1. Art and Diseño

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Transfected Leukocyte Integrin C D l l b/CD18 (Mac-1) Mediates Phorbol
Ester-Activated, Homotypic Cel1:Cell Adherence in the K562 Cell Line
By Dennis D. Hickstein, Eduardo Grunvald, Grace Shumaker, Dianne M. Baker, Anthony L. Back, Lisa J. Embree, Ester Yee,
and Katherine A. Gollahon
The C D I 1b/CD18 leukocyte integrin molecule mediates
diverse neutrophil adherence-related functions, including
cell:cell and cell:extracellular matrix attachments. To
study the individual role of this leukocyte integrin in cell
adherence in hematopoietic cells, we expressed the
C D I 1b/CD18 complex on the surface of K562 cells, a cell
line derived from an individual with chronic myelogenous
leukemia in blast crisis. We used an amphotrophic retroviral vector designated LCDI 8SN. harboring the complete
coding sequence for the CD18 subunit, to transfer the
CD18 cDNA into K562 cells and select stable cell lines.
The C D I 1b subunit in the expression plasmid pREP4 was
transfected into these K562/CD18 cells by electroporation
and stable cell clones were selected. These K562 cells possessed RNA and intracellular protein for each subunit, and
they expressed the CDI 1b/CD18 heterodimer on the cell
surface. When CDI 1b/CD18 expressing K562 cells were
stimulated with phorbol myristate acetate (50 ng/mL) for
2 4 to 48 hours, these K562 cells formed dense cekcell
aggregates. This homotypic aggregation required both activation of the C D I 1 b/CD18 complex and the induction of
the counter-receptor for C D I 1 b/CD18 on the conjugate
cell. This cell line will (1) enable the structure-function relationships between cell activation and homotypic adherence to be assessed, (2) provide the opportunity to identify
accessory molecules required for activation of the C D I 1 b/
CDI 8 complex, and (3)facilitate the identificationof novel
ligands for the CDI 1b/CD18 complex.
0 1993 by The American Society of Hematology.
T
plished using a Not I/Acc I11 fragment at the 5’ end, an Acc III/Xho I
fragment for the middle section, and an Xho I/Not I fragment at the
3’ end. The 4.1-kb CDl I b reconstruction contained the full-length
coding sequence for CD 1 1b. The CD 1 1b cDNA with Not I ends was
cloned into the Not I site of the expression vector PREP 4 (in vitrogen). The PREP 4/CDl lb antisense of “inverted” clone was constructed by cloning the CD 1 I b cDNA into the PREP 4 expression
vector with the 3’ end of the CDI Ib cDNA located adjacent to the
vector promoter. This construct served as a control.
Transfer of the CD18 and C D l l b cDNA into K562. Supernatant from the LCDl8SN producing PA3 17 packaging cell line was
used to infect K562 cells. After 24 hours of infection, the K562 cells
were incubated in the presence of G4 18 ( 1 mg/mL). The G4 18-resistant K562 cells were used in all subsequent experiments.
The PREP 4/CD1 l b plasmid was introduced into K562 cells,
and K562 cells infected with LCD I8SN, by transfection. For the
transfection, the K562 cells were electroporated using a BioRad
Electroporation “Gene Pulser” (BioRad,Richmond, CA).Approximately 1 X lo7 cells were suspended in 0.5 mL of HEPES buffer
(pH 7.5), placed in a 0.4 mL cuvette, and 100 pg of circular plasmid
DNA was added. Plasmid DNA was prepared using equilibrium
centrifugation in cesium chloride/ethidium bromide gradients.*
K562 cells were electroporated at 250 V and 960 pF. Cells were
HE NEUTROPHIL integrin complex CD 1 1b/CD 18
(Mac-1) has been shown to play a major role in both
homotypic and heterotypic neutrophil adherence in vitro’,’
and in vivo.) Specifically, the adherence of activated neutrophils to each othe8 and to endothelial monolayers is inhibited by monoclonal antibodies (MoAbs) binding to the
CD 1 1b/CD 18 complex.’
To facilitate understanding of the structural relationship
between the CD 1 1b/CD 18 complex and hematopoietic cell
adhesion, we transfected the cDNAs encoding the human
CD1 l b and CD18 subunits into K562 cells. We selected
K562 cells for these studies because (1) the cell line is derived from an individual with chronic myelogenous leukemia/blast crisis, and thus is an immature leukocyte cell line;
(2) K562 cells do not express any of the leukocyte integrins,
enabling CDl lb/CD18 expression to be studied in isolation; (3) K562 cells are susceptible to infection with retroviral vectors and to transfection by electroporation; (4)the
promoters that result in high levels of expression in K562
cells are well characterized and ( 5 ) K562 cells are nonadherent.4 Previous studies of the leukocyte integrins have
used transient transfections into adherent cell lines.
