induced upregulation of certain neutrophil surface antigens. Thus, diverse
signalling events are unaffected by DFP.
O ur analysis shows that the protease involved in the shedding o f L-selectin exhibits
many special characteristics which will aid in its eventual molecular characterization. The
protease does not appear to be secreted by the cell, since bystander lymphocyte L-selectin is
not shed upon neutrophil activation with FM LP (Figure 13). Thus, it may be in o r closely
associated with the cell membrane. DFP has the unique capacity to pass within membranes
(22), w hich may account for its ability to inhibit the protease. A nother interesting
characteristic o f the protease is that it is not active or its catalytic site is not accessible in
untreated leukocytes. Some type o f signal (activation or receptor crosslinking) is required
for DFP to bind and inhibit it.
In preliminary studies o f DFP reactive proteins expressed by leukocytes, we provide
direct evidence for molecules with the characteristics described above. A group o f proteins
in the 60-90 IcD m olecular weight range, which are bound by DFP, are selectively
expressed in the membranes o f activated neutrophils. Both PM A and FMLP induce
expression o f these molecules. Furthermore, they are not detectable in supernatant fluids
from activated cells (data not shown), which suggests that they are not released. W e know
o f no previously defined leukocyte protease with these characteristics. Determining if these
putative proteases m ediate the shedding o f L-selectin is a m ajor goal. W e are also
95
determining if the proteins are constitutively expressed on the surface o f leukocytes and
activation leads to conformational changes in them exposing DFP binding sites or if they
are translocated from intracellular pools.
Proteolysis has been previously shown to be important in a variety o f proinflam matory
activities o f leukocytes (1,2,4,5,13,25,27). W e extend those observations here and show
that a serine protease is involved in the regulation o f expression o f a leukocyte adhesion
molecule required for exit o f cells from the circulation. In other studies, we have found that
protease inhibitors also affect the expression o f M ac-1 (data not shown), another adhesion
protein important in leukocyte migration. Altering the regulation o f adhesion molecule
expression may be ah effective means o f inhibiting leukocyte extravasation, and, if so,
proteases may provide novel targets in the design o f anti-inflammatory drugs. W e are
currently attempting to isolate the protease involved in L-selectin shedding and raise
monoclonal antibodies that block its activity, as has been accomplished for other membrane
proteases (28). If this can be done, we can directly test the effectiveness o f blocking Lselectin regulation on leukocyte extravasation in vivo.
In conclusion, we provide the first demonstration that inhibition o f proteolysis blocks
the shedding o f L-selectin. This finding complements previous circumstantial evidence for
the role o f proteolysis in the regulation o f L-selectin expression and function. The specific
protease that appears to cause the shedding o f L-selectin has many unique characteristics,
such as being membrane-bound and requiring a signal (delivered by cross-linking o f LSelectin or activation o f the leukocyte) for its activity. Additional' characterization o f this
protease may provide insight into the regulation o f L-selectin expression which mediates
immunologically critical functions.
96
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100
CH AFFER §
CONCLUSIONS
The nonrandom recirculation o f lymphocytes and the emigration o f leukocytes to sites
o f inflammation require an initial leukocyte/endothelial interaction. This event is mediated
largely by L-selectin expressed on the surface o f all leukocytes and its counter-receptor,
which is either constitutively expressed on peripheral-lymph-node HEV or induced on
postcapillary venules at sites o f inflammation. L-selectin-m ediated rolling in vivo is
required for beta-2 integrin-mediated transmigration in vivo. Blocking o f L-selectin with
specific antibodies or L-selectin-lgG chimeras in vivo inhibits lymphocyte migration into
peripheral lymphoid tissues and neutrophil emigration into sites o f inflammation. These
findings indicate that L-selectin-mediated leukocyte/endothelial interactions are critically
im portant for leukocyte extravasation. The recent finding that L-selectin m ediates
lymphocyte binding to myelinated central nervous tissues suggests that L-selectin may be
involved in autoimmune responses. Also, the binding o f neutrophils, monocytes, and
lymphocytes to cytokine-activated glomerular endothelium in vitro is mediated by Lselectin, suggesting a possible role fo r the L-selectin-m ediated interactions in
glomerulonephritis. Therefore, L-selectin expression and regulation seems to be o f utmost
importance in both normal and disease conditions. Due to the importance o f this interaction,
regulation of L-selectin expression would require stringent control.
