CD15 expression remains high through- out the granulocyte maturation pathway and is transiently increased during the later stages. The background staining.
Differential during
surface
expression
granulocyte Fridtjof tBecton
of Pathology,
Dickinson
The
and
Leon
Gade
Institute,
Immunocytometry
Systems,
W. M. M. Terstappent University San Jose,
Abstract: Individual steps of granulocyte maturation, such as lineage commitment, proliferation, maturation, and migration from the marrow to the peripheral blood, may be influenced by distinct interactions with the bone marrow stroma. To identify candidates of membrane components involved in maturational stage-specific interactions, we studied changes in the expression of cell adhesion molecules along the granulocyte maturational pathway. Three-color flow cytometric measurements were used to measure levels of cell adhesion molecules along this pathway. The a chains of VLA-4 (CD49d) and VLA-5, the integrin 31 chain (CD29), and CD31 (PECAM-1) were expressed in high density on all early myeloid cells but down-modulated during postproliferative maturation. CD44 and L-selectin were expressed on CD34 myeloid progenitor cells and mature granulocytes but downmodulated during the intermediate stages of maturation. The granulocyte receptor for endothelial selectins, sLex, was specifically expressed by myeloid progenitor cells. sLex was down-modulated during the intermediate stages of granulocyte maturation but up-regulated again during terminal maturation. In contrast, CD67, a putative granulocyte adhesion molecule, was negative on progenitors, transiently up-regulated during the intermediate stages of maturation, and almost absent from the surface of mature granulocytes. These results show that each stage of granulocyte maturation is associated with the expression of a unique combination of cell adhesion molecules. L-selectin, CD44, and j31 integrins were regulated as previously described for immature lymphopoietic cells and may therefore play general roles in the compartmentalization and development of leukocytes. In contrast, sLex and cells and talization J. Leukoc.
molecules
maturation
LundJohansen*
*Department
of cell adhesion
CD67 were specifically expressed could be specifically important for of distinct phases of granulocyte Biol. 54: 47-55; 1993.
by myeloid compartmenmaturation.
of Bergen,
Haukeland
Hospital,
Bergen,
Norway,
and
Cahfornia
bone
marrow.
Studies
on
the
adhesive
properties
of periph-
eral blood leukocytes have revealed a number of molecules that mediate cell-cell and cell-matrix interactions. Families of molecules with similarities in structure and functional characteristics have been described, including carbohydrate epitopes, the selectins, the H-CAMs, the integrins, and the immunoglobulin supergene family [4-6]. Members of these families are important in controlling leukocyte traffic among the peripheral blood, lymphoid tissues, and sites of inflammation. Recent reports indicate that some of the same cell adhesion molecules mediate binding of early myeloid progenitor cells to bone marrow stromal components [7-12]. The distribution of these and other adhesion molecules among the subsequent stages of granulocyte maturation is, however, generally unknown. Information about the maturation-linked changes in granulocyte expression of cell adhesion molecules may be important for understanding mechanisms that control lineage commitment, cell growth, and retention of immature cells in the bone marrow. This study investigated the possibility that granulocytes may express different cell adhesion molecules depending on their state of maturation. Multidimensional flow cytometric analysis
was
used
to
define
the
neutrophilic
maturational
pathway [13]. The expression of the carbohydrate epitope sLe”, L-selectin (LAM-i, LECAM-i), and H-CAM (CD44); 32-integrins CDi8 (13-2-chain) and CDlia (LFA-1); 3-iintegrins CD29 (f31-chain), CD49d (VLA-4), and VLA-5; members of the immunoglobulin supergene family PECAM-i (CD31); and NCA-100 CD67) was assessed on the neutrophilic maturation pathway defined by the expression of CD34, CDilb, CD16, cell cycle analysis, and specific changes in light-scattering properties.
MATERIALS
AND METHODS
Reagents Key Words: bone monoclonal antibodies
marrow
.
flow
cytometry
.
hematopoiesis
Dulbecco’s magnesium myristate
icals from
INTRODUCTION Granulocytopoiesis stem cells into
the
involves granulocytic
of granulocyte-committed number of cell divisions segmented granulocytes. influenced by interactions and The
components of spatial proximity
of growth contacts also
factors [1-4].
be important
differentiation cell lineage
may be largely Interaction with for
retaining
regulated stromal granulocyte
cells
acetate
(St. Louis, Molecular
(PMA)
MO); Probes
Calf were
saline Serum
without (FCS),
purchased
from
7-amino-actinomycin (Eugene, OR).
calcium and and phorbol Sigma
Chem-
D (7AAD) Paraformaldehyde
was
of bone marrow and maturation
cells, which undergo a limited and finally enter the circulation as Each step in this process is likely between granulocyte precursors the bone marrow of hemopoietic
phosphate-buffered (PBS), Fetal
microenvironment. and local sources at the level components precursors
of cell may in the
Abbreviations: 7AAD, 7-amino-actinomycin D; FCS, fetal calf serum; forward light scatter; GAM-y-PE, phycoerythrin-conjugated goat anti-mouse immunoglobulin G; GAM-s-FI1C, fluoroscein isothiocyanateconjugated F(ab’)2 goat anti-mouse immunoglobulin M (is-chain specific); PBS, Duibecco’s phosphate-buffered saline; PMA, phorbol myristate; SSC, orthogonal light scatter.
