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Expression of the CDll/CD18, Leukocyte Adhesion Molecule 1,
and CD44 Adhesion Molecules During Normal Myeloid and Erythroid
Differentiation in Humans
By Geoffrey
S.Kansas, Michael J. Muirhead, and Morris 0. Dailey
We have used three-color flow cytometry to investigate
the pattern of expression of the CDI 1lCD18, CD44. and
leukocyte adhesion molecule 1 (LAM-1) adhesion molecules during myeloid and erythroid differentiation in humans. The earliest myeloidcells, identified as CD33'"CD15-,
were exclusively CD44hi but contained both leukocyte
function-associated antigen 1 (LFA-1hi) and LFA-1'" cells, as
well as LAM-1' and LAM-1- cells. This CD33'"CD15myeloid subpopulation expressed only low levels of CDI 1c
and failed t o express CD11b, CD14, or any lymphoid (CD3,
CD16, CD19) antigens or glycophorin. Commitment to
monocyte differentiation, suggested by the presence of an
LFA-lhi CDI IC+
subset within the CD33'"CDI 5- subpopulation, was clearly signaled by upregulation of CD33; these
monocyte-lineage committed cells were exclusively CD33hi,
CDUhi, CDllahi, CDllc', and exhibited a broad range of
intensity of CDI 5 expression. Later stages of monopoiesis
were identified by acquisition of CDI 1b, and subsequently
of CD14. Myeloid cells committed t o granulopoiesis re-
mained LFA-1'". and underwent a sharp upregulation of
CDI 5 along with downregulation of both CD33 and CD44.
Successive stages of granulocyte development were marked
by expression of CD11b and, subsequently, of CD16. The
earliest cells capable of erythroid differentiation were
CDMhi, LFA-1'". and LAM-I+. Both LFA-1 and LAM-1 were,
lost before the onset of glycophorin (glyco) expression,
whereas CD44 expression remained high on glyco+ cells,
which also expressed CD45. CD44 expression was intermediate on glyco' CD71' cells, and low on glyco' CD45XD71cells, similar t o normal, circulating erythrocytes. Our results allow us to phenotypically define discrete stages in
the normal development of monocytes, neutrophils, and
erythrocytes. The expression of LFA-1. LAM-1, and high
levels of CD44 on the most primitive hematopoietic cells
detectable by flow cytometry suggests that at least some
of these molecules are critically involved in leukocyte
adhesion during development.
0 1990 by The American Society of Hematology.
A
HEV is suggested by reports from several groups.26-28Unlike
both LFA-1 and LAM-1, CD44 is expressed not only on all
classes of leukocytes but also on red blood cells (RBC),
fibroblasts, keratinocytes, and other types of epithelial
CENTRAL FEATURE of effective host defense is the
ability of leukocytes to leave the bloodstream and enter
tissues in response to immune or inflammatory stimuli. The
principle point of regulation of leukocyte extravasation from
blood into the various tissues is a t the level of adhesive
interactions between circulating leukocytes and vascular
endothelium. Numerous examples of these interactions have
been described. Among the most prominently studied is the
interaction between T and B lymphocytes and the specialized
high endothelial cells which line the postcapillary venules of
most secondary lymphoid organs (high endothelial venule
[HEV]), an event of crucial importance to lymphocyte
recirc~lation.l-~
In addition, the binding of neutrophils,
monocytes, lymphocytes, and other leukocytes to endothelium in vitro has been extensively characterized in numerous
laboratorie~.~-'~
These studies have permitted the identification of three
families of leukocyte surface glycoproteins that participate in
adhesion to one or more types of endothelium: the CD11/
CD18 heterodimers, the CD44 family, and the leukocyte
adhesion molecule 1 (LAM- 1) molecule(s). The leukocyte
function-associated antigen 1 (LFA-1) molecule (CD1 l a /
CD18), which is expressed on all leukocytes, appears to play
a role in the adhesion of all classes of leukocytes to a diverse
array of target cells,"-13 consistent with the wide expression
of intercellular adhesion molecule 1 (ICAM- 1),14the principle ligand for LFA-1.I5*l6Both the MAC-1 (CDllb/CD18)
and p150,95 (CDllc/CD18) molecules, whose expression is
confined to myeloid cells and a subset of lymphocytes, appear
to function as receptors for C3bi," as well as for a currently
undefined ligand on the surface of endothelial cells (EC).'*,I9
The LAM-1 molecule is expressed on all classes of
leukocytes," and is believed to play an important role in the
adhesion of both lymphocytes and neutrophils to several
types of endothelium,21*22
similar to its murine homologue,
MEL-14.23-25A role for CD44 in lymphocyte adhesion to
Blood, Vol76, No 12 (December 15). 1990: pp 2483-2492
cell^.^^.^'
Previously, we have described the pattern of expression of
LFA-1, LAM-1, and CD44 during B lymphopoiesis in
human^.^' In this report, we have examined the expression of
these LAMS during the normal development of monocytes,
neutrophils, and erythrocytes. The results allow us to define
discrete stages of development for each of these hematopoietic lineages. In addition, our data can be integrated with
those of others to provide a comprehensive and coherent
picture of phenotypic changes during hematopoiesis in humans.