Gene transfer of CD18 and C D l l b into K562 cells resulted in surface expression of the CD 1 1b/CD 18 complex.
Treatment of the doubly transfected K562 cells with phorbo1 myristate acetate (PMA) induced homotypic cel1:cell
adherence. The generation of this cell line expressing the
CD 1 1b/CD 18 complex provides the opportunity to identify
accessory molecules required for activation of the C D 1 1b/
CD 18 complex, and to identify novel ligands or counter-receptors for the CD 11b/CD 18 complex.
MATERIALS AND METHODS
Construction of expression vectors for CD18 and C D l l b . We
used an amphotrophic retroviral vector, designated LCD 18SN,
harboring the complete coding sequence for the CD 18 subunit, to
transfer the CD18 cDNA into K562 cells. The construction of this
vector and the packaging of this vector have been described.s.6
The full-length CD1 l b cDNA was reconstructed from overlapping cDNA for CDll b in X gt 1 1.7 This reconstruction was accomBlood. Vol82, No 8 (October 15). 1993: pp 2537-2545
From the Medical Research Service, Seattle VeteransAfairs Medical Center, Seattle; and the Department of Medicine, University of
Washington School of Medicine, Seattle.
Submitted December 28, 1992; accepted June 18, 1993.
Supported by National Institutes of Health Grant No. DK 43530
(D.D.H.),the March of Dimes Birth Defects Foundation (D.D.H.),
and the Veterans Afairs Career Development and Merit Review
Programs (D.D.H.).A.L.B. is supported by a C.I.A.Awardfrom the
National Institutes of Health (KO8 HL02637).
Address reprint requests to Dennis D. Hickstein, MD, Medical
Service (11I), Seattle VA Medical Center, 1660 S Columbian Way,
Seattle, WA 98108.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-49 71/93/8208-0001$3.00/0
2537
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HICKSTEIN ET AL
2538
A
The MoAbs used were anti-CD18 (MoAb 60.3), anti-CDIlb
(MoAb 60.1 or MoAb OKMI), anti-CDI la (MoAb RR 3.1), antiCDI IC (MoAb LeuMS), and anti-murine leukemia virus envelope
protein (MoAb 9E8). In flow cytometry, an increment of 20 U on
the log scale on the abscissa represents a doubling of fluorescence
promoter
5.4 kb
intensity. In selected studies, PMA was incubated with cell populations at a concentration of 50 ng/mL for 48 hours before flow cytometry analysis.
Western blotting. Cell lysates were generated from K562 cells,
K562
cells infected with the retroviral construct LCDl8SN, K562
pREP4/CDllb
cells infected with LCDl8SN and transfected with the PREP expression vector containing the CDI Ib cDNA, and normal polymorphonuclear leukocytes (PMN). The individual lysates were
subjected to gel electrophoresis on a 7% acrylamide reducing gel,
transblotted to nitrocellulose, and reacted with the anti-CD18 or
antLCD1 1b rabbit polyclonal antibodies. Blotting patterns were esPREP4
tablished by exposure to the chromogen 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium. In previous studies, we have
described the monospecific nature of the anti-CD 18 antibodysa6and
the anti-CDI Ib a n t i b ~ d y . ~
SV-40
Northern blotting. Total cellular RNA was isolated from K562
Ori
cells, LCDl8SN infected K562 cells, LCDISSN + pREP4/CDI Ib
K562 cells, and LCD18SN + PREP 4/CD1 Ib antisense or "inFig 1. Vectors used to express the CD18 and C D l l b subunits.
verted" clone, using the guanidine HCl method." In the Northern
(A) The amphotrophic retroviral vector LCDl8SN was created by
cloning the CD18 cDNA into the Hpa I site of MSN. (B) pREP4/
blotting studies, 10 pg of total RNA was electrophoresed on a formCDl 1b was created by cloning the full-length C D l l b cDNA into
aldehyde-agarose gel, transferred to nitrocellulose, and hybridized
the Not I site in the polylinker of the episomal expression vector
to a nick-translated "P-labeled 1.8-kb cDNA corresponding to the
pREP4. RSV LTR indicates the Rous sarcoma virus LTR.