FSC,
Reprint cytometry Received
Journal
requests: Systems, January
Fridtjof 2350 5,
Lund-Johansen, Qume
1993;
of Leukocyte
Drive, accepted
Biology
Becton San March
Jose, 29,
Volume
Dickinson CA
Immuno-
95131.
1993.
54, July
1993
47
and all salts used in laboratory-made solutions were of analytical grade. Microwell plates (polystyrene, V-bottomed) were from Nunc (Copenhagen, Denmark). The monoclonal antibodies used in this study are listed in Table 1. All monoclonal antibodies were diluted in PBS-FCS containing 0.1% sodium azide and were titered to obtain maximal fluorescent as determined by
Polyclonal
flow
staining with cytometry.
secondary
minimal
cell
aggregation
antibodies
FITC-conjugated F(ab’)2 goat anti-mouse 1gM (j-chain specific) (GAM--FITh), was generously provided by Jackson Immunoresearch (West Grove, PA). Phycoerythrin (PE)conjugated goat anti-mouse IgG (y specific); (GAM-y-PE) was from Southern Biotechnology Inc. (Birmingham, Ala.). The solution used for staining monoclonal antibody-labeled
cells
consisted
EDTA,
and
of
PBS
10 g/ml
containing GAM-’y-PE
Isolation of nucleated blood leukocytes Bone marrow undergoing
10%
goat
serum,
or 7.5 g/ml
bone marrow
2 mM
GAM-j-FITh.
cells and peripheral
and cardiac
venous blood was obtained from patients surgery according to the guidelines of the ethical board at Haukeland Hospital. The samples were immediately diluted i:iO in a solution containing 0.8% NH4C1, 0.08% NaHCO3, and 0.08% EDTA (pH 6.8, 20#{176}C) to lyse erythrocytes. by centrifugation PBS-FCS.
Three-color Bone with
and
(5
x i0) to CDiib
monoclonal
Ig
mAb
1
IgGI
Leu-M7
IgG1
1gM 1gM 1gM
DAKOCDI5 Leu-Ml Vepl3
M onocional
antigens.
LFA-l, CR3, CR3,
V.
fl2 integrin 32 integrin 32 integrin
MEM-48
IgGi
68-5A5 K20.3 IOM-31 Leu-M9
IgGl IgG2b IgGl
CD18
integrin
j32
CD29
integrin
/31
CD31
PECAM-l
IgG1 IgGl IgG2b IgG2b
CD33 CD34 CD44 CD44
HCAM HCAM
IgG2a
CD44
HCAM
IgG2b
MEM-ll2
IgGl
CD54
B.l3.9
IgGI
CD67
NCA-95/CGM6
CD67
NCA-95/CGM6
CD67
NCA-95/CGM6 L-Selectin
IgGl
1gM
GIOF5 80H3 Leu-8 CSLEX-1 FH6
IgGl
48
VLA-2, VLA-4, VLA-5,
Journal
V.
R. Vilella Immunotech
/31
Immunotech
ACB
integrin
difucosyl
antibody;
Ig,
provided
of Leukocyte
BDIS BDIS#{176} Anstee Horejsi
integrin flu integrin
31
V.
Horejsi
E.
van der Schoot J.S. Thompson Immunotech BDIS
ACB
sLe’
1gM 1gM
kindly
R. Vilelia BDIS Immunotech
ICAM-l
IgG2a
mAb, monoclonal “PerCP conjugate
L. Ashman BDIS DAKO BDIS H. Krafft Meda Rex V. Horejsi
FcyRIII FcyRIII integrin 132
CD49b CD49d
Horejsi Coulter
IgGl
IgG1
F. Symington
sLe’
immunogiobulin.
by Ken Davis,
Biology
Volume
Staining for measurement of two-color immunofluorescence and DNA content The
bone marrow cells were stained with antibodies as described above except that CD34-PerCP was not used. Immunostained cells were resuspended in 50 tl of ice-cold PBS containing 0.5% paraformaldehyde and fixed for 30 mm on ice. Two hundred microliters of PBS was then added to each well, and the cells were centrifuged. The fixed cells were resuspended in 10 tl of PBS and whirl-mixed before 10 l of PBS containing 20 mg/ml n-octyl-/3-D-glucopyranoside and 20 g/ml ofthe DNA stain 7AAD were added [14, iS]. After 30 mm of incubation on ice, 200 ,.tl of PBS containing 10 g/ml 7AAD was added. The cells were incubated at 4#{176}C overnight and measured by flow cytometry.
