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RAPID COMMUNICATION
Detection of an L-Selectin Ligand on a Hematopoietic Progenitor Cell Line
By Susan M. Oxley and Robert Sackstein
L-selectin, the peripheral lymph node ”homingreceptor,“ is
an adhesion protein that mediates lymphocyte binding t o
lymph node high endothelial venules. Ligands for this protein have been identified only on endothelial cells, and recent murine studies indicate that CD34 on endothelial cells
is an L-selectin ligand.Toinvestigate
whether CD34 expressed on hematopoietic cells functions as an L-selectin
ligand, we used an in vitro binding assay t o examine lymphocyte adherence t o KGla, a CD34+human hematopoietic
progenitor cell line. We observed specific L-selectin-mediated adherence of lymphocytes t o KGla: the binding was
calcium-dependent, was strictly inhibited byanti-L-selectin
antibodies and by carbohydrate ligands of L-selectin, and
was abrogated by inductionof L-selectin shedding from the
lymphocyte membrane by treatment with phorbol esters.
However, blocking studies using anti-CD34 antibodies, and
experiments using KGla cells sorted for CD34 expression
and COS-7 cells transfected with full-length CD34 cDNA indicate that the ligand on KGla is notCD34; moreover, RPM1
8402, a CD34+cell line, does not support lymphocyte adherence in the binding assay. Treatment of KGla with the enzymes neuraminidase, chymotrypsin, and bromelain abrogated lymphocyte binding t o t h e cells, indicating that the
ligand isa glycoprotein. These experiments show thatCD34
on hematopoietic cells is not an
L-selectin ligand andprovide
the firstevidence of a ligand forL-selectin present on a nonendothelial cell.
0 1994 by The American Societyof Hematology.
T
HE PERIPHERAL lymph node “homing receptor,” Lcharacterization of L-selectin and its ligands among progeniselectin (CD62L), is a -75-kD glycoprotein that meditor cells is of considerable interest because adhesion proteins
regulate cell-cell and cell-stromal interactions fundamental
ates attachment of lymphocytes to lymph node (LN) high
to hematopoiesis. To investigate whether L-selectin and heendothelial venules (HEV), an adhesive interaction that is
the first step in the migration of lymphocytes from blood into
matopoietic CD34 function as an adhesive receptor-ligand
lymphoid tissues.’.’This protein is recognized by a variety of
pair, we performed in vitro binding studies of lymphocytes
monoclonal antibodies (MoAbs) in h u m a n P and is a memto KGla, a primitive CD34+ human cell line derived from
ber of the selectin family of adhesion molecules, which
an acute myeloid
These studies show highly
includes P-selectin (CD62P) and E-selectin (CD62E). Selecspecific adherence of lymphocytes to KGla cells mediated
tins share a common structure consisting of an amino-termiby L-selectin on the lymphocyte, but not involving CD34 as
nal calcium-dependent lectin domain, an epidermal growth
the corresponding ligand. The results indicate the presence
factor domain, a variable number of repeat sequences bearing
of a ligand for L-selectin on the surface of this hematopoietic
homology to complement regulatory and catalytic proteins
progenitor cell line and provide the first evidence of L-selecbinding C3b or C4b, a transmembrane portion, and a Ctin-mediated adhesion between lymphocytes and a nonenterminal cytoplasmic
The lectin domain of these prodothelial cell type.
teins directs their adhesion to carbohydrate molecules presMATERIALS AND METHODS
ent on the cell surface.
The adhesive interaction between lymphocytes and HEV
Cell lines. Cell lines used in these studies were obtained from
has been extensively analyzed using anin vitro binding
the following sources: KGla and Nalm 16, gift of Dr William E.
Janssen; HL60, K562, and Raji, gift of Dr Lynn Moscinski; COSassay.’ This assay is performed under shear at 4”C, whereby
7, gift of Dr Kenneth Zuckerman (all from H. Lee Moffitt Cancer
binding mediated by L-selectin is maximized and effects of
Center, Tampa, FL); and RPMI 8402, gift of Dr Daniel G. Tenen
other adhesion molecules are minimized.’.’0 The interaction
(Harvard Medical School, Boston, MA). All cells were cultured in
of L-selectin with its corresponding ligand(s) onHEV is
RPMI 1640 (GIBCO-BRL, Gaithersburg, MD) supplemented with
calcium-dependent” and requires the presence of sialic
acid12.13 and sulfate14 on the ligand(s). In vitro adherence of
lymphocytes via L-selectin can be inhibited by carbohydrates
From the Division of Bone Marrow Transplantation, H . Lee MO$
such as mannose-6-phosphate (man-6-P), PPME (phosphojitt Cancer Center, and the Departments of Internal Medicine and
mannan monoester core from Hansenulu hostii, a phosphoof Pathology and Laboratory Medicine, University of South Florida
mannosyl-rich polysaccharide), and fucoidin (a sulfated, fuCollege of Medicine, Tampa, FL.
cose-rich polysaccharide).15,16
Submitted August 4, 1994; accepted September 7, 1994.