CD18 subunit,12a 0.7-kb cDNA corresponding to the CDl Ib subunit,' or to the 2-kb chick @-actinPst I cDNA fragment.I3
Analysis of CD1I b/CDl&mediated cel1:cell adhesion. To assess the ability of the reassembled CDl 1b/CD18 heterodimer on
placed in Dulbecco's modified Eagle's medium (DMEM) after elecK562 cells to mediate cel1:cell adhesion, K562 cells, K562 cells
troporation and incubated at 37°C in 5%C02/95%air in the contininfected with LCDlSSN, K562 cells transfected with the CDI l b
uous presence of hygromycin at 100 pg/mL. Individual hygromycDNA, and K562 cells expressing the CDI lbjCD18 complex were
cin-resistant CDI I b clones were isolated by limiting dilution.
incubated in the presence of PMA (50 ng/mL) for 4, 24, and 48
Immunofluorescence flow cytometry. Individual cell populahours. At the end of the incubation period, the cell populations
tions of K562 cells resistant to (3418 and hygromycin were anawere photographed using a Nikon inverted microscope (Nikon
lyzed for surface expression of the CD1 I/CD18 leukocyte integrin
Corp, Melville, NY). Adhesion of K562 cells was scored according
heterodimers using subunit specific MoAbs and indirect immunofluorescence followed by flow cytometry as described p r e v i o u ~ l y . ~ ~ to
~ ~the extent of cluster formation or aggregation in response to
LCD18SN
B
I
K562
K56BCDlB
K562JCD18
+CDllb
K56BCD18
+CDllb
flnverted)
CDlla
Fig 2. Immunofluorescence flow cytometry of
K562 cells and human neutrophils. The cell populations designated at the top consist of (1) K562
cells, (2)
K562 cells infected with the retroviral construct LCD18SN. (3)K562 cells infected with
CDllb
(MAb60.1)
z
P
2
NeulroDhil
CDllb
(MAbOKMI)
CDllc
CDl0
Relative fluorescence intensity (log scale)
LCDl8SN and transfected with the C D l l b cDNA
in the expression vector pREP4, (4) K562 cells infected with LCDl8SN and transfected with the
C D l l b cDNA in the antisense orientation in the expression vector pREP4, and ( 5 )normal human neutrophils. The cell populations designated at the top
were incubated with a control MoAb 9E8 (---) or
with an MoAb specific for the individual C D l l or
After incubation with the
CD18 subunits (-).
MoAbs, cells were incubated with a fluoresceinated sheep anti-mouse antibody and analyzed by
flow cytometry. Relative fluorescence intensity is
shown on the abscissa on a log scale from 0 to 200,
where each mark represents a fourfold increase in
fluorescence intensity. Cell number appears on the
ordinate.
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2539
TRANSFECTED LEUKOCYTE INTEGRIN C D l l B/CD18
K562
K562/CD18+ CD11 b
Nikon fluorescent microscope was used to capture images of the
samples. In addition, photographs were taken in the plates with a
Nikon inverted fluorescent microscope using Ektachrome ASA 400
color film. Filters were the G2 and B2 filters from Nikon.
RESULTS
Relative flourescsnce intensity
(log scale)
Fig 3. Analysis of surface expressionof the C D l l b subunit. Indirect immunofluorescence (as in Fig 1) was used to determine surface expression of the C D l l b subunit in K562 cells (top left panel),
K 5 6 2 cells infected with LCD18SN and transfected with PREP 4/
C D l l b (top right panel), and two different clones of K562 cells
transfected with PREP 4/CD11 b alone. The K562 cells infected
with LCDl8SN represent clones selected in G418, and the K562
cells transfected with pREP4ICDll b were selected in hygromycin.
as described in the Materials and Methods. Each mark on the relative fluorescence intensity scale on the abscissa represents a sixfold increase in fluorescence intensity.
PMA. The degree of aggregation was scored with scores ranging
from 0 to 5+, where 0 indicated that essentially no cells were in
clusters, 1+ indicates less than 10%of cells in aggregates, 2+ indicates less than 50% of cells in aggregates, 3+ indicates up to 30%of
cells in loose clusters, 4+ indicatesup to 100%of cells in aggregates,
and 5+ indicates up to 100%of cells in large, very compact aggregate~.’~,’~
The blocking MoAbs used in the adhesion assays consist of
MoAb MHM 23 (anti-CDl8), MoAb H52 (antLCD18), MoAb 904
(anti-CD1 Ib), MoAb 60.1 (anti-CD11b), and MoAb OKMl (antiCDI Ib). All MoAbs were used at saturating concentrations. The
anti-CD1 Ib MoAb LPM19c (Dako Corp, Carpintena, CA) was
used in separate blocking experiments (see Fig 7).16
Two-color analysis of homotypic adherence of K562 cells. K562
cells or K562/CD18 + CDI Ib cells were harvested and resuspended at IO’ cells/mL. Cationic membrane probes from Molecular Probes (Eugene, OR) were used to mark the two cell lines.”