Flow cytometry
and data analysis
Measurements were performed with a FACScan flow cytometer [Becton Dickinson Immunocytometry Systems (BDIS), San 4ose CA] Data analysis was performed using Paint-AGate lus software or LYSYS II (BDIS). All experiments were performed at least six times with duplicate samples.
Characterization
Source
33-3B3 IOP49b L25 IOP49e
1gM monoclonal antibodies to CDiib and CD16 VEP13) were used combined with IgG monoclonal antibodies to other antigens. After being labeled with monoclonal antibodies for 30 mm, the cells were washed twice in PBS-FCS and incubated with 10 jl of the secondary antibody solution. The cells were then washed twice, incubated with 10 jl of PBS with 50% mouse serum, washed again, and stained with CD34-PerCP. During the entire staining procedure, the cells and all solutions used for washing and staining were kept on ice. Viability of immunostained cells was higher than 98%, as determined by propidium iodide exclusion (data not shown). +
RESULTS
3G8
HPCA-2 Bric222 MEM-85
Alterna-
Antibodies
Molecule
CDlla CD1 lb CD1 lb CDI3 CD15 CD15 CD16 CD16 CD18
IgGl IgG
I
.
simultaneously (i4.B6.E2 + 3G8)
to other
CD
class
MEM-25
were incubated and CDi6
antibodies
TABLE
Mol 14.B6.El
pelleted once in
immunofluorescence
marrow cells IgG antibodies 1gM
The nucleated cells were then i8Og for 5 mm and washed
at
tively,
(Mol
BDIS.
54, July
1993
The
sequential
of the granulocytic maturational
stages
maturation of myeloid
cells
pathway differen-
tiated into the granulocyte lineage can be identified by simultaneous determination of forward light scatter, orthogonal light scatter, and expression of three cell surface antigens [13]. We modified this technique to permit the assessment of cell adhesion molecules expression during granulocyte maturation. Forward light scatter (FSC), related to cell size; orthogonal light scatter (SSC), related to cell granularity; PerCP fluorescence from monoclonal antibodies to CD34 present clonal
on progenitor cells; antibodies to CDilb
and and
PE fluorescence CD16, present
from monoin increasing
densities on the cell surface starting from the myelocyte stage and metamyelocyte stage, respectively, were determined simultaneously from bone marrow cells. Shown in Figure 1 is a typical example of this assessment of granulocyte maturation after ammonium chloride lysis of erythroid cells. Progenitor cells (depicted green) were identified by the presence of CD34, absence of CDilb and CD16, low-to-intermediate SSC, and high-to-intermediate FSC [16]. Progenitor cells with low FSC/SSC were excluded from analysis (i.e., not colored), because these cells are committed to the B-cell lineage [16]. The green-colored cells, which mainly represent nonlymphoid progenitors, constituted 0.7-i.0% of the total bone marrow population. Of these, cells that express the highest levels green-colored myeloid-commited
of CD34 are the most population 68 ± 7% (mean ± SEM,
immature [16]. Of the were CD33 bright, i.e., n = 6). CD34 blasts
monoclonal antibodies to CDiib and CD16 (panels only with CD11b (panels E and F) are compared. in panels A-D that have higher PE-staining intensity
B 800
ci) 400 ri Cl)
3
.i
cells in the lower panels express CD16 in addition to CD1Ib. These have low FSC and represent the most mature cells, i.e., band forms and segmented granulocytes [13]. Arrows in Figure 1 indicate the maturational pathway of neutrophils. Cells not belonging to the granulocyte lineage are depicted gray. To further validate the analytical approach, we examined the expression of CD15 on the cell populations illustrated in Figure 1. Figure 2A shows that progenitor cells (green) are negative or weak for CD15. Panel B shows that blast cells (red; intermediate SSC) are CD15 weak to intermediate and
10
200
10
10
c) 400
800
10
10
CD11B
FSC
10 CD16-PE
+
D 800
800
400
that Panel
c)
200
400
i
out
1
2
10
CD11B
+
CD16-PE
CD11B
10 CD16-PE
+
of terminal metamyelocyte above permit tion pathway
F 800
800
the
L) 400 400
2
10
3
10
10
CD11B
-PE
1
10
1. Four-dimensional flow cytometric analysis tion in normal bone marrow. Panels A-D illustrate marrow stained with CD11b PE (14.B6.Ell), (HPCA-2)
stained
with CDllb (CD34”, CDllb/CD16) progenitors
scatter
were
(FSC).
and
panels
Blasts
and
by
E and
eliminating
promyelocytes
10
10
F shows
of granulocyte maturathe analysis of a bone CDI6 PE(3G8). and the
same
PE and CD34 PerCP without CDI6 were colored green, whereas
excluded
3
CD11B-PE
Fig.