Ligands for L-selectin have thus far been characterized
Supported in part by grants from the Florida Division ofthe
American Cancer Society and The George W. Jenkins Foundation.
on murine endothelial cells using a murine L-selectin-IgG
This material is based on work supported under a National Science
chimera molecule as a probe.” This approach has identified
Foundation Graduate Fellowship (S.M.O.).
two proteins, GlyCAM-1 (Sgp50)’’and CD34 (Sgp90),”
Address reprint requests to Robert Sackstein, MD, PhD, Division
present on endothelial cells. GlyCAM-1 is a novel sialomuof Bone Marrow Transplantation. Room 3151, H. Lee Mofltt Cancer
cin and its role as a ligand for L-selectin is its only known
Center, 12902 Magnolia Dr, Tampa, FL 33612.
function.” Although present on endothelial cells in most
The publication costs of this article were defrayed in part by page
tissues:’ CD34 is best known for its expression on the earlicharge payment. This article must therefore be hereby marked
est multilineage colony-forming hematopoietic stem cells.”
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
Hematopoietic progenitor cells characteristically express
indicate this fact.
both L-selectin and CD34,’3 and there is growing evidence
0 1994 by The American Society of Hematology.
that L-selectin mayplay a role in h e m a t o p o i e s i ~ .The
~ ~ ~ ~ ~ 0006-4971/94/8410-004.5$3.00/0
Blood, Vol 84, No 10 (November 15), 1994 pp 3299-3306
3299
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3300
OXLEY AND SACKSTEIN
Table 1. Expression of Surface Molecules on Cell Lines Used in t h e Lymphocyte Adherence Assay
Relative Expression of Membrane Proteins.
Cell Line
KGla
RPM1 8402
HL60
Nalm 16
K562
Raji
Lymphocyte
Adherence
Yes
No
No
No
No
No
Lineage
CD34
LFA-1
VLA-4
CD44
Sialyl Le'
Myeloid
Lymphoid
Myeloid
Lymphoid
Erythroid
Lymphoid
++++
++++
++++
++++
++++
c+++
-
++++
C++
-
-
-
+c++
+
-
-
-
++++
-
-
-
+++
++
-
-
-
++++
++, 36% to 65% positive; +++, 66% to 95% positive; ++++,
+++
Abbreviations: -, 0% to 5% positive; +, 6% to 35% positive;
Percentage of positive cells a s determined by flow cytometric analysis.
10% heat-inactivatedfetalbovineserum
(FBS) in a humidified
chamber at 37°C with S% CO2 in air.
Prcpnratior? of /ytnphoc.vtes. Humanperipheralbloodlymphocytes(PBL)wereisolated
by Ficoll densitygradientfromblood
drawn in sodium citrate. To obtain rat thoracic duct lymphocytes
(TDL), thoracic ducts of
rats were cannulated as described" and
LN wascollected in phosphate-bufferedsaline(PBS) with 0.1%
CD43
+I++
++++
++++
+++L
+++
i
296% positive.
penicillinlstreptomycinand S UlmL heparin. PBLorTDLwere
washedthreetimes
in RPM1 I640 medium without bicarbonate
(GIBCO-BRL). pH 7.4. and suspended at 1 X 10' cellslmL in the
above-described medium with S% FBS and kept on ice until use in
the adherence assay.
Lymphocyte cd/wrmce crssc~y. Theprocedurefor
the in vitro
binding of human or
rat lymphocytes to KGla was adapted from
Fig 1. Lymphocyte-KGla adherence and LAM1-3
inhibition. Cytospin preparationsofKGla cellsshowing adherence of lymphocytes (solidcircles, original
magnification x 250). (A) Lymphocytesadhere t o
KGla in the presenceof CD45 or isotype controlAbs.
(B) Lymphocytebindingassay
in the presence of
LAM1-3 Ab (anti-L-selectin).
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DETECTION OF AN L-SELECTIN LIGAND ON KGla
the rat lymphocyte-lymph node binding assay that has been described
in detail elsewhere.s~28
Cytospin preparations of KGla or other cell
lines were made on a Cytospin 3 Cytocentrifuge (Shandon Lipshaw,
Pittsburgh, PA). Frozen rat LN sections 8-pm thick were mounted
on slides, and lymphocyte binding to LN HEV served as a positive
control in all experiments. Slides were air-dried, fixed in 3%glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA) in
PBS, rinsed with PBS, incubated in 0.2 m o m L-lysine (Sigma
Chemical CO, St Louis, MO) to block unreacted glutaraldehyde, and
then rinsed and held in RPMI 1640 with 1% FBS at 4°C until use
in experiments.
Lymphocyte suspensions (200 pL) were overlaid onto cytospin
or LN sections in duplicate and placed on a rotating platform (80
rpm) at 4°C for 30 minutes. Slides were then rinsed in cold PBS to
remove nonadherent lymphocytes, fixed in 3% glutaraldehyde, and
stained with methyl green-thionin. Slides were examined under the
light microscope for adherence of lymphocytes to KGla orLN HEV.
Treatment of lymphocytes with potential inhibitors. Lymphocytes in RPMI 1640 medium with 5% FBS were preincubated (30
minutes on ice) and the assay was performed in the presence of the
following inhibitors: 1 mmol/L EDTA (no preincubation period); 10
mmol/L D-mannose-6-phosphate (Sigma); 10 pg/mL PPME (kindly
provided by Dr M.E. Slodki, USDA, Peoria, IL); and 5 pg/mL
fucoidin (Sigma).