MATERIALS AND METHODS
Cells. Normal human low-density bone marrow (BM) cells were
isolated by Ficoll-Hypaque density centrifugation of bone marrow
aspiratesfrom normal healthy adult donors in the University of Iowa
Bone Marrow Transplant Program. All procedures conformed to
From the Departments of Pathology, Microbiology, and Medicine, University of Iowa College of Medicine, Iowa City.
Submitted February 8,1990; accepted August 17.1990.
Supported by Grant No. A12273045 from the National Institutes of Health (NIH). G.S.K. was supported by F32 A107820-01
from the NIH.
Address reprint requests to Geoflrey S. Kansas, PhD. Division of
Tumor Immunology, Dana Farber Cancer Institute, 44 Binney St,
Boston, M A 021 15.
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 1990 by The American Society of Hematology.
0006-4971/90/7612-0003%3.00/0
2483
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2484
KANSAS, MUIRHEAD, AND DAILEY
Table 1. MoAb Reagents Used
Surface Marker.
Distribution
My9
MMA
OKT3
4G7
3GB
lOF7MN
LFA-l/CDl l a
LFA-l/CDl l a
CD44
LAM-1
CD33
CD15
CD3
CD19
CD16
Glyco
OKT9
GAP8.3
Transferrin Receptor/CD7 1
CD45
All leukocytes
All leukocytes
Leukocytes, fibroblasts, EK
Leukocytes
Myeloid; M > G
Myeloid; G > M
T cells
B cells
NK cells and neutrophils
RBC and their precursors
Proliferatingcells; erythroid precursors
All leukocytes
Monocytes, neutrophils, lymphocyte
subset
Monocytes
Monocytes
Progenitor cells
MoAb
Form Used
~~
G25.2
TS1.22
515
Leu8
CD1 l b
C D l IC
CD14
CD34
OKM 1
LeuM5
63D3
MY10
8, APC
F
F, B, APC
B , APC
B
F
F
F
F
F. B
B
F. B
B, APC
B , APC
F, B , APC
Purified
Reagents were prepared as described in Materials and Methods.
Abbreviations: F, FITC: B, biotin; APC, allophycocyanine; NK, natural killer; EK, epidermal keratinocytes.
*Cell surface marker identified by the indicated MoAb. Neither LAM-1 nor glyco have yet been assigned to a CD.
slides, stained with Wright-Giemsa, and morphologically assessed
by light microscopy to determine the cellular composition of each
subpopulation. Histochemical stains for nonspecific esterase (NSE)
and myeloperoxidase (MPO) were also performed on sorted, cytocentrifuged BM subpopulations.
established guidelines regarding informed consent and use of patient
cells, and were specifically approved by an Institutional Review
Board, in accordance with assurances with, and approved by, the
Department of Health and Human Services. Cells were washed once
in RPMI-1640 containing 10% fetal calf serum, and aliquoted for
immediate staining, as described below.
Monoclonal antibodies (MoAbs). Purified LeuM5 and purified
MY 10 MoAb were generously supplied by Becton Dickinson (Mountain View, CA). All other MoAbs (Table 1) were prepared as ascites
fluid in pristane-primed Balb/c mice, purified from ascites fluid
using the MAPS I1 Kit (BioRad) according to the manufacturer's
instructions, and conjugated to fluorescein isothiocyanate (FITC),
biotin or allophycocyanine (APC) using standard methods, as
described (Table I)."All FITC, biotin-, and APC-conjugated
MoAbs were pretitered on normal human BM cells, prepared as
described above, to determine optimal concentrations for staining.