These reagents were DiO, octadecyl C I8 oxacarbocyanines (green),
and DiI, octadecyl C18 indocarbocyanines (red). These reagents
were dissolved in dimethylsulfoxide (DMSO) at 100 mg/mL and
the K562 cells were stained with DiO (green) and the K562/CDl8
+ CDI I b cells (labeledas doubles)were stained with DiI (red) ( 100
pg/mL at 37°C for 2 hours). The cells were then washed and resuspended in media at 5 X I05/mL. One aliquot of each of the two cell
populations was treated with PMA at a concentration of I O ng/mL
(these cells are labeled as + in Fig 8). A second aliquot of each cell
population was not treated with PMA (these cells are labeled as - in
Fig 8). After 48 hours of incubation, the following 1:1 mixtures of
cells were made in a 6-well plate: K562 (-) and doubles (+); K562
(+) and doubles (-); and K562 (+) and doubles (+). Wet mounts
were made of the samples and a Nikon video camera attached to a
Vectors used to transduce CD18 and CDllb into K562
cells. Two different vectors using two different methods of
gene transfer were used to introduce the CD 18 and CD 1 1b
cDNAs into K562 cells. An amphotrophic retroviral vector
containing the CD18 subunit (LCD18SN) was used to
transduce the CD 18 subunit into K562 cells (Fig 1A). In this
vector, the CD18 subunit is expressed from the Moloney
long terminal repeat (LTR).’*The CD11b subunit wasintroduced into K562 cells using the pREP4 expression plasmid
(Fig 1B). This vector contains the Epstein-Barr virus (EBV)
origin of replication and replicates episomally. It also contains a hygromycin-selectable marker. In this construct, the
CDI lb cDNA is expressed from the Rous sarcoma virus
promoter.
Surface expression of leukocyte integrins on K562
cells. Expression ofthe individual CDI 1 and the common
CD 18 subunit was analyzed using subunit-specific MoAb
and indirect immunofluorescence followed by flow cytometry (Fig 2). None of the leukocyte integrins are expressed on
K562 cells or K562 cells transduced with the CD18 subunit
alone (Fig 2, columns 1 and 2). However, both CD1 l b and
CD 18 are expressed on the surface of K562 cells transduced
with cDNAs for both CD18 and CD1 lb (Fig 2, column 3).
Introduction of the CDI Ib subunit in the antisense orientation (CD1 lb inverted) into CDl 8-expressing K562 cells
failed to result in surface expression of the CDI Ib/CD18
heterodimer (Fig 2, column 4). Levels of expression of
CD 1 1b/CD 18 on human neutrophils were more uniform
than the levels present on the CD 1 1b/CDIg-transduced
K562 cells, consistent with the variability in copy number
of CDl Ib introduced into K562 cells using the episomal
based vector pREP4. Human neutrophils contain CDl la/
CD 18, CD 1 1b/CD 18, and CD 1 1c/CD 18; in contrast, the
CDI lb + CD18 transfected K562 cells express only
CDI lb/CD18.
Table 1. Adherence of Transfected K 5 6 2 Cells
in Response to PMA
K562
K562/CD 18
K562/CD11 b (low)
K562/CD1 l b (high)
K562/CD18 CD1 l b
+
+
+
- PMA
+ PMA
PMA
+Anti-CD1 1b
PMA
+Anti-CDlB
010
010
010
+1/+1
+2/+1
+1/+2
+2/+1
+4/+3
+1/+1
+2/+1
+1/+2
+2/+1
+4/+3
+1/+1
011
111
+2/+2
+1/+2
+2/+1
+4/+3
K562 cells were incubated without (-PMA) or with PMA (+PMA) at
5 0 ng/mL for 48 hours. The MoAbs used were anti-CD18 (MoAb MHM
23 and MoAb H52) and anti-CD1 l b (MoAb 904, MoAb 60.1, and
MoAb OKM1). These MoAbs were added to the PMA-treated cells as
described in the Materials and Methods. The scoring system for aggregation ranges from 0 (no aggregation) to 5+ (100% of cells in large,
compact aggregates), as described in the Materials and Methods.
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HICKSTEIN ET AL
2540
A
B
aD
c
0
z
0
\
V
N
N
u)
W
v)
Y)
Y
Y
ez+ ,
2
E
- 200 KD
e-
1
2
3
4
- 116 KD
- 97KD
- 66KD
1
2
3
4
Fig 4. Westem blotting studies of K562 cells and m a l PMN. Cell lysates were obtained from K562 cells (lane 1). K562 cells infected
with the LCD18SN retroviral ~ ~ n s t (lane
~ c t2), K562 cells infected with the LCDlSSN retroviral construct and transfected with the
PREP-CD11b vector (lane 3). and normal PMN (lane 4).The individual cell lysates were separated by sodium dodecyl sulfate-polyactylamide
gel electrophoresis, transferred to nitrocellulose, and reacted with an anti-CD18 rabbit polyclonal antibody (A) or an anti-CD11 b rabbit
polyclonal antibody (E) followed by an alkaline phosphatase-linked antirabbit antibody and chromogen, as described in the Materials and
Methods. Molecular weight standards appear on the right. Arrows indicate the size of the PMN CD18 and CD11 b subunit.