Antibody blocking experiments. Lymphocytes (1 X lo7 cells/
mL) were preincubated on ice for 20 minutes with MoAbs at 1.0
pg/mL and used in the binding assay without further washing. The
following MoAbs were used: LAMI-3 (anti-L-selectin; kind gift of
Dr. Thomas Tedder, Duke University, Durham, NC, and also obtained from Coulter Corp, Hialeah, FL); anti-CD45 (leukocyte common antigen; Becton Dickinson, San Jose, CA); and IgG, (isotype
CONTROL
LAM 1-3
CD45
C
RELATIVE FLUORESCENCE INTENSITY
Fig 1. (Cont'd) (C) FACS profiles of lymphocytesused in the binding assay after incubation with isotype-matchedIgG control, LAM13, or anti-CD45 Abs, followed by GAM-FITC. Resuttsshown are representative of three independent experiments.
3301
control; Coulter). In some experiments, prepared KGla slides were
incubated with 0.2 pg of anti-CD34 Abs (HPCA-I [clone Mylo]
and HPCA-2 [clone 8G12; Becton Dickinson], QBENDlO [AMAC,
Westbrook, ME], and 12.8 [kindly provided by Dr Pat Roth, Coulter
Corp]) in RPMI 1640 with 5% FBS for 30 minutes before the binding
assay.
Phorbol myristate acetate (PMA)treatment of lymphocytes. Lymphocytes were suspended at 1 X 10' cells/mL in cell culture medium
and incubated for I hour at 37°C with or without 10 ng/mL PMA
(GIBCO-BRL). Cells were then washed twice in PBS and used in
either the lymphocyte binding assay or analyzed for surface antigens
by flow cytometry (see below).
Enzyme treatment of KGla or L N . Cytospin preps of KGla or
LN frozen sections were glutaraldehyde-fixed and then treated with
various enzymes before the binding assay. For treatment with neuraminidase (sialidase), slides were rinsed twice with enzyme buffer
(50 mmol/L NaAc, 154 mmom NaCl, 9 mmol/L CaCI,,pH 5 . 5 )
and then incubated for 30 minutes at 37°C with 50 pL of buffer
(control) or undiluted neuraminidase (1.2 U/mL; Boehringer Mannheim, Indianapolis, IN). In protease studies, slides were incubated
with RPMI 1640 alone or RPMI 1640 containing enzymes: 100 U/
mL chymotrypsin (Sigma; 15 minutes at 37°C) or 0.1% bromelain
(Sigma; 30 minutes at 37°C). To assess specificity, the protease
inhibitors phenylmethylsulfonyl fluouride (PMSF; 1.0 mg/mL;
Sigma) and chymostatin (900 pg/mL; Boehringer Mannheim) were
coincubated with chymotrypsin (100 U/mL) for 15 minutes at 37°C.
After enzyme treatments, slides were washed three times with RPMI
1640 and placed in RPMI 1640 with 1% FBS until use in thebinding
assay.
KGla cells in suspension (4 X IO' cells/mL) were incubated with
0-sialoglycoprotein endopeptidase (Accurate Chemical and Scientific Corp, Westbury, NY; 0.24 mg/mL, 37°C for 30 minutes) and
washed three times with 2% FBS in PBS, and cytospin preparations
were made for use in the binding assay. To verify the activity of
the enzyme, cells were tested for the cleavage of CD34 byflow
cytometry using QBENDlO MoAb.
Antigen expression by flow cytometry. Flow cytometric analysis
was performed using the following commercially available MoAbs
together with isotype-matched controls: TQ1 (anti-L-selectin),
LAMl-3 (anti-L-selectin), 4B4 (anti-VLA-4) (all from Coulter
Corp); QBENDlO (anti-CD34; AMAC); anti-CD44, LFA-1-p (antiCD18), LFA-I-a (anti-CD1la), HPCA-2 (anti-CD34), anti-CD45,
Leukosialin (anti-CD43), anti-Sialyl-Le" (all from Becton Dickinson). Cells (1 X IO6) in 100 pL of PBS with 2% FBS were incubated
on ice for 25 minutes with Ab as per the manufacturer's recommendations, washed three times, and analyzed on a FACStafLUS(Becton
Dickinson).
Fluorescence activated cell sorting of KGla cells. KGla cells
were stained with anti-CD34 MoAbs (QBENDIO-FITC in 2 experiments, HPCA-2-PE in 1 experiment) and positive and negative expressing cells were sorted on a FACStdLUS
flow cytometer equipped
with an argon laser tuned at 488 nm (Becton Dickinson). Sorted cell
populations were restained with anti-CD34 Ab directed at epitopes
not used for sorting and were analyzed to determine the efficiency
ofthe sort. Cytospin preparations were made of the positive and
negative sorted fractions and were used in the lymphocyte binding
assay.
Transfection of COS-7 with CD34 cDNA. COS-7 cells were transiently transfected with human full-length CD34 cDNA in pCDM8
plasmid (a gift from Dr Daniel Tenen, Boston, MA) using a diethylaminoethyl (DEAE) Dextran transfection method.29Briefly, COS-7
cells were incubated for 4 hours at 37°C with 10 mL of transfection
solution containing 20 to 40 pg of plasmid DNA, 10%Nu Serum
(Collaborative Biomedical Products, Bedford, MA), 400 pg/mL
DEAE Dextran (Sigma), and 100 pmol/L chloroquine (Sigma) in
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3302
OXLEY AND SACKSTEIN
Dulbecco'smodifiedEagle'smedium(GIBCO-BRL).