Avidin-PE was obtained from Fisher-Biotech (Pittsburgh, PA).
Immunofluorescence staining andflow cytometry. The methods
for two- and three-color staining, flow cytometry, and data analysis
have all been described in detail elsewhere." FITC-, biotin-, and
APC-conjugated isotype matched irrelevant control MoAbs were
included in all experiments, and were used to define appropriate
quadrants for data analysis (see below). In some cases (see Results),
electronic cell sorting was performed to isolate specific subpopulations of BM cells. Sorted cells were then cytocentrifuged onto glass
- 3
W'
?=
RESULTS
Definition of myeloid subpopulations. The CDl5 and
CD33 myeloid antigens have distinct patterns of expression
on monocytes, neutrophils, and their precursor^.^*-^^ Twocolor analysis of BM cells for the correllated expression of
CD15 and CD33 showed four distinct subpopulations: one
which was CD33'"CD15- (designated population 1); one
which was CD33h'CD15f (population 2); one which was
CD33"/-CD1Sh' (population 3); and a fourth that expressed
neither of these markers (Fig 1). Populations 1, 2, and 3,
which contained 1% to 5%, 4% to 12%, and 15% to 25% of the
total cells, respectively, were individually isolated by electronic cell sorting to greater than 90% purity, and WrightGiemsa stained cytocentrifuge preparations were examined
by light microscopy. Population 1 was quite heterogeneous,
containing undifferentiated blast cells, early myeloid cells,
early erythroblasts, and a few monocytes, but no lymphoid
'I
m
m
2
0
1
n
~
0
0
Forward Scatter
1
2
3
CD15 (FITC)
4
Fig 1. Definition of myeloid subpopulations.
(A) Typical light scatter contour map of normal
low-density BM cells. (B) Correllated expression of
CD33 and CD15 on all viable cells defines three
distinct subsets of myeloid cells in normal human
BM. For (B) and the other data in this report,
scatter gates were set to include all viable cells,
excluding only dead cells, mature erythrocytes,
and platelets.
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ADHESION MOLECULES IN HEMATOPOIESIS
2485
Table 2. Differential Cell Counts of Myeloid B M Subpopulations
Population 1
Cell Type
It
Myeloblast
Promyelocyte
Myelocyte
Metamyelocyte
Bandlmature neutrophil
Monoblast
Promonocyte
Monocyte
Erythroblast$
Undifferentiatedblast
Basophil
Eosinophil
Lymphocyte/lymphoblast
0
0
0
0
22
22
14
19
20
2
0
0
Population 2
Population 3
0
0
0
0
0
16
23
56
0
4
1
0
0
3
3
11
35
48
0
0
0
0
0
0
0
0
Lin- CD33-.
0
0
0
0
0
0
0
0
27
73
0
0
0
BM Subpopulations were defined as in Fig 1, cytocentrifugedonto glass slides, stained with Wright-Giemsa, and examined morphologically. Results
given are for one of three typical experiments.
*Defined as described in the legend to Fig 5, except that antibody to CD33 was added to the cocktail of MoAbs used for electronic cell sorting. See
Figs 5, 6, and 7 and text for complete description of these cells.
tPercent of each indicated BM subpopulation.
$Large basophilic cells showing little or no evidence of hemoglobinization. Because more differentiated, hemoglobinizedcells were never seen in these
BM subsets, they have not been listed in the table.
cells or later erythroid cells (Table 2). In contrast, population
2 exclusively contained cells in various stages of monocyte
development, and population 3 contained exclusively granulocytic cells (Table 2). Consistent with these morphologic
observations, population 2 contained approximately 60%
NSE+ cells and only faint diffuse MPO staining typical of
monocytes, and population 3 contained greater than 90%
cells displaying a spectrum of intensity of MPO staining; less
than 5% of population 1 cells exhibited staining with either
NSE or MPO (Table 3). Thus, populations 1, 2, and 3
consisted of cells belonging to mixed, monocytic, and granulocytic lineages, respectively.