The individual CDI I and CD18 subunits appear to require heterodimer formation for efficient surface expression.I9To evaluate the ability of individual CDI I b subunits
to become surfaceexpressed independent of CD18. we
compared the surface expression of CD I I b on K562 cells
transduced with CDI I b alone with that of K562 cells transduced with both subunits (Fig 3). Efficient surface expression of CDI Ib required CD18 (Fig 3, upper right panel).
However, selected clones of K567 cells transduced with
CD 1 I b alone were capable of surface expression of CD I I b
in the absence of CDI 8 (Fig 3. lower right panel). These two
CDI Ib-expressingclones were used in functional studies to
assess homotypic aggregation (Table I). In those studies. the
cell populations are labeled K562/CDI Ikl (low) and
K562/CD I I k 2 (high).
,fnulj*.visol'inirucidlrrlar proicin,fi)rCDI Ih and CDI8 in
KS62 ccll.~. To analyze the molecular weights of the
CDI Ib and CD18 subunit in K562 cells. Western blotting
studies were performed. The CD18 subunit in K562 cells
had an approximate molecular weight of 90 Kd (Fig 4A.
lane 2). The CD18 subunit is not processed to the higher
molecular weight 100-Kd membraneexpressed form in the
absence ofCD1 I b. consistent with previous~tudies.'~
When
CDI I b is introduced. approximately 25% of the CD18 is
processed to a higher molecular weight form (Fig 4A. lane
3). indicating that heterodimer formation is important for
processing. Despite the increase in molecular weight of
CD18 in thepresenceofCD1 Ib, theCD18 protein in K562
never reaches the size of the membranecxpressed form of
CD18 prescnt in neutrophils. The CDI Ib subunit in K562
cells appears to be the same molecular weight as that of the
CDI Ib subunit in human PMN (Fig 4R. compare lanes 3
and 4).
Ana/wis orCDl Ih and CD18 mRNA in K562 cell.^. The
levels and sizes of CD18 and CDI I b mRNA in the transduced K562 cell clones were approximately equal on
Northern blotting(Fig 5). This indicated that both the retroviral vector used to introduce CD18 and the episomal
pREP4/CDI Ib resulted in production of their respective
mRNAs of appropriate size. These results are consistent
with previous studies showing the utility of the promoters
used in these constructs to express heterologous genes in
hematopoietic cells.
R c y m s c * d K M 2 cells io P.V.4. To assess the effect of
PMA on the surface expression of the leukocyte integrins.
flow cytometry studies were performed (Table 2). There was
no increase of surface expression of the CD 1 I b/CD I8 complex on the CDI 1 b/CDI %expressing K562 cells in response
to PMA.
Firnctii)nnl activiiy (?ftiii~
CDI Ih/CD18 iicicrodimcr.The
transfected K562 cells were examined for their ability to
mediate cell:ccll adherence reactions in response to PMA.
K562 cells. K562 cells infected with LCDl 8SN, and K562
cells expressing high levels of surface CDI I b formed a few
small clumps in response to PMA (Fig 6. right-hand column). In contrast. K562 cells expressing surface CDI Ib/
CD I8 formed dense aggregates in response to exposure to
PMA for 48 hours (Fig 6.bottom right panel). To quantitate
the homotypic cell:cell aggregation. cell aggregates were
scored according to the amount ofclumping. asdcscribed in
the Materials and Methods. The K562 cells expressing the
CDI I b/CDI 8 heterodimer aggregated to a much greater
extent than the K562 cells transduced with either subunit
alone (Table I). MoAb M H M 23 and MoAb H52 directed
against the CDI 8 subunit. and MoAb 904. MoAb 60.1. and
MoAb OKM I directed against the CDI I b subunit failed to
block this homotypic adherence (Table I). Only MoAb
LPM 19c. directed against the CDI I b subunit. blocked homotypic aggregation (Fig 7). MoAb LPM I9c was not used
in the experiments shown in Table I. Without MoAb
LPM I9c. the K562 cells expressing CD I 1 b/CD I8 showed
3+ to 4+ aggregation in response to PMA. whereas the aggregation was reduced to 2+ in the presence of LPM19c.
The MoAb R6.5. directed against domain 3 of intercellular
adhesion molecule-I (ICAM-I ). also failed to block this homotypic adhesion (data not shown).'"
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TRANSFECTED LEUKOCYTE INTEGRIN CD1 lelCD18
2541
6 6
00
+ +
C D l lb
CD18
Kb
DISCUSSION
Kb
4.7
3.4
-
-
I
1 2 3 4
Actin
Kb
Actin
I
tion (Fig 8, top panels). When the doubles (red)were treated
with PMA and added to untreated K562 cells (green), the
clumps that formed werc entirely red (Fig 8. middle panels).