Cells were
then rinsedand treated with10%dimethyl sulfoxide (DMSO; Sigma)
in PBSfor 2 minutesatroomtemperature,rinsedinPBS,
and
incubated in tissue culture media for 3 days. In one set of experiments, trypsinization was avoided by growing transfected cells directly on glass slides for subsequent use in the binding assay or for
analysis of CD34 expression by fluorescence microscopy. In other
experiments, COS-7 cells grownon 10-cm plates were removedwith
trypsinEDTA (0.25%/1 mmoVL;GIBCO-BRL) and then analyzed
for CD34expression by flowcytometry.Thesetrypsinizedcells
werethenplaced on slides by cytospinforuse in the lymphocyte
binding assay.
RESULTS
Lymphocytes bind to KGla. Lymphocytes (both PBL
and TDL) adhered specifically and reproducibly to KGla,
but not to RPMI 8402, HL60, Nalm 16, K562, or Raji cell
lines in the in vitro binding assay (Table 1). All experiments
were performed in parallel with LN frozen sections as positive controls. Lymphocyte binding to KGla was observed
under conditions identical to those whereby L-selectin mediates binding of lymphocytes to LN HEV.
Lymphocyte binding to KGla is mediated by L-selectin.
To directly examine whether lymphocyte attachment was
mediated by L-selectin, PBL were preincubated with the
anti-L-selectin MoAb LAM1-3, anti-CD45, or IgG, isotype
control Abs. The LAMl-3 Ab completely inhibited lymphocyte binding to KGla and LN control, whereas CD45 and
isotype control MoAbs did not affect binding (Fig 1A and
B). To quantify the relative amounts of Ab attachment to
lymphocytes, Ab-treated lymphocytes were incubated with
goat-antimouse fluorescein isothiocyanate (F1TC)-conjugated secondary Ab and analyzed byflow cytometry. Although the amount of anti-CD45 Ab on lymphocytes was
significantly greater than that of LAM1-3 as indicated by
mean channel fluorescence (Fig lC), LAMl-3alone blocked
lymphocyte adherence to KGla andLN HEV, indicating
that this effect was specific and not secondary to charge or
steric alterations of the lymphocyte membrane.
The effect of enzyme treatment of KGIa on lymphocyte
binding. Pretreatment of both KGla and LN control sections with neuraminidase (60 mu), chymotrypsin (100 U/
mL), or bromelain (0.1%) before the binding assay abrogated
binding of lymphocytes, whereas treatment with buffer or
medium alone did not alter binding capacity. In addition, the
effects of chymotrypsin were confirmed by coincubation
with the protease inhibitors chymostatin and PMSF, which
prevented chymotrypsin effects on lymphocyte binding.
However, pretreatment of KGla with 0-sialoglycoprotein
endopeptidase had no effect on lymphocyte binding despite
complete enzymatic removal of the CD34 epitope recognized
by QBENDlO MoAb.
Lymphocyte binding to K G l a is calcium dependent. Lymphocyte binding to KGla and to LN control sections was
completely inhibited by the presence of EDTA, indicating a
calcium requirement for lymphocyte-KGla binding.
Man-6-P, PPME, andfucoidin inhibit lymphocyte binding
to KGla. The specificity of lymphocyte-KGla binding was
investigated by treating PBL or TDL with carbohydrate inhibitors of L-selectin-HEV interactions before the adherence
assay. Man-6-P (10 rnmoYL), PPME (10 pLg/rnL), and fucoidin (5 pg/rnL) all inhibited lymphocyte binding to both
KGla and LN control sections.
PMA treatment of lymphocytes results in the loss of binding to KGIa. PBL were incubated for 1 hour at 37°C with
10 ng/mL PMA and then used in the lymphocyte binding
assay. PMA-treated PBL were unable to bind to either KGla
or LN HEV, whereas control PBL showed high amounts of
binding.
LOSSof surface L-selectin was assessed by flow cytometric
analysis of TQ1 levels in control and PMA-treated PBL.
PMA-treated lymphocytes showed a dramatic decrease in
TQ1 mean channel fluorescence (to levels less than 10% of
that of untreated cells) in three separate experiments (data
not shown). PMA-treated PBL were also analyzed for expression of CD44, LFA-1 (both a and p chains), and VLA4, and expression of these adhesion molecules after PMA
exposure was identical to expression on control PBL (data
not shown).
Pretreatment of KGla with anti-CD34 antibodies did not
inhibit adherence of lymphocytes. Cytospin preparations of
KGla were preincubated with anti-CD34 Abs and the binding assay wasperformed in the presence of the Abs. MoAbs
to four different CD34 epitopes were used alone or in combination, including the clones MylO, QBEND10, 8g12, and
12.8, in amounts ranging from 0.2 to 17 pg/slide. Anti-CD45
(irrelevant control) and IgG, (isotype control) Abs were also
tested. None of the anti-CD34 Abs inhibited lymphocyte
binding to KGla, despite immunohistochemical evidence of
extensive Ab binding to the glutaraldehyde-fixed KG I a sections (data not shown).