Phenotype of myeloid subpopulation. We next examined these three myeloid subpopulations for their expression
of CDlla, CDllb, CDllc, LAM-1, CD44, and CD14 (Fig
2). CDlla expression was largely bimodal in population 1,
with nearly all cells exhibiting either a CDl lah’or a CD1 la’”
phenotype; a minority of population 1 cells were CD1 la-. In
contrast, populations 2 and 3 were unimodally CDllahiand
CDl la”, respectively, suggesting that these populations may
be derived from the population 1 cells with the corresponding
phenotypes. Very few CDllb+ cells were detectable in
population 1, whereas the majority of cells in both populations 2 and 3 were CDl lb+. In addition, the peak level of
CDllb staining on population 2 is greater than that on
population 3, reflecting the relative cell surface density of
CD1 l b on mature monocytes and neutrophils. A clear subset
of population 1 cells and essentially all population 2 cells
Table 3. Histochemical Staining of Myeloid BM Subpopulations
NSE
MPO
Population 1
Population 2
Population 3
5 (0-7)
3 (0-5)
6 0 (50-70)
0 (0-1)
0 (0-0)
80 (65-90)
BM subpopulations were defined as in Fig 1, cytocentrifuged onto
glass slides, and stained for NSE or MPO. Mean (range) of three
experiments.
were CDllc+, but few, if any, population 3 cells expressed
CDllc, suggesting that population 2 cells may be derived
from the CDl IC+ subset of population 1 cells. LAM-1 was
expressed on the majority of cells in both populations 1 and 2,
but on only a minority of population 3 cells; some of these
differences may be due to a selective loss of LAM-1 from
population 3 cells.” CD44 expression was uniformly high on
both population 1 and population 2 cells, but sharply lower,
although still positive, on population 3 cells. CD14 expression
was limited to a subset of population 2; no cells expressing
this monocyte marker were detected in either population 1 or
population 3. This constellation of phenotypes is consistent
with the morphologic and histochemical observations given
above (Tables 2 and 3).
Additionally, it should be noted that, for each of these
myeloid subpopulations, the expression of each of these
markers followed a uniform hierarchy. Specifically, for
population 1: % CD44h’ > % CDllahi, % CDllc+ > %
CDllb+. Similarly, for population 2: % CD44hi,% CDl lah’,
% CDllc+ > % CDllb+ > % CD14+. As discussed below,
these relationships are important for determining the order
in which these cell surface antigens are acquired during
development.
Coordinate expression of CDl I C and upregulation of
CDlla. Taken together, the data in Fig 2 and Table 2
suggest that a subset of population 1 cells, in addition to those
in population 2, are committed to monocyte differentiation,
and that this commitment is indicated by the simultaneous
appearance of CDllc and upregulation of CDlla. To
explore this question more closely, the correlated expression
of CD1 l a and CDl IC was examined on only those cells that
did not express lymphoid (CD3, CD16, CD19) or erythroid
(glycophorin [glycol) markers. This “color gate” essentially
excludes the CD33-CD15- cells in Fig lB, and therefore
defines a heterogeneous subset of BM cells that includes
myeloid cells (ie, populations 1, 2, and 3 in Fig lB), glyco-
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2486
KANSAS, MUIRHEAD, A N D DAILEY
CDI 1a
NEG. CTRL.
CDllc
CD11 b
LAM-I
CD44
CD14
I
1
L
POPULATION I
CD33I0CD15-
POPULATION II
hi
CD33 CD15+
E
!
!
!
POPULATION 111
5"i
CD3310'*CD1
0
I
l
1
2
l
3
4
0
I
l
l
1
2
3
4
0
1
2
3
4
T
0
1
2
3
4
0
1
2
3
4
0
1
I
2
3
4
0
1
2
3
4
Fig 2. Phenotypicanalysis of BM myeloid subpopulations. The indicated BM subsets were identified by correllated expression of CD33
and CD16. as in Fig 1B. end expression of the indicated cell surface markers was analyzed in the third color (APC). Direct comparison of
these three subpopulations is facilitated by differential "color gating" within the same sample. These three myeloid subpopulations exhibit
distinct patterns of expression of CDl 1a, C D l l b. C D l l c. LAM-1, CD44. and CD14.
erythroid cells, and early progenitor cells. The results (Fig 3)
demonstrate that only two populations of cells are detectable:
a CD1 la'"CD1 lc- subset, and a CD1 lahiCD1lc+ subset.
This result suggests that upregulation (or high levels of
expression) of CDl l a and expression of CD1 ICoccur simultaneously and on the same cells within this heterogeneous
BM subset, and that this phenotypic change is a very early
event associated with commitment to differentiation along
the monocyte pathway.
Downregulation of CD44 during erythroid deveIopment.
Although the absence of any markers specific for glycoerythroid cells make analysis of the phenotype of these cells
difficult, the data described below indirectly suggest that
............