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L selecdn expression is known to rapidly downregulate upon activation o f leukocytes
with phorbol esters or chemotactic agents in vitro. Based on this observation, a prevailing
hypothesis is that L-selectin shedding allows the bound leukocyte to release from the
endothelial cell and proceed with transmigration into sites o f inflammation. Since the loss
o f L-selectin prevents the interaction o f the leukocyte with the endothelium, L-selectin
shedding may be a protective mechanism to prevent activated neutrophils from damaging
normal endothelial cells.
However, L-selectin-mediated leukocyte/endothelial interactions occur at sites other
than those involving acute inflammation: fo r example, the lym phocyte PLN-HEV
interaction during the normal lymphocyte recirculation. Lymphocytes should be able to
release from the L-selectin-mediated binding to endothelium in the absence of inflammatory
or activating agents to enter into secondary lymphoid organs in the periphery. Also, all
leukocytes express L-selectin and therefore have the potential to roll on the endothelium,
but only neutrophils emigrate into sites o f acute inflammation. These observations suggest
that leukocytes, such as eosinophils and monocytes, should be able to release and reenter
circulation at sites o f acute inflammation without themselves being activated. Therefore, an
alternate activation-independent mechanism o f L-selectin shedding may be involved in these
settings.
M y hypothesis is that L-selectin/ligand interactions may be the primary signal fo r the
release o f L-selectin, either independent o f or synergistic with activation. H us dissertation
has presented the various experiments perform ed to test my hypothesis. Based on
experim ental results presented in the earlier chapters, I have reached the following
conclusions:
I . A ctivation is not absolutely required for L-selectin shedding. Rapid activation-
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independent shedding o f L-selectin can be induced at 4°C with chemical crosslinkers. Shed
L-selectin can be detected by ELISA in the supernatant o f leukocytes treated with chemical
crosslinkers in the presence o f azide.
2. Specific crosslinking o f L -selectin w ith anti-L -selectin antibodies induces
downreguladon o f L-selectin expression which cannot be accounted for by activation alone.
C rosslinking o f L -selectin, but not other surface antigens, induces L-selectin
downreguladon while levels o f activation, as measured by M ac-1 upregulation, are similar
under both treatment conditions.
3. The sulfated polyfucose fucoidin, which binds L-selectin, inhibits in vitro lymphocyte
binding to PLN -H EV and potently blocks in vivo rolling o f leukocytes, in d u cin g .
downreguladon o f L-selectin expression. W hether fucoidin is crosslinking L-selectin is not
known; it may be that receptor perturbation and/or crosslinking induces L-selectin
downreguladon. L-selectin downreguladon mediated by fucoidin treatment is independent
of activation because similar levels of leukocyte activation are observed following treatment
with all polysaccharides tested; but only fucoidin induces L-selectin downreguladon. Also,
lym phocytes interacting w ith im m obilized fucoidin under non-static conditions
downregulate L-selectin expression.
4. Ligand interactions in vivo induce L-selectin downreguladon. The mouse pre-B cell
line, transfected with L-selectin cDNA, downregulates L-selectin following 15 m in
trafficking in vivo. This downreguladon is due to L-selectin-dependent interactions in vivo
since pretreatm ent o f the L l/2 transfectants with function-blocking anti-L-selectin
antibodies completely blocks the in vivo downreguladon of L-selectin.
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5. Crosslinking-induced downregulation is not a common feature o f selecting since the
expression o f E selecdn is not altered following chemical crosslinking. Also, L l/2 cells
transfected with E-selectin cDNA do not downregulate L-selecdn expression following 15
min o f in vivo trafficking.
6. The serine protease inhibitor DFP completely blocks activation- and crosslinkinginduced L-selectin shedding. Various other protease inhibitors, including other serine
protease inhibitors, did not inhibit L-selectin shedding. This could be due to the ability o f
D FP to pass through the plasma membrane. However, there is very high variability in the
lot preparations of DFP in its ability to block L-selectin shedding. Each fresh lot o f DFP
should be individually tested to determine the optimum concentration to be used in these
assays.