Other surjke antigens on KGla donot appear to mediate
binding. The surface expression of several antigens on
KGla, RPMI 8402, HL60, Nalm 16, K562, andRajiwas
analyzed byflow
cytometry (Table I). LFA-I, VLA-4,
CD44, Sialyl Le", and CD43 were allexpressed by KG la and
at least one other cell line that did not support lymphocyte
adherence. Of note, although RPMI 8402 cells express CD34
at levels comparable to KGla, there was no adherence of
lymphocytes to these cells in the binding assay.
CD34+ and CD34-KGla cellssupported
equivalent
amounts of lymphocyte binding. CD34' and CD34- KG1 a
cells were separated by fluorescence-activated cell sorting
and cytospin preparations of eachpopulationweremade.
The in vitro adherence of lymphocytes was identical in the
CD34+ and CD34- populations despite an enrichment of
greater than 90% and lessthan 10% CD34' cells in the
respective populations (Fig 2 ) .
CD34-transfected COS-7 cellsdid not support lymphocyte
adherence. COS-7 cells were transfected with CD34 and
tested in the in vitro binding assay; both trypsinized and
intact transfected COS-7 cells failed to support lymphocyte
adherence. Byflow cytometric analysis, transfected cells
were approximately 60% positive for CD34 expression and
the mean channel fluorescence was greater than that of KG 1 a
control cells (Fig 3). Intact, untrypsinized COS-7 cells
transfected with CD34 also strongly expressed CD34 (-90%
positive as estimated by fluorescence microscopy).
The results of experiments using chemical and antibody
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DETECTION OF AN L-SELECTIN LIGAND ON KGla
3303
CONTROL
7
A
CD34
7
RELATIVEFLUORESCENCEINTENSITY
A
Fig 2. Lymphocytes adhere to CD34- KGla cells.
KGla cells were sorted by FACS before the binding
assay into CD34' and CD34- fractions using MoAb
HPCA-2-PE. Sorted cell fractions were restained for
CD34 using MoAb QBENDIO-FITC and analyzed as
shown in (A). Positive and negative sorted fractions
were greqter than 90% and less than 1046 positive
forCD34,respectively. The lymphocyte adherence
assay was performed on the sorted cells, and nodifferences in lymphocyte adherence were evident
among the CD34* and 0 3 4 - populations. Lymphocytes (solid circle)
adherent to theCD34- fraction are
shown in (B). Results shown are representative of
three independent experiments.
treatments of lymphocytes and KGla are summarized in
Table 2.
DISCUSSION
The lymphocyte-HEV adherence assay an
is in vitro approximation of physiologic adhesion mediated by L-selectin and has
been a fundamental tool in studying the function of L-selectin
in its native state on the surfaceof lymphocytes. In the studies
reported here, this binding assay was adapted to examine lymphocyte-hematopoieticcelladhesion,andtheresultsprovide
the first evidence of L-selectin-dependent adhesive interactions
between lymphocytes and nonendothelial cells.
Several independent lines of evidence indicate that lymphocyte binding to KG 1a is mediatedprimarily, if not solely,
by L-selectin. First, an anti-L-selectin MoAb (LAMI-3)
previously shown to block L-selectin-mediated adherence
to LN HEV,3" completely inhibited PBL from binding to
KGla or LN HEV, whereas anti-CD45 and isotype control
Abs did not block lymphocyte binding. Second, L-selectinmediated binding is a calcium-dependent event, and lymphocytes were unable to bind to KGla in the presence of the
calcium chelator EDTA. Third. carbohydrates such as man6-P, PPME, and fucoidin (all known to bind to L-selectin
andto inhibit lymphocyte binding to HEV in the in vitro
inhibited lymphocyte adherence to KGla. Lastly,
it is known thatPMA treatment of lymphocytes causes shedding of membrane L-selectin via a protein kinase C activationpathwayand
corresponds tothe loss of lymphocyte
binding to LNHEV in the in vitro assay.' In these studies,
PMA-treated PBLwere no longer able tobindto
KGla.
Other adhesion molecules. such as LFA- 1. CD44. and VLA4, remained unchanged on the surface of PBL after PMA
exposure. indicating that these molecules do not play a primary role in the binding to KGla.
The nature of the ligand was investigated by determining
the effects of various enzyme treatments of KGla on the
binding capacity. Previous studies have shown that ligand
expression of sialic acid is essential for L-selectin-mediated
binding of lymphocytes to LN HEV." In this study, neuraminidase-treated KG 1 a showed a complete loss of lymphocyte binding, indicating that sialic acid residues are also a
necessary component on the KG l a L-selectin ligand;as such.
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3304
OXLEY AND SACKSTEIN
CONTROL
El-
_ , , ,
CD34
i”
l h , ,
TRANSFECTED
COS-7
5
7
L
TRANSFECTED
RELATIVE FLUORESCENCE INTENSITY
Table 2. Lymphocyte Adherence to KGla
Mean (SEM) of Binding
(% of untreated
control)*
Lymphocyte treatmentt
EDTA
Man-6-P
Fucoidin
PPME
LAM1-3 MoAb
Anti-CD45 MoAb
MoAb
IgG, control
PMA
KGla treatmentt
MoAbsS Anti-CD34
Anti-CD45 MoAb
MoAb
lgG, control
CD34’ sort
CD34- sort
Neuraminidase
Neuraminidase
control
buffer
0-sialoglycoprotein
endopeptidase
Bromelain
Chymotrypsin
Chymotrypsin, PMSF, chymostatin
0.3 (0.3)
5.7 (1.0)
1.4 (0.4)
(0.5)
1.9 (0.4)
98.7 (6.3)
115.1 (9.0)
1.1 (0.3)
116.2
98.0
101.8
102.8
104.1
(7.7)
(3.6)
(8.5)
(3.5)
(4.2)
(0.7)
100.5 (6.7)
(2.31 98.4
3.8 (0.4)
6.7 (0.7)
94.0 (3.8)
* Number of lymphocytes adherent to confluent
area of KGla were
counted by light microscopy using an ocular grid under
250X magnitwo fields per
fication. Quantitation was performed by examining
slide, minimum of two slides perexperiment, three separate experiments. Results are presented
as percent binding compared
with corresponding untreated control sections.