CD34PerCP
2
cells
with
(CDllb/CD16-
low
experiment
and
SSC
most
maturation. This occurs at the stage [17]. Although the markers the identification of the granulocyte starting from cells in the progenitor
mature
cells,
it is impossible
myelocytedescribed maturastage up
to
to
now
PE. Progenitor lymphoid-commited forward
(red; high SSC) are CDi5 bright. CD15 expression remains high throughmaturation pathway and is transiently
identify the exact point of transition from the proliferative to postproliferative stage. To identify this point, bone marrow cells were stained with CD11b/CD16 PE and the DNA dye 7AAD [14]. Shown in Figure 3 are the results of a typical experiment. Cells in the S and G2/M phases of the cell cycle are depicted black whereas all other cells are depicted gray. Most cells in cell cycle are CDi1b.k5/CD16. As explained above, there is a continuous increase in CDlib/CD16 along the later part of granulocyte maturation pathway. The most mature proliferating cells can therefore be identified as those with the
C13
-
promyelocytes C shows that the granulocyte
increased during the later stages. The background staining ofthe green-, red-, and orange-colored cells stained with an isotype control antibody is shown in the panels D-F of Figure 2. These results are similar to those previously reported [13]. An important hallmark during granulocyte maturation is the point when the cells no longer divide and enter the stage
3
10
A-D) or The cells than do
cells
highest
angle
light
levels
LI
of CDlib/CD16.
These
cells
longing to any of the mentioned populations were not colored and remain gray. Arrows in the panels A-D indicate the granulocyte maturational pathway. Multiple two-variable displays were used to identify the position of the colored cell clusters (PaintaGaten software) and 30,000 cells are displayed in the figure.
the
transi-
::J
intermedi-
ate to high) were colored red. Granulocytic cells gradually mature as their CDllb and CD16 densities increase and are colored orange. Cells not be-
mark
In I-
LI
111213 10
CD34.PerCP
SSC
10
CD11B
10
CD16.PE
+
LI (depicted red) were identified by the absence of CD34, absence of CDlib and CD16, intermediate SSC, and intermediate-to-high FSC (Fig. 1 panels A and C) [13]. Their identity as myeloblasts was confirmed by showing that 88 ± iO% of the cells in this population were CD33 bright (mean ± SEM, n = 6). Promyelocytes (also depicted red) are distinguished from blast cells by higher SSC (Fig. 1, panels A and C) [13]. Myelocytes, metamyelocytes, bands, and segmented neutrophils (depicted orange) were identified by the absence of CD34, increasing densities of CDiib and CD16, high Metamyelocytes, press
similar
differential is illustrated
SSC,
and intermediate-to-low band forms, and segmented
FSC (Fig. neutrophils
levels of CDiib but can be distinguished expression of CD16 and different FSC [13]. in Figure 1 where two samples stained
Lund-Johansen
1). exby This with
and
F-
LI F-
i0
10
200
CD34-PerCP Fig. 2. Five-dimensional ofCDl5 (panels A-C) granulocyte
FLS and granulocytic as described. 1)
Terstappen
cells Cells
Cell
adhesion
at not
from
10#{149} i0
800
cells
different
by
CDllb/CDI6
(green). levels
belonging
the
CD11B
flow cytometric analysis of differential an isotype control antibody (panels
depicted
Progenitor
excluded
were
and
maturation
SSC.
400
SSC
and
blasts
and
of
maturation
to any
of these
CD34
expression D-F) during expression
promyelocytes (orange)
populations
CD16-PE
+
and
(red),
and
were
identified
(gray
in Figure
analysis.
molecules
during
granulocytopoiesis
49
i
800
40O4j;
400
B
CD34 800
400
CD11B
CD16-PE
+
C
F-
10 2
In
LI
101
‘2
10
CD11B
10
CD16-PE
+
‘i
2
10
10
3
10
Four-dimensional
CD11B
and
of maturation.
CDllb
CDI5
An
S and
on bone
Panel
(3G8),
D
cell
shows
cycle
and (black)
cells
CD15,
with
was with
identified
versus
a
up-
that
VLA-4,
and
DNA
content
showed
VLA-4
remains high until proliferation ceased (right black population in panel D). During terminal there was a sharp decrease in CD49d expresmature granulocytes virtually lacked CD49d D). Monoclonal antibodies to integrin /31 chain VLA-5 showed staining patterns similar to that
and
CD29
(Figure
(Figure 4B). First, no inverse the expression of these antimature granulocytes expressed CD29 than of CD49d (Figure of the immunoglobulin superwas regulated similarly to 4B).