0
1
2
3
4
C D l l a (APC)
CorrelFig 3. Coordinate upregulation of CD1l a and CDl IC.
lated expression of C D l l e and C D l l c is shown for those cells
which do not express CD3, CD16, CD19, or glyco: gating for this
subset is shown in the inset, the histogram for which was
generated by staining cells with a "cocktail" of FITC-conjugated
MoAbs to CD3, CD16, CD19, and glyco. Upregulationof C D l l a and
occur simultaneously and on the same cells
expression of CDl IC
during myelopoiesis.
these early committed erythroid cells are CD44hiLFA1-LAM-l-. No cells coexpressing glyco and either LFA-1 or
LAM-1 were detectable (not shown), consistent with the loss
of these markers being an early event in erythropoiesis. In
contrast, CD44 was expressed at high levels on a subset of
glyco+ cells (Fig 4A); further analysis showed that these
glyc0+CD44~'
cells coexpressed CD45, a marker found on all
leukocytes and early erythroid cell^^^.^' (Fig 4C). Glyco+
cells that expressed intermediate levels of CD44 were transferrin receptor/CD71+ (Fig 4B). Glyco+ cells expressing
neither CD45 nor CD71 expressed low levels of CD44,
similar to that found on normal, circulating RBC. Thus,
CD44 expression declines gradually and in a stepwise fashion
during normal erythropoiesis.
Progenitor cells. Because adequate numbers of BM cells
for electronic cell sorting and subsequent functional studies
were unavailable, we attempted to identify progenitor cells
by expression of CD34, a 115-Kd surface structure whose
expression on virtually all progenitor cells of multiple hematopoietic cell lineages has been documented by several
Unfortunately, the low level of CD34 expression
consistently prevented the generation of data sufficiently
reliable for color gating. As an alternative, progenitor cells
were putatively identified as those cells that failed to express
markers found on lymphoid (CD3, CD16, CD19), erythroid
(glyco), or myeloid (CD14, CD15) cells. In addition to this
"Lin-" phenotype, these cells exhibit other properties consistent with those reported for CD34+ cell^^*-^': they are present
in low numbers (1% to 2% of normal marrow); they have high
forward and low orthogonal light scatter characteristics; and
a subset of these Lin- cells express CD33 (Fig 5). Morphologic examination of Wright-Giemsa stained cytocentrifuge
preparations of these Lin- cells showed them to consist of a
mixture of myeloid and undifferentiated blasts and early
erythroblasts (not shown). Collectively, these observations
make it likely that these Lin- cells are identical to, or overlap
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ADHESION MOLECULES IN HEMATOPOIESIS
......................
0
1
2
3
o
4
i
2
3
CD44 (APC)
CD44 (APC)
4
O
i
i
3
4
CD44 (APC)
Fig 4. Downregulation of CD44 during erythropoiesis. (A) CD44 exhibits three levels of expression on glyco' BM cells. (B) Correllated
expression of CD71 and CD44 on glycot cells; gating for glyco for this contour map and that depicted in (C) is shown in the insert in (B).
Most glyco'CD71' cells are CD44". but a minor subset of the glyco+CD71+cells are CDUhi. IC) Correllated expression of CD45 and CD44
on those BM cells which are glyco'. Glyco'CD45'
cells are exclusively CDUhi. The data in (B) and (C) also demonstrate that
glyco'CD45-CD71- cells are exclusively CD44'". Thus, expression of CD44 is downregulated during normal erythropoiesis.
considerably with, the CD34+ progenitor cell subpopulation(s) described by other^.^'-^'
We then examined the Lin-CD33+ and Lin-CD33- cells
for their expression of CDl la, CD1 lb, CD1 IC, LAM-1, and
CD44 (Fig 6 ) . Comparison of these results with those
0
1
2
3
4
Lin markers (FITC)
0
1
2
3
4
CD33 (PE)
C
obtained for the myeloid subpopulations described above
(Fig 2) shows that the Lin-CD33+ cells and the
CD33'"CD15- cells (population 1) have identical phenotypes
with respect to each of these five markers, strongly suggesting that these two independently identified BM subsets are
one and the same. Although both the Lin-CD33+ and
Lin-CD33- were exclusively CD44h'and essentially CDl 1b-,
only the Lin-CD33+ subset contained cells expressing CD1 IC.