This indicated that the untreated K562 cellswere not participating in the aggregates, and it suggested that a ligand for
CDI Ib/CD18 needed to be induced on the K562 cells by
PMA. When both cell populations were treated with PMA.
the aggregates consisted of both red and green cells. indicating that both activating the CDI I b/CD18 complex and inducing a ligand for CDI 1 b/CDI 8 were required for the aggregation (Fig 8, bottom panels).
Kb
2-
I
1 2 3 4
Fig 5. Northem blotting of K562 cells. Total cellular RNA (20
pg/lane) was extracted from the designated cells, separated on
formaldehyde-agarose gels, transferred to nitrocellulose, and sequentially hybridized to "P-labeled cDNA probes correspondingto
CDl 1b, CD18, and B-actin. The lanes are K562 cells (lane 1). K562
cells infected with the LCDl8SN retroviral construct (lane2). K562
cells infected with the LCDl8SN retroviral construct and transfected with the PREP-CD11b vector (lane 3).and K562 cells infected with the LCDl8SN retroviral construct and transfected with
the pREP4 expression vector harboring the C D l l b cDNA in the
antisense orientation (inverted) (lane4). The size of the transcripts
are indicated on the left.
T~tw-cokor staininx of nontransclttccd and transdtrccd
KS62 cd1.s. To address the question of whether PMA was
"activating" the CDI I b/CD18 complex to mediate homotypic adhesion. or whether PMA was inducing a ligand or
counter-receptor for the CDI I b/CDI 8 complex. two-color
studies were performed using K562 cells and K562 cells
transduced with both subunits (termed doubles). Cells were
incubated in the absence of PMA (-) or in the presence of
PMA (+), and then the labeled cells were mixed and assessed. These results are shown (Fig 8). When K562 cells
(green)were treated with PMA and incubated with doubles
(red) that were untreated. the few small clumps that formed
were green, suggesting that the CDI Ib/CD18 complex
needed to be "activated" by PMA to mediate cell aggrega-
The inflammatory response requires the recruitment of
circulating leukocytes to the site of infection. The recruitment of neutrophils or P M N requires that the neutrophil
possess the ability to participate in a variety ofadherence-related functions. To participate in these functions. neutrophils use a number of different adhesion molecules.2"'' The
leukocyte integrin CDI I b/CD18 adhesion molecule playsa
major role in mediating these adherence functions.
The evidence for the role of CDI 1 b/CD18 in neutrophil
adherence is twofold. MoAbs directed against the CDI I b/
CD18 complex block ( I ) the binding of C3bicoated particles to the neutrophil.24(2) the adherence of neutrophils to
(3) homotypic neutrophil aggregation.'
endothelial
and (4) neutrophil adherence in vivo.-' Secondly. children
born with genetic defects in CDI 8 that prevent surface expression oftheCD1 I/CD18 complexsuffersevererecurrent
bacterial infections because of the inability of leukocytes to
migrate to the site of infection.'"
There are two special features ofCDl I b/CDI %mediated
adherence. First. there is evidence that the epitop on
CDI Ib/CD18 that binds C3bi is distinct from the epitope
that binds to the endothelial celL2' Second. adhesion mediated by CDI I b/CD18 requires "activation" of the integrin heterodimer. Activation can be accomplished experimentally by phorbol esters. or by various inflammatory
mediators (ie. tumor necrosis factor. C5a. f-met-leu-phe).
Table 2. Expression of Leukocyte lntegrins on K562 Cells
in Response to PMA
K562 cells
K562 cells + PMA
K562/CD18
K562/CD18 + PMA
K562fCD18fCDllb
K562/CD18/CD1 l b + PMA
K562/CD18/CDll b (inverted)
K562/CD18/CDll b (inverted) + PMA
Control
CDt8
CDttb
3.6
7.2
5.4
15.3
3.8
6.9
6.2
14.5
33.1
30.3
5.5
6.9
5.0
7.1
6.2
15.9
81.5
53.1
7.2
6.3
3.0
9.2
4.6
7.3
Each cell population was incubated with an irrelevant first antibody
(control), an anti-CD18 MoAb. or an anti-CD11b MoAb followed by a
fluorescein isothiocyanate-linked sheep antimouse antibody and analyzed by flow cytometry. Results are expressed as the percentage of
cells above the backgroundfluorescence obtained using only a fluorescein-linkedsecond antibody. Cells were stimulated with 50 ng/mL PMA
for 48 hours.
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HICKSTEIN ET AL
2542
+ PMA
K562
K562/
CDllb
Fig 6. Adhesion of K562 cells in response to
PMA. K562 cells. K562 infected with the
LCDl8SN retroviral construct, K562 cells transfected with pREP4/CDllb, and K562 cells infected with the LCDl8SN retroviral construct and
transfectedwith pREP4/CDll b, were incubated in
the absence (left-hand panels) or presence (righthand panels) of PMA at 5 0 ng/mL for 4 8 hours. At
the end of the incubation period, the cells were
photographed using a Nikon inverted microscope
and scored for aggregation as described in the Materials and Methods.