t Experimental details as described i n text.
Combination of HPCA-1, HPCA-2, 12.8, and QBENDIO MoAbs.
*
Fig3.CD34
transfection of
COS-7cells.COS-7cells
were
transfected with either CD34pCDM8 or pCDM8 (mock) and
then analyzed by FACS
and compared with KGla for CD34expressionas shown. Absused
were isotype-matched lgGl
control and anti-CD34 MoAb
QBEND10. Lymphocytes did not
adhereto CDBdt~ansfectedW - 7
cells,despitehigherlevels
of
CD34expressionascompared
with KGla cells.
lymphocyte adherence to KGla involves carbohydrate motifs and is not based strictly on protein-protein interactions.
This finding, combined with the results of protease experiments, indicates that the KGla ligand is a glycoprotein. To
examine whether 0-linked glycosylations on the ligand play
a central role in the adhesive interaction, KGla were digested
with the enzyme 0-sialoglycoprotein endopeptidase that specifically cleaves proteins at sites of 0-linked sialo-glycosylation3’ and that has been shown to differentially cleave epitopes of the CD34 molecule.32The data show that treatment
of KGla in suspension with the enzyme actively destroyed
CD34 epitopes, yet had no effect on lymphocyte adherence.
These results suggest that ligand sialic acid residues critical
to binding may be present on N-linked rather than on 0linked glycosylations.
CD34 has been reported to be a ligand for L-selectin
based on the finding that a murine L-selectin-IgG chimera
molecule precipitated CD34 from a murine lymph node lysate.” The results of our study indicate that CD34 as expressed on KGla is not a functional ligand for lymphocyte
L-selectin, because no difference in lymphocyte binding to
sorted CD34- and CD34+ KGla cells was observed. Titration studies using varying proportions of KGla and HL60
have shown that the amount of lymphocyte adherence is
directly proportional to the percentage of input KGla cells,
indicating that differences in lymphocyte binding to the positive and negative sorted fractions would have been evident if
CD34 were the ligand. It is unlikely that a particular binding
epitope of CD34 was selected, because this experiment was
performed using two different anti-CD34 MoAbs to sort the
KGla. Two forms of CD34 on KGla have been reported
(“truncated” and “full-length”)33; however, these differences do not account for the data here because sorting was
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DETECTION OF AN L-SELECTINLIGANDON
KGla
also performed using QBEND10, which recognizes both
forms.
In addition to sorting experiments, evidence that CD34 is
not the L-selectin ligand on KGla is derived from MoAb
blocking studies and adherence assays using other CD34+
cells. None of the anti-CD34 MoAbs tested, or any combination thereof, was able to block lymphocyte binding to KGla.
Furthermore, lymphocytes did not adhere to another primitive CD34+ cell line, RPM1 8402, and transfection of CD34
into COS-7 cells did not confer lymphocyte binding capacity.
Although potential glycosylation differences of the CD34
molecule expressed by these cell types could affect their
ability to support lymphocyte adherence, this explanation is
unlikely in light of equivalent adherence observed among
the sorted CD34+ and CD34- KGla cells. Taken together,
the data presented here indicate that the CD34 glycoform
present on hematopoietic cells is not a ligand for L-selectin.
Moreover, flow cytometric analysis of the various cell lines
used in the binding assay provides evidence that membrane
structures such as LFA-1, VLA-4, CD44, Sialyl LeX, and
CD43 do not play a primary role in lymphocyte adherence
to KGla because each of these molecules was also present
on at least one other cell line tested that did not show lymphocyte binding.
In the present study, we used direct cell-cell interactions
to detect the presence of an L-selectin ligand on a hematopoietic cell. Other studies directed at identifying L-selectin ligands have relied on molecular approaches using a murine
L-selectin-IgG chimera molecule, synthesized in a human
embryonal kidney cell line, as a probe.” Of note, studies
using this chimera have failed to show binding of the molecule to KGla cells.34In general, tissue- and species-specific
patterns of glycosylations are well
and such
differences can affect the biologic activity of proteins expressed in different ~ e l l s .Because
~ ~ , ~ it~ is known that glycosylation of L-selectin varies among different cells expressing
the protein,“042such differences may account for the observation here that native L-selectin, expressed on lymphocyte
membranes, selectively binds to a corresponding ligand on
KGla cells, whereas the chimera apparently does not. Similarly, differences in glycosylation of CD34 among endothelial cells and hematopoietic cells may account for the differential capacity of this protein to participate in L-selectin
interactions among these cell types.