7. A ctivation and crosslinking exposes a protease involved in L-selectin shedding.
Presence o f DFP is required during crosslinking or activation o f leukocytes to block
shedding. Pretreatment o f leukocytes with DFP, followed by washing away o f excess
DFP, does not block L-selectin shedding. Since D FP is an irreversible serine protease
inhibitor, the protease involved in L-selectin shedding is being exposed only upon
activation or crosslinking.
8. Activation o f neutrophils and lymphocytes leads to exposure o f 60-90 kD DFP-binding
membrane-associated glycoproteins. The 90 kD DFP-binding glycoproteins are present on
both neutrophils and lymphocytes, whereas the 60 kD DFP-binding protein is present only
on lymphocytes. Importantly, the 60-90 kD DFP-binding proteins are not secreted upon
activation of leukocytes.
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9. The protease involved in L-selectin shedding is not released from the leukocyte surface
since activation o f neutrophils -with FM LP leads to L-selectin shedding on the neutrophil
only and not on the bystander lymphocyte cell surface; or, if the protease is released, it
loses proteolytic activity.
In summary, I have shown that overt activation is not absolutely required for L-selectin
shedding. Receptor perturbation or crosslinking and ligand interactions in vivo induce Lselecdn downreguladon. Soluble L-selectin can be detected in normal plasma samples. A
DFP-inhibitable, membrane-associated protease is involved in L-selectin shedding. Based
on these findings, I propose the following model for L-selectin-mediated endothelial
interactions in vivo.
M odeL
Leukocyte rolling on the endothelium is mediated by L-selectin. This event slows the
leukocyte in circulation and brings it into close proximity to the endothelium, thereby
allowing any cytokines or chemokines that may be present to exert their action on the cell.
The rolling interaction can occur only on endothelium where the L-selectin ligand is
expressed.
In PLN-HEV, which constitutively express a ligand for L-selectin, all leukocytes are
capable o f rolling, but emigration o f cells into the lymphoid organ would require cellspecific signals delivered by the endothelium. Therefore, lymphocyte subset-specific
signals in PLN-HEV would induce necessary emigration pathways (upregulation o f as yet
unknown adhesion molecules required for transmigration) in the lymphocyte subset
expressing receptors for the signalling molecules. Other rolling leukocytes, such as
neutrophils, would release from the endothelium due to crosslinking-induced shedding of
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L-selectin and reenter circulation.
Similarly, all leukocytes have the potential to roll on inflamed endothelium at sites o f
acute inflammation. Only neutrophils emigrate into the underlying inflamed site, whereas
other leukocytes release from the L-selectin-mediated endothelial binding event and reenter
circulation. In this setting, according to our model, neutrophil-specific chemoMnes present
on the endothelium w ould induce pathw ays required fo r m igration (for exam ple,
upreguladon o f C D l 1/CD 18) only on neutrophils. Other rolling leukocytes would shed Lselecdn, due to crosslinking mediated by its ligand and reenter circulation.
This model allows the use o f the same receptor (L-selectin) on all subsets of leukocytes
to interact with and sample the endothelium prior to tissue- and site-specific tight adhesion
and transm igration. The presence o f leukocyte subset-specific chemokines allow s
preferential recruitment o f leukocyte subsets into distinct sites; for example, lymphocytes
into secondary lymphoid organs, neutrophils into acutely inflamed tissues, and eosinophils
into sites o f allergic reactions. Therefore, regulation o f L-selectin could be mediated by
either activation or crosslinking, depending on the microenvironment.
Testing o f my hypothesis requires the identification and purification o f the vascular
ligand for L-selectin. The interaction o f leukocytes on purified ligand coated on to glass
slides will be a means to determine the effect o f L-selectin/endothelial-cell ligand on Lselectin expression. This experiment will show whether ligand interaction in the absence o f
other factors, such as vascular cytokines, is by itself sufficient to induce L-selectin
downregulation. W e have recently tested PNad, immunoisolated and adsorbed onto the
inner surface o f glass capillary tubes, for its ability to induce L-selectin downregulation
under shear. Preliminary data from these experiments showed that PNad by itself can
support leukocyte rolling under conditions mimicking vascular flow. Importantly, mouse
bone-marrow neutrophils and human L-selectin transfectants allowed to roll on PNad did
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loose L-selectin expression in the absence of activation, as measured by M ae-I expression.