10
Neutrophil
maturation
content,
at different and
stained were
CD11b/CD16
of VLA-4,
13
DNA
aspirate
and
permeabiiized
ofthe
of
marrow marrow
GAM--y-PE
associated
CD16-PE
+
analysis
bone
were
phases
fluorescence.
expression
CD16 cells
G2M
cytometric
erythrocyte-lysed
(i4.B6.Ell),
Antibody-labeled the
flow
increase
is
CDllb/CD16,
A Fig. 3. CDllb/CDI6,
an
of VLA-4/ depicted cell surface
VLA-5
FI
with
commitment
of VLA-4 with two exceptions correlation was found between gens and CD34, and second, higher levels of VLA-5 and 4B). Interestingly, a member gene family, CD31/PECAM-i,
LI
F-
lineage
of VLA-4 (panel A). The expression CD49d was high on blast cells and promyelocytes, red (panel B). Combined measurement of expression border of differentiation sion, and (panels C, (CD29) and
800
3
concurrently
that
regulation
FSC
LI
diminished
suggesting
stages
labeled
with
:‘
GAM--FITh. 7AAD. by
Cells
high
nonspecific
in C
7AAD
0
102
control C
monoclonal
same
antibodies
experiment.
Figures
1 and
(TEPC-128)
Results
are
for
taken
from
bone
the
marrow
cells
same
stained
experiment
in
the
as those
4
2.
tion point between the proliferative and postproliferative stages of granulocyte maturation (i.e., the right border of the black-colored population on any plot displaying CDilb/
CD16
on the x-axis)
(Fig. 3 B-D). As shown in Figure 3, the which requires permeabilization of the not affect the staining of CDi5, CDilb, with Figure 2). The expression of and 7AAD staining were used in all further exillustrating the changes in the expression of cell molecules associated with transition from the to the postproliferative stage of maturation. 4A schematically illustrates the granulocyte pathway as defined by FSC, SSC, CDiib,
B
:‘
staining with 7AAD, cell membrane, does and CD16 (compare
CDiib/CD16 periments adhesion proliferative Figure
maturational CD16, and
expression. In addition, a dotted line mdicates the point of transition from the proliferative to the postproliferative stage of maturation. In the assessment of cell adhesion molecule expression during granulocyte maturation, this schema was used to facilitate the visualization of the changes that occur during granulocyte maturation. In Fig. 5-10 the green, red, and orange colors are assigned to cell populations identical to those in Figure 1 and permit comparison of the changes of the various cell adhesion molecules during granulocyte maturation. In the panel D of all the figures, the CDiib/CDi6 expression is plotted against
C 0
in Figures
5-10 are taken from the therefore directly comparable. The of the six experiments performed.
50
molecules, and the cells black. All the diagrams same experiment and are results are representative
and VLA-5 progenitor high levels
Journal
102
C
4
101
c .;
i03
C
C 0
i0
C
4
101
Nil
Fig.
4. Schematic
expression
in
the
of Leukocyte
Biology
in The
Volume
Figure 5, expression
54, July
1993
cxof
representation
of CD34, expression
of
CDllb, of
the
CD16, cell
granulocyte FLS,
adhesion
and
maturation SSC,
molecules
panel
as A.
CD29,
defined
The
by
changes
CD31,
VLA-4,
VLA-5, CD44 and L-selectin during this maturation is indicated in panel B, whereas that of CD18, CDIIa, CD67 and sLex is indicated in panel C. Maturation is plotted along the X-a.xis and four stages NI-NIV are distinguished.
cells, depicted green of VLA-4 (CD49d).
NW
Maturation
to
CD34 pressed
i#{248}
CD34
the expression of the cell adhesion that are in S and G2/M are depicted
VLA-4
101
in
In
NIl,
and
the
following
orange
to
CDIlb/CDI6
staining
tion
the
between
NIh
figures
the
and
NIV,
represent
proliferative
cells
NIV. and
colored
of which
The
dotted
postproliferative
green the
cells
belong with
line indicates stages
of
to the
NI,
red
brightest
the transimaturation.
A
two
B
i0.-I
Progenitor
10
‘2
10
‘3
10
200
400
800
ssc
CD34-PerCP
C
that 2
10*
10 +
5. Five-dimensional flow cytometric a chain (CD49d) during cells (green), blasts and promyelocytes ent levels of maturation (orange) were 1gM monoclonal antibodies to CDIIb -FITh were used. Panels A-C show
CD34,
CDllb
PerCP,
GAM--y-PE.
Panel
4-PE
(L.25.3)
with
7AAD.
are are
+
Proliferating
cells
10’
(black)
excluded
from
+
of differential
GAM-s-FITh,
3. Cells not identified 1) were
analysis
and
CDllb+CDI6-FITC marrow cells after were
as
expression
anti-CD49d/VLA-4
was cells,
expressed depicted
in high green
L-selectin
was
down-modulated
suggesting
that
in Figure
8,
expressed
the
was
upregulated
somewhat
later
than
CD18
4C).
subset
(53
± 5%,
mean
± SEM,
n
=
6)
of
progenitor
9, was positive for monoseparate three-color staining CD13, all sLex/CD34 dualpositive bone marrow cells expressed high densities of CD33 and CD13 (not shown). Although there were CD33 + and CD13+ cells in the sLex negative progenitor population, the two former antigens were expressed in higher density on the sLe” + cells (not shown). These results suggest that expression of sLex is specific for myeloid progenitor cells and possi-
A
+
identified
as
A-C,
blasts, whereas
are taken from experiments.