K562/
CD18
CDllb
Stimulation of neutrophils with chemoattractants is required to activate binding of these integrins to ICAM-I .25*27
Stimulation of neutrophil integrin avidity is a rapid response that occurs within minutes. and does not require an
increase in integrin surface expression.’*
Studies in which other integrins have been transfected
into heterologous cells provide a useful background for interpreting our results. Intracellular signals have been shown
to influence the functional activity of the CDI Ia/CD18
complex. Lymphoid cell lines expressing CDI la/CD18 do
not spontaneously aggregate in culture: however. treatment
with phorbol esters induces a rapid aggregation of these
cell^.'^ Phorbol ester stimulation is not accompanied by an
increase in surface expression of CDI la/CD18.” Instead,
exposure to phorbol esters convertsCDI Ia/CDI 8 to a highaffinity state.29In addition to treatment with agents that
activate intracellular protein kinase C, the adhesion of the
leukocyte integrin CDI Ia/CD18 (LFA-I) for ICAM-I substrates is enhanced by cross-linking of the T-cell receptor.”
These observations support the idea that signals from the
cytosol lead to changes in the extracellular domain of
CDI la/CD18, and these changes result in a CDI Ia/CDI8
complex with an increased adherence for ICAM-I.
When the CDI la and CDI 8 subunits were cotransfected
into Coscells. the CDI 1a/CD18 heterodimer wasconstitui-
tively active and not functionally regulated. Although Cos
cells expressing the dimer bound to ICAM-I. these cells did
not respond to PMA with a further increase in affinity for
ICAM-I
In addition. the CD18 subunit was absolutely
required for expression of the CDI la subunit in Cos cells.”’
These studies indicated that the CDI Ia/CD18 complex on
fibroblastoid cells is not functionally regulated.
Upon transfection into leukocyte adherence deficiency
(LAD) ERV B-lymphoblastoid cell lines, the transfected
CD18 subunit forms a heterodimer with the endogenous
CDI la subunit. and this complex can be activated by PMA
to mediate binding to ICAM-I.” This binding activity is
modulated by cytoplasmic domain sequences.
Studies of the platelet integrin Ilb/llla also provide insight into theCDI I b/CDI 8 leukocyte integrin. The Ilb/llla
complex binds to soluble fibrinogen only after cellular stimulation.’* In llb/llla. this affinity change is due to alterations in the conformation of the receptor.” When Chinese
hampster ovary (CHO) cell lines were cotransfected with
Ilb/llla in expression vectors, surface expression of the heterodimer was achieved. The Ilb/llla-transfected cells did
not bind soluble fibrinogen unles the Ilb/llla complex was
activated using an MoAb that binds to the extracellular domain of llla and “activates” the receptor.-” When a Ilb subunit containing a deletion of the cytoplasmic domain was
.’””’
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2543
TRANSFECTED LEUKOCYTE INTEGRIN CD11B/CD18
K562/CD18
-
Fig 7. MoAb LPM19c blocking studies. K562
cells expressing the CD11 b/CD18 heterodimer
were unstimulated (top panel), stimulated with
PMA for 48 hours in the absence of LPMl9c (middle panel). and stimulated with PMA in the presence of MoAb LPMl9c (lower panel). At the end of
the incubation period, the cells were photographed
as described in Fig 6. The aggregation of the
C D l l b/CD18-expressing K562 cells scored 3 to
in
4
in the absence of MoAb LPMl9c and 2
the presence of MoAb LPMl9c.
+
CDllb
+
C D l lb
PMA
K562/CD18
+
+
PMA
K5621CD18 + CD1 1 b
+
PMA
+
LPM19c
+
+
transfected with Illa. the resulting heterodimer was constituitively active. These results indicate that sequences in the
cytoplasmic domain of Ilb control the affinity state of Ilb/
Illa. It is postulated that these sequences bind to an intracellular molecule that maintains the receptor in a low-affinity
conformation.
To characterize the role of the CDI I b/CDI 8 complex in
mediating adherence reactivity. we used gene transfer to
reassemble the two subunits of this heterodimer on K562
cells.4We selected the KS62 cell line because it is a nonadherent, immature leukocyte cell line that lacks all members
of the leukocyte integrin family. In addition, KS62 cells are
susceptible to retroviral-mediatedgene transduction as well
as transfection by electroporation. We aswssed the KS62
cells after gene transfer and showed the presence of RNA,
intracellular protein, and surface expression of the CDI I b
and CD18 subunits. We then showed that this heterodimer
enabled K562 cells to aggregate in response to PMA. However. aggregation required the addition of PMA as an agonist. and the homotypic cell:cell adhesion required 24 to 48
hours for maximum effect.