In addition, the Lin-CD33+ cells were CD1 lahior CD1 la'"
and nearly all LAM-1 +,whereas the Lin-CD33- cells
displayed C D l l a + and C D l l a - as well as LAM-I+ and
LAM-1- subsets. Examination of the CD33-Lin- cells for
the correlated expression of these two markers demonstrated
that these markers defined principally two subsets: a
CDlla+LAM-l+ subset, and a CDlla-LAM-1- subset.
Few cells expressing only C D l l a or LAM-1 were detected
(Fig 7). Morphologic examination of these Lin-CD33- cells
(Table 2) showed them to be composed exclusively of early
erythroblasts and undifferentiated blast cells. Collectively,
these data suggest that the Lin-CD33-CDl la-LAM-1subpopulation includes the earliest committed erythroid
progenitor cells.
DISCUSSION
Forward Scatter
Orthogonal Scatter
Fig 5. Definition and partial characterization of the Lin- BM
subset. (A) BM cells were stained with a cocktail of FITCconjugated MoAbs against CD3. CD14. CD15. CD16, CD19. and
glyco. Lin- cells, indicated by the arrow in (A), constituted 1.4% of
the cells in this sample. (B) A subset of Lin- cells expressed CD33;
the dotted line represents the negative control. (C and D) Lin- cells
(dotted lines) have higher average forward light scatter measurements (C), but equally low orthogonal light scatter measurements
(DL compared with all BM cells (solid lines).
The adhesion of bloodborne leukocytes to vascular endothelium at sites of inflammation or immune response, and the
relatively organ specific interaction of lymphocytes with
HEV, each represent important events in the regulation of
leukocyte traffic. At least three classes of adhesion molecules
participate in one or both of these types of leukocyteendothelial interactions: the CD11 /CD18 heterodimers, the
CD44 family, and the LAM-1 molecule(s). In this report, we
have examined the pattern of expression of these adhesion
molecules during the normal differentiation of monocytes,
granulocytes, and erythrocytes in humans.
Our data suggest that commitment to the monocytic,
granulocytic, or erythroid lineages is in each case associated
with the appearance, in a subset of the CD331°CD15CDl la'"/+CDIlb-CD1 lc-LAM-1 +CD44"' progenitor cell
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2488
KANSAS, MUIRHEAD, AND DAILEY
Neg Ctrl
C D l lb
CDlla
I
CD1 IC
C D44
LAM- 1
LinCD33+
LinCD330
1
2
3
4 0
1
2
3
4 0
1
2
3
4 0
1
2
3
3
I
l
l
1
2
3
4 0
1
2
3
4
Fig 6. Phenotypicanalysis of Lin- cells. Lin- cells were identified as in Fig 4. CD33 expression distinguished two subpopulations of Lincells, which differed in their expressionof CD1l a , CD1IC,
and LAM-1. but not C D l l b or CD44. Note that the phenotype of Lin-CD33' cells
here is essentially indistinguishable from that of the CD33"CD15- cells (population 1) in Fig 2, indicating that these two independent
methods identify the same subset of BM cells.
population (see below) of a specific, distinct phenotypic
change. For monocytes, this phenotypic change appears to be
the simultaneous acquisition of CD1 IC and upregulation of
C D l l a (Fig 3). This change precedes the upregulation of
CD33, because only some of population 1 (CD33'"CDlS-),
but all of population 2 (CD33h'CD15+),is CD1 lahiCD1lc+
(Fig 2). Similarly, upregulation of CD33 precedes surface
expression of CDl 1b, because only a subset of population 2 is
CD1 Ib+. The sequential appearance of CDl IC and CDl l b
during monopoiesis contrasts with the simultaneous appearance of these markers on 12-0-tetra-decanoylphorbo1-13acetate (TPA)- or retinoic acid-treated U937 and HL60
cells,42possibly reflecting differences between normal immature myeloid cells and long-term in vitro leukemic cell lines.
The final stage of monocyte differentiation is marked by the
appearance of CD14 (Fig 2). The sequential stages of
i
2
3
C D l l a (APC)
Fig 7. Correllated expression of LAM-1 and C D l l a on
Lin-CD33- cells. Lin-CD33- cells were identified as in Fig 4,
except that My9-FITC (anti-CD33) was added to the cocktail of
lineage markers. Two principal subsets of Lin-CD33- cells were
seen: a CD1l a + L A M - l + subset, and a CD1la-LAM-1 - subset. A
small transitional population of cells is also visible, suggesting
coordinate loss of C D l l a. LAM-1, and CD33 from Lin- cells.
monocyte development defined by our studies are summarized in Fig 8A.