L-selectin ligands have been recognized heretofore only
on endothelial cells. The detection of an L-selectin ligand on
a nonendothelial cell expands the physiologic implications
of L-selectin function beyond its well-characterized role in
regulating leukocyte trafficking. Future studies will be directed at molecular characterization of the L-selectin ligand
on KGla cells and its distribution in human cells. Because
the KGla cell line represents a relatively primitive, “stem”
cell-like stage in human hematopoiesis, this ligand may be
expressed on at least a subset of bone marrow hematopoietic
progenitor cells. Insofar as L-selectin is expressed on
“stem” cells, it is possible that adhesive interactions mediated via this L-selectin-ligand pair, among progenitors
themselves or between lymphocytes and progenitors, may
play a role in hematopoietic events.
3305
ACKNOWLEDGMENT
We thank Drs Katrina Allen and William Janssen for helpful
suggestions and advice. We are also grateful toAmy Zuber, Xiu
Min Xu, Ling Fu, Paul Fallon, and Christine O’Connell for excellent
technical assistance.
REFERENCES
l . Gowans JL, Knight ET: The route of re-circulation of lymphocytes in the rat. Proc R SOCLond B 159:257, 1964
2. Marchesi VT, Gowans JL: The migration of lymphocytes
through the endothelium of venules in lymph nodes: An electron
microscope study. Proc R Soc Lond B 159:283, 1964
3. Gatenby PA, Kansas GS, Xian CY, Evans RL, Engleman EG:
Dissection of immunoregulatory subpopulations of T lymphocytes
within the helper and suppressor sublineages in man. J Immunol
129:1997, 1982
4. Reinherz EL, Morimoto C, Fitzgerald KA, Hussey RE, Daley
JF, Schlossman SF: Heterogeneity of human T4’ inducer T cells
defined by a monoclonal antibody that delineates two functional
subpopulations. J Immunol 128:463, 1982
5. Tedder T F , Penta AC, Levine HB, Freedman AS: Expression
of the human leukocyte adhesion molecule, LAMI, identity with the
TQ1 and Leu-8 differentiation antigens. J Immunol 144532, 1990
6. Bevilacqua MP, Nelson RM: Selectins. J Clin Invest 91:379,
1993
7. Rosen SD: Cell surface lectins in the immune system. Semin
Immunol 5:237, 1993
8. Stamper HB, Woodruff JJ: Lymphocyte homing into lymph
nodes: In vitro demonstration of the selective affinity of recirculating
lymphocytes for high-endothelial venules. J Exp Med 144:828, 1976
9. Shaw S, Luce GEG, Quinones R, Gress RE, Springer TA,
Sanders M E Two antigen-independent adhesion pathways used by
human cytotoxic T-cell clones. Nature 323:262, 1986
10. Spertini 0, Luscinskas F W , Kansas GS, Munro JM, Griffin
JD, Gimbrone MA, Tedder T F Leukocyte adhesion molecule-l
(LAM-], L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion. J Immunol 147:2565, 1991
11. Woodruff JJ, Katz IM, Lucas LE, Stamper HB: An in vitro
model of lymphocyte homing 11. Membrane and cytoplasmic events
involved in lymphocyte adherence to specialized high-endothelial
venules of lymph nodes. J Immunol 119:1603, 1977
12. Rosen SD, Singer MS, Yednock TA, Stoolman LM: Involvement of sialic acid on endothelial cells in organ-specific lymphocyte
recirculation. Science 228: 1005, 1985
13. True DD, Singer MS, Lasky LA, Rosen SD: Requirement for
sialic acid on the endothelial ligand of a lymphocyte homing receptor. J Cell Biol 111:2757, 1990
14. Imai Y, Lasky LA, Rosen SD: Sulphation requirement for
GlyCAM-I, an endothelial ligand for L-selectin. Nature 361555,
1993
15. Stoolman LM, Rosen SD: Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation. J Cell Biol
96:722, 1983
16. Stoolman LM, Tenforde TS, Rosen SD: Phosphomannosyl
receptors may participate in the adhesive interaction between lymphocytes and high endothelial venules. J Cell Biol 99:1535, 1984
17. Watson SR. Imai Y, Fennie C, Geoffroy JS, Rosen SD, Lasky
LA: A homing receptor-IgG chimera as a probe for adhesive ligands
of lymph node high endothelial venules. J Cell Biol 1102221, 1990
18. Imai Y, Singer MS, Fennie C, Lasky LA, Rosen SD: Identification of a carbohydrate-based endothelial ligand for a lymphocyte
homing receptor. J Cell Biol 113:1213, 1991
19. Baumhueter S , Singer MS, Henzel W, Hemmerich S, Renz
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
3306
M, Rosen SD, Lasky LA: Binding of L-selectin to the vascular
sialomucin CD34. Science 262:436, 1993
20. Lasky LA, Singer MS, Dowbenko D, Imai Y, Henzel WJ,
Grimley C, Fennie C, Gillet N. Watson SR, Rosen SD: An endothelial ligand for L-selectin is a novel mucin-like molecule. Cell 69:927,
1992
2 1. Beschomer WE, Civin CI, Strauss LC: Localization of hematopoietic progenitor cells in tissue with the anti-My-IO monoclonal
antibody. Am J Pathol 1 l9:1, 1985
22. Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF,
Shaper JH: Antigenic analysis of hematopoiesis 111. A hematopoietic
progenitor cell surface antigen defined by a monoclonal antibody
raised against KG-la cells. J Immunol 133:157, 1984
23. Terstappen LWMM, Huang S, Picker LJ: Flow cytometric
assessment of human T-cell differentiation in thymus and bone marrow. Blood 79:666, 1992
24. Terstappen LWMM, Huang S, Bui N, Picker LJ: Induction
of human hematopoietic stem cell outgrowth by L-selectin ligation.