described
in
the
or granulocytic results
a single
from
all
experiment
lineage
commitment
density on the majority of in Figure 6 panel A. concurrently with CD34, is associated with de-
CD34-PerCP
C’)
blast cells (red; cells (red; high progenitor cells, suggesting an extensive down-modulation of L-selectin during early granulocyte maturation (panel C). Expression of L-selectin remained low during granulocyte maturation until the stage where proliferative activity ceased (panels C and D). At the point of transition between the proliferative and postproliferative stages of maturation (right border of black population in panel D), L-selectin was up-regulated. Mature granulocytes expressed levels similar to those of the progenitor cells (panels C and D).
in Figure to that
7 is the staining observed for L-selectin
and blast cells expressed homogeneously CD44 as opposed to L-selectin (compare The staining patterns observed for CD44
of CD44, except that
which was progenitors
high densities of Figures 6 and 7). were reproduced with
Lund-Johansen
and
Terstappen
SSC
C
creased levels of L-selectin (panel A). Most intermediate SSC) and all promyelocytic SSC) were weaker for L-selectin than were
similar
green
and CD44
progenitor
Shown
depicted
versus anti-CD49d/VLApermeabilization and staining
progenitors,
panels
222
cells, depicted green in Figure clonal antibodies to sLex. In experiments with CD33 or
granulocyte maturation. Progenitor (red), and granuiocytic cells at differidentified as described except that (Mol) and CD16 (Vepl3) and GAMstaining of each subpopulation with
shown in panel D. The results representative of six performed
L-selectin L-selectin
D shows
of all bone
legend to Figure ( gray in Figure cells and
+CDI6
10
CD11B CD16-FITC
of the VLA-4
LFA-1
A
1’2’3
Fig.
Bric
sLex
101
CD11B CD16-FITC
cells,
(Figure
10
10
and
maturation was accompanied by a slight decrease in CD18 expression (panels C and D). CDila (LFA-l, a chain) expression of bone marrow cells was similar to that of CD18 except
[D
10
MEM-85
integrin fl2-chain (CD18) heterogeneously (panel A). Blast cells (red; intermediate SSC) were also heterogeneous for CD18 (panel B). Most promyelocytic cells (red; high SSC), however, expressed lesser amounts ofCDl8 than did progenitor cells, suggesting a down-modulation of /32 integrins during early granulocyte maturation (panel B). CD18 staining intensity increased concurrently with CD11b expression and reached its maximum when proliferation ceased (panel C, right border of black population panel D). The terminal
101. ‘1
antibodies,
integrins
f32
.:p
i02.
102.j
other monoclonal shown).
( not
:D
1 2
2
10
10
101.
101
1
10
10
2’3
CD11B CD16-FITC Fig. of
6. Five-dimensional L-selectin
Bone
marrow
Figure
5.
Cell
flow
10
10*
adhesion
were
cytometric
labeled
molecules
10’
CD11B CD16-FITC
+
(Leu-8/LAM-l/LECAM-1)
cells
10
with
leu8
during
analysis
of differential
during
granulocyte
instead
of CD49d.
granulocytopoiesis
+
expression maturation.
See
legend
to
51
bly
that
tion
the than
antigen
appears
CD13
do
later
and
during
CD33.
myeloid
Most
differentia-
blast
cells,
(red;
intermediate SSC) expressed high densities of sLex (panel B). Promyelocytes (red; high SSC) were homogeneously sLex bright (panel B). Combined measurement of DNA content, CDiib/CD16, and sLex expression, showed that sLex was down-modulated during the intermediate stages of granulocyte maturation (panel D). After termination of proliferative
activity
to those
C
rD
10
‘1’2’3
10
10
The CEA-related surface of CD344
2
up-regulated cells (panels
10’
CD11B + CD16-FITC
10’
10”
CD11B CD16.FITC analysis Bone
See
of granulocytic cells with monoclonal was also seen with the monoclonal antirecognizes the difucosylated form of shown).
CD67 progenitor
antigen cells,
was not depicted
of differential marrow cells
legend
creased population
+
proliferation panel D).
ceased opposite
The
5.
B .t::
10
102 I
10
10 Ii
10
‘2
2
_____
.
.
.
..
1
::
L)
2
.
‘3
10
10
200400
800
ssc
C
200
400
800
[#{246} C
1 2
S
10
10
:j
CD34-PerCP
2
1o3 2
101
101.
2
I
10
10 1
-l-i
10
I
10
10
10’
+
CD11B
(integrin
labeled
Journal
flow
f32 chain)
with
10’
cytometric during
CD18
analysis granulocyte
(68-5A5)
instead
10’
+
of differential
CD11B
Biology
maturation.
Bone
of CD49d.