The activity of the CDI I b/CDI 8 complex on K562 cells
requires some explanation. First. the complex does enable
K562 cells to display homotypic cell:cell adherence in response to PMA. The rapid response to PMA may require
additional cytosolic factors that are not present in KS62
cells as a result of the relative immaturity of these cells.
Instead. these accessory molecules may need to be induced
by PMA. In previous studies using transient transfection
assays,Coscellscotransfectcdwith CDI I b/CDI 8 expressed
the heterodimer on the surface ofapproximately 43%ofthe
cells: however. these transfected cells did not interact with
ICAM-1.” One explanation for thisobservation is that both
K562 and Cos cells lack factors required to “activate” the
CDI 1 b/CDI 8 complex to mediate adherence. For example.
the FcRdllA cDNA mediated a phagocytic response when
transfected into P388DI (a mouse macrophage cell line).
but not when transfected into CHO cells.34Thus, the
FcRdllA-mediated phagocytic response, and the associated
calcium flux. require macrophage signaling machinery.
which is not present in fibroblasts. Our transfection studies
of CDI Ib/CD18 in KS62 cells are consistent with parallel
studies using the integrins CDI Ia/CD18 and platelet Ilb/
llla in that transfection of both subunits reconstitutes surfaceexpression. but activation ofthe integrin is required for
functional adherence.
The observation that homotypic adherence of the transfected KS62 cells requires 2 4 to 48 hours for maximum
effect, unlike neutrophil aggregation. which occurs within
minutes after the addition of PMA, suggests that PMA may
be acting by inducing a ligand on the conjugate cell. The
leukocyte integrin CDI Ib/CD18 binds to the third amino
terminal Ig-like domain of ICAM-I. and this binding a p
pears to be influenced by the extent of glycosylation on the
ICAM-I m01ecuIc.~~
Of note. this binding is of low affinity.
The ICAM-I molecule was not upregulated on K562 cells
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
HICKSTEIN ET AL
2544
Phase
Doubles (red)
K562 (green)
Doubles hellow)
K562 + PMA
Doubles - P M A
K562 - PMA
Doubles + PMA
+
Fig 8. Two-color analysis of homotypic aggregationof K562 cells. K562 cells were stained with DiO (green) and K562/CD18 CD1
cells (labeledas doubles) were stained with Dil (red) at 37°C for 2 hours. The cells were then washed, resuspended in media, and treai
with PMA ( ) or left untreated ( ). After 48 hours of incubation,the following 1:1 mixtures of cells were made in a 6-well plate: K562 (
and doubles ( 1; K562 ( ) and doubles ( -); and K562 ( + ) and doubles (+ ). Wet mounts were made of the samples and a Sony Trinii
video camera attached to a Nikon fluorescent microscope was used to capture images of the samples. The left-hand column represei
phase microscopy, the middle panel images the red cells, and the right-hand panel images the green cells.
+
+
+
-
using PMA (data not shown). Because the CD 1 1b/CD I8
complex does not bind to ICAM-2:7 an endothelial molecule related to ICAM-1:'
and ICAM-3 is not present on
K562 cells, a novel counter-receptor may be mediating this
adhe~ion.~',~~
In the MoAb blocking studies, only MoAb LPM19c
blocked the PMA-induced aggregation of the CD 1I/CD 18expressing K562 cells. MoAb LPM19c, which binds to the I
domain ofCD1 Ib, blocked neutrophil awegation in a previous study.37A number ofother MoAbs, which bind to the
I domain of CD11b and reduce binding to fibrinogen,
ICAM-1, and iC3b, only minimally influence neutrophil
homotypic adhe~ion.'~
These differences in the patterns of
MoAb inhibition indicate the presence of several different
functional domains within the I domain. Of note, the
CD1 lb/CDl8 counter-receptor responsible for neutrophil
homotypic adhesion has not been characterized.
These studies provide an expenmend system in which
the structure-fundion relationships between CDIlb/CD18
cell activation and homotypic adherence can be
using a hematopoietic cell line rather than a fibroblastic or
Provide the
to
lymphoid
line, and
identify the structural determinants of heterodimer formation and surface expression of the CDI lb/CD18 complex.
In addition, identification of the accessory molecules required for CD 1 1b/CD 18 activation, and identification of
ligand or counter-receptor for the CD 1 1b/CD 18 complex
may be facilitated by using this CD 1 1b/CD 18-expressing
K562 cell line.
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1993 82: 2537-2545
Transfected leukocyte integrin CD11b/CD18 (Mac-1) mediates phorbol
ester-activated, homotypic cell:cell adherence in the K562 cell line
DD Hickstein, E Grunvald, G Shumaker, DM Baker, AL Back, LJ Embree, E Yee and KA Gollahon
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