Blood 82:109a, 1993 (abstr, suppl 1)
25. Kobayashi M, Imamura M, Uede T, Sakurada K, Maeda S,
Iwasaki H, Tsuda Y, Musashi M, Miyazaki T: Expression of adhesion molecules on human hematopoietic progenitor cells at different
maturational stages. Stem Cells 12:316, 1994
26. Koeffler HP, Billing R, Lusis AJ, Sparkes R, Golde DW: An
undifferentiated variant derived from the human acute myelogenous
leukemia cell line (KG-l). Blood 56:265, 1980
27. Bollman JL, Cain JC, Grindlay JH: Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the
rat. J Lab Clin Med 33:1349, 1948
28. Sackstein R, Falanga V, Streilein JW, Chin YH: Lymphocyte
adhesion to psoriatic dermal endothelium is mediated by a tissuespecific receptoriligand interaction. J Invest Dermatol 91:423, 1988
29. Selden RF: Transfection using DEAE-dextran, in Ausubel
FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA,
Struhl K (eds): Current Protocols in Molecular Biology, v01 2. New
York, NY, Wiley, 1992, p 9.2.1
30. Spertini 0, Kansas CS, Reimann KA, Mackay CR, Tedder
TF: Function and evolutionary conservation of distinct epitopes on
the leukocyte adhesion molecule-l (TQ1, Leu-8) that regulate leukocyte migration. J Immunol 147:942, 1991
3 I . Abdullah KM, Udoh EA, Shewen PE, Mellors A: A neutral
glycoprotease of Pasteurella haemolytica A1 specifically cleaves 0sialoglycoproteins. Infect Immun 6056, 1992
OXLEY AND SACKSTEIN
32. Sutherland DR, Marsh JCW, Davidson J , Baker MA, Keating
A, Mellors A: Differential sensitivity of CD34 epitopes to cleavage
by Pasteurella haernolytica glycoprotease: Implications for purificntion of CD34-positive progenitor cells. Exp Hematol 20:590, 1992
33. Krause DS, Fackler MJ, Smith OM, May WS: Two forms of
CD34 protein are expressed in human KMT2 and KG 1 a cells. Blood
82: I loa, 1993 (abstr, suppl 1)
34. Majdic 0, Stock1 J, Pick1 WF, Bohuslav J, Strobl H.
Scheinecker C, Stockinger H, Knapp W: Signaling and induction of
enhanced cytoadhesiveness via the hematopoietic progenitor cell
surface molecule CD34. Blood 83:1226, 1994
35. Yamashita K, Hitoi A, Tateishi N, Higashi T, Sakamoto Y,
Kobata A: Organ-specific difference in the sugar chains of gammaglutamyltranspeptidase. Arch Biochem Biophys 225:993, I983
36. Cullen SE, Kindle CS, Shreffler DC, Cowing C: Differential
glycosylation of murine B cell and spleen adherent cell Ia antigens.
J Immunol 127:1478, 1981
37. Yamashita K, HitoiA, Tateishi N, Higashi T, Sakamoto Y.
Kobata A: The structures ofthe carbohydrate moieties ofmouse
kidney gamma-glutamyltranspeptidase: Occurrence of X-antigenic
determinants and bisecting N-acetylglucosamine residues. Arch Biochem Biophys 240:573, 1985
38. Cowing C, Chapdelaine JM: T cells discriminate between la
antigens expressed on allogeneic accessory cells and B cells: A
potential function for carbohydrate side chains on la molecules. Proc
NatlAcad Sci USA 80:6000, 1983
39. Huff TF, Uede T, Iwata M, Ishizaka K: Modulation of the
biologic activities of IgE-binding factors. 111. Switching of a T cell
hybrid clone from the formation of IgE-suppressive factor to the
formation of IgE-potentiating factor. J Immunol 131: 1090, 1983
40. Lewinsohn DM, Bargatze RR, Butcher EC: Leukocyte-endothelia1 cell recognition: Evidence of a common molecular mechanism
shared by neutrophils, lymphocytes, and other leukocytes. J Immunol
138:4313, 1987
4 I . Ord DC, Ernst TJ, Zhou LJ, Rambaldi A, Spertini 0, Griffin
J, Tedder TF: Structure of the gene encoding the human leukocyte
adhesion molecule-l (TQI, Leu-8) of lymphocytes and neutrophils.
J Biol Chem 265:7760, 1990
42. Griffin JD, Spertini 0, Ernst TJ, Belvin MP, Levine HB,
Kanakura Y, Tedder TF: Granulocyte-macrophage colony-stimulating factor and other cytokines regulate surface expression of the
leukocyte adhesion molecule-l on human neutrophils, monocytes,
and their precursors. J Immunol 145576, 1990
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1994 84: 3299-3306
Detection of an L-selectin ligand on a hematopoietic progenitor cell
line
SM Oxley and R Sackstein
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