Commitment to granulocyte differentiation is marked by
acquisition of high levels of CD15 (Fig 1 and Table 2);
whether upregulation of CD15 occurs as a result of this
hapten43being expressed on a particular molecule or set of
molecules, including CD1 l a and CD1 lb,44is unknown. High
levels of expression of CD15 are associated with decreasing
levels of CD33 (Fig 1) and lower levels of CD44 (Fig 2 ) ,
along with an increase in orthogonal light scatter measurements (data not shown). As we have shown previously, this
lower level of CD44 expression, compared with monocytes, is
characteristic of mature, bloodborne neutrophil^.^^ The initiation of these phenotypic changes precedes expression of
CDl l b on these granulocytic cells, but CD1 l b is expressed
on most cells before the CD33-CD44I0 phenotype is achieved
(Fig 2). The final stage of granulocyte differentiation is
signaled by the acquisition of CD16. Although CD16 is
typically absent from BM neutrophil^^^ but present on blood
neutrophils, this may relate to its expression being on only
those BM neutrophils whose high density excludes them
from retention on Ficoll-Hypaque gradients. The relatively
late expression of C D l l b and CD16 (for granulocyte^)^^ or
CD14 (for monocytes) offers an interesting parallel in the
development of these two myeloid lineages (Fig 8).
Because no markers specific for glyco- erythroid cells
exist, determining the phenotype of these cells is difficult.
However, the absence of CD1 l a and LAM-1 on glyco+ BM
cells suggests that the loss of these markers is an early step in
erythropoiesis. Consistent with this, the Lin-CD33- population, which contains early erythroblasts (Table 2 ) , also
contains a CDlla-LAM-1- subset (Fig 6). These cells are
uniformly CD44h' (Fig 5 ) , and this high level of CD44 is
maintained on the subset of glyco+ cells that coexpress CD45
(Fig 7). Downregulation of CD44 on these cells is associated
with the appearance and subsequent disappearance of CD71
(Fig 7). The gly~o+CD44~"CD45-CD71phenotype of these
later erythroid cells is identical to that of mature RBC (Fig
8C). It should be noted that our results suggest that glyco
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2489
ADHESION MOLECULES IN HEMATOPOIESIS
I
:D44
Fig 8. Summary of phenotypic changes during the normal
differentiation of (A) monocytes, ( 8 ) granulocytes, and ( C ) erythrocytes. The approximate relative surface intensities of the indicated
adhesion molecules and lineage markers are represented on the
vertical axis. Differentiation proceedsfrom left to right. The mark on
the vertical axis denotes the lowest level of surface staining
detectable. Phenotypically distinct stages of differentiation are
indicated and separated by dashed lines, and the phenotype of
mature, bloodborne cells is indicated. (***I As discussed in the text,
LAM-1 expression during this phase of granulocyte development is
.IM-'
uncertain, but probably similar to earlier and later cells, as indicated.
See text for details and complete explanation.
:D71
:D45
;D33
.FA-I
expression "extends back" farther in the erythroid lineage
than was observed by others.36The basis for this discrepancy
remains unclear. However, our results regarding the sequential appearance of CD45 and CD71 during erythropoiesis are
consistent with this previous report, and support our hypothesis that CD44 is downregulated during erythropoiesis in
humans.
Our results confirm and extend an array of previous
observations regarding the phenotype of various classes of
human hematopoietic progenitor cells. Other investigators
have shown that myeloid, erythroid, and mixed colonyforming cells (CFC) express CD33,32 CD34,38-41CDl
and LAM-1,22 but not CD11b47; CD15 is detectable by
complement-mediated depletion on more differentiated myeloid CFC.33 It has also been shown that precursors of all
classes of CFC express CD34 but not CD33, and have
distinct light scatter proper tie^.^' These results are in excellent agreement with our observations regarding myeloid
subpopulations defined as Lin- or by differential expression
of CD15 and CD33 (Figs 1, 2, and 5). Although inadequate
numbers of BM cells prevented us from directly quantitating
the CFC potential of the various BM subsets defined in this
report, the close concordance of their detailed phenotype
with that reported by numerous groups for several types of
CFC argues strongly that the Lin- BM subset identified in
these studies includes the CD34' progenitor cell population
defined by
Our observations therefore suggest (1)
that the earliest hematopoietic cells have the phenotype
CD33-CD34+(Lin-)CDl lalo/+LAM-1+CD44hiCD1
1bCD1 IC- (but see below); and (2) that acquisition of CD33 by
these cells4' is associated with commitment toward mixed
myeloid/erythroid differentiation. Thus, we predict that cells
capable of giving rise to mixed myeloid/erythroid or myeloid
colonies, operationally defined as CFC-mix and CFC-GM,
respectively,will have the phenotype CD33IoCD34+CD1lalo/+
CD1 lb-CDl IC-LAM-1+CD44hi. Cells with this precise
phenotype are easily detectable as a subset of population 1
(Figs 1 and 2), and constituted 0.3% to 0.8% of total marrow
cells in our studies.