See
Volume
10’
10
10’
CD16-PE
+
CD11B
10’ +
10”
CD16-PE
expression
Fig.
marrow
legend
to
of with
of Leukocyte
10
10”
CD16FITC
CD16FITC 8. Five-dimensional
1
10
li
CD11B
52
of black of CD67 granulocyte decrease of C and D). G1OF5, and shown).
3
CD34-PerCP
cells were Figure 5.
on the in Figure
(right border regulation
versus sLex, L-selectin, and CD44 during maturation was further evident by the significant CD67 during terminal differentiation (panels Three CD67 monoclonal antibodies, B.13.9, 80H3, gave similar results (Figure 10 and not
expression were labeled
to Figure
until
1
of CDI8
expressed green
B
A
Fig.
to levels similar C and D). The
10 and not expressed on nongranulocytic cells (not shown). Blast cells (red; SSC intermediate) were also CD67 negative, whereas promyelocytes (red; SSC high) expressed CD67 in high densities (panel B). In contrast to what was observed for sLex, L-selectin, and CD44, the expression of CD67 in-
10
Fig. 7. Five-dimensional flow cytometric of CD44 during granulocyte maturation. with CD44 (33-3B3) instead of CD49d.
was
CD67
101
10
sLex progenitor
on
differential staining antibodies CSLEX-1 bodies FH6, which sLe” [i8] (data not
ssc
CD34-PerCP
seen
54, July
1993
9. Five-dimensional sLex sLex
during
flow
granulocyte
(CSLEX-l)
cytometric maturation.
instead
of
CDI5.
analysis Bone See
of differential marrow
legends
cells to
Figures
expression were
stained
2 and
5.
A
pression of these molecules. with more than one monoclonal
B
______
bone marrow aspirates. The expression of the
1 io210
,
.
;
--‘1 10
-
12
. .
.
I __1
,
13
10
200
10
CD34-PerCP
400
800
2
Ii
10
CD11B CD16FITC
with
10.
Five-dimensional during
CD67
13
1
10
10
analysis Bone
of CD49d.
See
legend
of differential marrow to
expression
cells Figure
+
were
labeled
5.
DISCUSSION Cell adhesion molecules are important in the homing and activation of mature leukocytes. Less is known, however, about their expression and biological roles during differentiation and maturation of cells in the bone marrow. The expression of LFA-1, f31 integrins, CD31, sLe”, and CD67 during granulocyte maturation has been poorly characterized or, as for CD44, differently reported [19, 20]. This may be at least partially due to the problems associated with the identification and characterization of subsets of cells of different lineages and their respective maturational stages. Here we defined the granulocyte maturational pathway by gradual coordinate changes in the expression of CD34, CDilb, CD16, FSC, and SSC (Figs. 1 and 4). The validity of this analytical approach has been demonstrated by morphology and functional properties of cells sorted along this pathway [13]. After in vitro stimulation of progenitor cells [21] and virgin T lymphocytes [22], the static flow cytometric images of
maturational
confirming
pathways the
usefulness
have of
this
analytical
been
recapitulated, technique
in
as-
sessing hematopoiesis. The granulocyte maturational pathway is indicated by arrows in Figure 1. Different maturational stages were depicted red, green, and orange and shown as such in all figures. The expansion of the neutrophil pool is confined to the neutrophils expressing low levels of or no CD11b and no CD16 as shown by simultaneous determination of cell cycle analysis and cell surface antigens (Figure 3 and the lower right corner of other figures). Expression of cell adhesion the variables to probe for
($1),
CD31
transition stages.
point These
from may
the be
proliferative important
to the for the
of hematopoietic progenitor cells to bone marrow stromal components [7-12, 23]. In concordance with this, we show that CD34 progenitor cells express high levels of CD44, L-selectin, VLA-4 (CD49d), VLA-5, and integrin /31 chain (CD29). However, the different distribution on the subsequent maturational stages of granulocytes suggests that /31 integrins have functional roles distinct from those of CD44 and L-selectin. VLA-4 and VLA-5 were expressed in high density on all immature myeloid cells until the stage where proliferation ceased. Both a chains and their as-
.7
10
CD11B CD16FITC
+
maturation.
instead
k
10
flow cytometric
granulocyte
(B13.9)
CD29
sion
10
CD67
CD49d,
six
maturation. Because most cells at this stage ofmaturation do not traffic between the bone marrow and blood, changes in their expression of adhesion molecules are most likely important for the control of differentiation, expansion, and maturation of myeloid cells. CD44, /31 integrins, and possibly L-selectin mediate adhe-
j of
were reproduced and in at least
selective release of postproliferative cells into the circulation as well as for controlling terminal differentiation. Figure 4 shows that there is complex regulation of individual cell adhesion molecules also during the proliferative stages of
C
Fig.