Subsequent preparations o f PN ad did not induce L-selectin dow nregulation. This
inconsistency could be due to the presence o f multiple glycoprotein species in each PNad
preparation: Each preparation o f PNad may not contain all the different glycoprotein species
required to induce L-selectin downregulation.
W e have also tested whether crosslinking o f L-selectin is synergistic to activation o f the
leukocyte. Treatment o f neutrophils with the chemotactic peptide FM LP induces activation
as m easyred by M ac-1 upreguladon and L-selectin downregulation. Sim ilar levels o f
activation (as m easured by M ac-1 expression) can be induced with a ten-fold low er
concentration o f the activating agent FMLP if activation is done together with L-selectin
crosslinking. These results suggest that crosslinking may be synergistic to activation in
modulating the surface expression o f adhesion molecules.
W e have also attem pted to raise anti-idiotypic antibodies against anti-L-selectin
antibodies in an attempt to define vascular ligands for L-selectin. This attempt was initiated
by an interesting observation during our crosslinking and polysaccharide experiments. W e
observed that one o f the anti-L -selectin antibodies, D R EG -56, recognizes the
polysaccharide fucoidin. We knew that the vascular ligand for L-selectin was fucosylated
and also that fucoidin blocked rolling in vivo and lymphocyte binding to PLN-HEV in
vitro. W e immunized mice with the DREG-56 antibody in an attempt to raise anti-idiotypic
antibodies which theoretically may see the vascular ligand for L-selectin. Interestingly,
three o f the four immunized mice died due to undetermined causes. This suggested to us
that the mice may be making auto-antibodies against the mouse vascular endothelial ligand
for L-selectin since there is about 70% identity between the mouse and the human Lselectin. The serum from the remaining mouse had high titers against the endothelium in
both human and bovine lymphoid tissues. W e have done a fusion and cloned hybridomas
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producing anti-human endothelial-cell antibodies and are currently attempting to determine
the molecules identified by these.
Availability o f a purified L-selectin ligand will allow further characterization o f the
pathways involved in the regulation o f L-selectin expression. Transfection and expression
o f the L-selectin ligand on cells other than endothelial cells will be a means to study the
effect o f L-selectin-ligand interaction on L-selectin expression. Here the effect of different
cytokines on subsets o f leukocytes can also be studied. Availability o f purified ligand will
be a means to determine if ligand interaction will, induce intracellular signalling through Lselectin. Thus the identification o f the ligand for L-selectin will be very .important-not only
for the testing o f my hypothesis, but also to understand the function o f L-selectin on
different subsets of leukocytes.
Identification o f the putative protease involved in L-selectin downregulation is another
means to understand regulation o f L-selectin expression. W e are currently in the process o f
making antibodies against the DFP-inhibitable protease. We have immunized mice with
human leukocytes activated (with the phorbol ester PMA) in the presence o f DFP. This
immunization protocol is interesting because the leukocytes used for immunization are
activated, but still have high levels o f surface L-selectin. Therefore, if indeed a previously
hidden membrane-associated protease is being exposed upon activation, then the protease
should be now bound by DFP but still exposed on the surface. Interestingly, serum from
two immunized mice recognize a major 90 kD glycoprotein on human leukocytes by
W estern blot. The next step is to do a fusion and attempt to raise monoclonal antibodies
against the putative protease involved in L-selectin shedding. Identification o f the protease
will allow detailed analysis o f the regulatory mechanism involved in L-selectin expression.
Effect o f blocking the protease in vivo and under conditions mimicking vascular flow in
vitro can be studied. Questions, such as whether L-selectin shedding is required for
rolling, can be addressed.
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In conclusion, identification o f a purified ligand for L-selectin o r identification o f the
protease mediating L-selectin shedding are o f crucial importance to better understand the
regulatoiy mechanics o f L-selectin shedding.
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