CD44,
results antibody
(PECAM-i), VLA-5, VLA-4 (CD49d), L-selectin, sLe”, CD67, CD18 (32), and CDiia (LFA-1) during granulocyte maturation is illustrated in Figure 4 and shows that the cxpression of each of these cell adhesion molecules is differentially regulated during granulocyte maturation. Notably, there were large changes in the expression of nearly all the molecules at the postproliferative
SSC
The
molecules was determined simultaneously with defining the granulocyte maturational pathway maturation-linked changes in cell surface ex-
Lund-Johansen
and
sociated integrin /31 chain (CD29) were down-modulated during the final stages ofmaturation and present in low density on peripheral blood granulocytes (Figures 4 and 5). In separate experiments these molecules were not up-regulated even after activation of peripheral blood granulocytes with 10 ng/ml PMA for 10 mm (not shown). Fibronectin and VCAM-i are the ligands for VLA-4 and VLA-5 and are present in the bone marrow matrix and on bone marrowderived endothelial cells. It is therefore possible that VLA-4 and VLA-5 participate in the retention of granulocyte precursors in the bone marrow. The similar distribution pattern of CD31/PECAM-i is interesting, because CD31 is synergetic with /31 integrins in T-cell adhesion [24]. The wide distribution of CD44, L-selectin, and /31 integrins among progenitor cells harmonizes with the lineage nonspecific inhibitory effects of monoclonal antibodies to these antigens on lymphopoiesis and hemopoiesis in longterm bone marrow cultures [7, 8, 10-12]. The regulation of these molecules during granulocyte maturation is similar to the expression pattern during lymphocyte maturation and suggests similar roles of these molecules in the various cell lineages [25]. Additional molecules will likely be identified that and
specifically lymphoid
mediate progenitor
interactions cells. The
of myeloid, heterogeneous
erythroid, staining
of progenitor cells and blasts with monoclonal antibodies to CD18 and CD11a shown here and in two earlier studies suggests LFA-1 as a possible candidate of such a lineagerestricted progenitor cell adhesion molecule (Figure 8) [12, 19]. Interestingly, our results also identified subsets of progenitor cells with different expression of sLe’ (Figure 9). This oligosaccharide is a receptor for P-selectin and Eselectin on cytokine-stimulated endothelial cells [26-29]. High expression of sLex on myeloid progenitor cells may therefore indicate that sLexselectin interactions are important for development in myeloid direction. Expression of the
Terstappen
Cell
adhesion
molecules
during
granulocytopoiesis
53
GPI-linked CD67 antigen was also restricted to myeloid cells. Although the function of granulocyte CD67 is unknown, its relation to the carcinoembryonic antigen family and the functional characteristics of an identical molecule on colonic epithelial cells suggest a role in cell adhesion [30-32]. CD67 may have a dual function in bone marrow cells and activated cells because it is stored intracellularly in the specific granules of mature granulocytes and highly upregulated upon activation [33, 34]. The lineage and maturational stage-restricted surface expression of sLex and CD67 suggest that they may participate for development of myeloid cells. at distinct stages of granulocyte
in interactions important The two molecules function maturation, because their
expression was almost inversely regulated during granulocytopoiesis (Figs. 4, 9, and 10). sLe’, CD44, /31 and /32 integrins, L-selectin, and CD31 all recognize ligands expressed by subsets of resting or cytokinestimulated endothelial cells [5, 7, 8, 10, 12, 23, 26, 27, 29, 35, 36]. Because most molecules were differently regulated during granulocyte maturation, it seems unlikely that they all mediate binding to the same cells (Figure 4). The results therefore give rationale for investigating possible heterogeneity among stromal cells expressing the counter receptors, such as VCAM-1, E-selectin, P-selectin, CD3I, and cellassociated fibronectin and hyaluronic acid. There may also be heterogeneity in the adhesive properties of individual molecules on different cells, because our results on phenotypic expression of adhesion molecules during myelopoiesis can not explain previously reported adhesive properties of myelopoietic cells [37, 38]. Alternatively, multiple molecules may be required for the binding to stromal layers and purified matrix components. The asynchronous changes in surface levels of multiple molecules that occurred throughout granulocyte maturation could be more important for cxplaining differential cell adhesiveness than variability in the expression of each molecule individually. In sLe’,
conclusion, L-selectin,
this study shows CD44, CD67,
differential and /31 and
expression /32 integrins
of
during granulocyte maturation. The independent regulation of the expression of these molecules during granulocyte maturation suggests that granulocyte maturation is associated with continuous changes’n cell adhesive properties. An attractive hypothesis is that the bone marrow is divided into specialized compartments that permit lineage differentiation, lineage expansion, lineage maturation, and egress to the peripheral blood. The profile of cell adhesion molecules on the surface of the bone marrow cells would then guide the cells
to
these
specialized
stromal
naling
in
cell
5. Springer,
Cell 69,
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ACKNOWLEDGMENTS We Dr.
thank Johanna
the
antibody donors and Dr. Robert Bjerknes, Olweus, and Professor Ole-Didrik Lareum for
reviewing the manuscript. grants from Inger-Margrethe
This and
study was Per Jger
supported and The
by Blix
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