The subset of the Lin-CD33- cells which is C D l l a LAM- 1- is usually greater than the fraction clearly identifiable as erythroblasts. Therefore, it is possible that early
hematopoietic cells that are precursors to CFC of all classes
are contained within this CDl la-LAM-1 - fraction, and that
expression of these two adhesion molecules, like that of
CD33, is associated with commitment toward a myeloid or
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2490
KANSAS, MUIRHEAD, AND DAILEY
erythroid pathway of differentiation. Consistent with this,
the earliest detectable committed pre-B cells (CD19+CD10h’CD20-) are LAM-1- and express only low levels of
CDl la,3’ suggesting that the immediate precursor of the
committed pre-B cell is LAM-1 -, or alternatively, that
commitment to B lymphopoiesis is associated with a loss of
LAM-1 expression. In the absence of direct data from
long-term BM culture systems, the precise phenotype of the
most primitive cells remains uncertain, as does the profile of
adhesion molecules expressed on these cells.
Little is known about cell adhesion during hematopoiesis.
Although good evidence exists for a role for cell surface
fibronectin receptors and stroma-associated fibronectin in
hematopoietic cell adhe~ion,4~”’
it seems unlikely that either
this adhesion system, or that involving h e m ~ n e c t i n ,can
~~
fully account for all aspects of hematopoietic cell adhesion.
Theexpressionof LFA-1 (CDlla/CD18), LAM-1, and high
levels of CD44 on the most primitive hematopoietic cells
suggests that these molecules may be involved in this process,
possibly by positioning developing cells in appropriate microenvironments within the marrow. Although LFA-1 plays
an important role in most cell-cell adhesion events involving
leukocytes, a role for LFA-1 in the adhesion of immature
hematopoietic cells to BM stromal cells has not, to our
knowledge, been demonstrated. Because the expression of a
molecule per se does not guarantee that it is functional in any
particular context, a role for LFA-1 as a hematopoietic cell
adhesion molecule awaits empirical confirmation.
LAM-1 (Leu8/TQl), the human homologue of MEL14,53.54is a member of the recently defined selectin gene
family, which, in addition to LAM-1 and MEL-14, also
includes the ELAM- 1 molecule,55 expressed on activated
endothelium, and PADGEM/gmpl40 (CD62), expressed on
activated platelets and e n d ~ t h e l i u mLAM. ~ ~ 1 is thought to
play a crucial role in the binding of both lymphocytes to
lymph node (LN) HEV and neutrophils to activated
endothelium,2’s22similar to what has been demonstrated for
MEL- 14 in the m o ~ s e . ~Although
~ - ’ ~ direct evidence implicat-
ing LAM-1 in adhesion events relevant to hematopoiesis has
yet to emerge, the lectin-like nature of lymphocyte-HEV
interactions involving LAM-1 and MEL-1 457-60
is consistent
with previous reports demonstrating a role for specific sugar
residues in the adhesion of immature myeloid cells to BM
stroma.61-62Collectively, these observations suggest that
LAM- 1 mediates numerous important and diverse adhesion
events throughout the lifespan of human leukocytes.
Several groups have reported inhibition of lymphocyte
binding to HEV in the Stamper-Woodruff frozen section
assay by MoAbs to CD44.27,28.63
These data, in addition to
certain close biochemical a n d immunohistologic
similarities:6329 had originally led others to propose that
CD44 constituted the human homologue of the MEL- 14
.~~
more recently,
defined LN HEV r e c e p t ~ r . ~ ’However,
molecular gene ti^^^,^' and other studies have firmly established that CD44, previously also known as Hermes, In(1u)related p80, and Pgp-1,66 is homologous to murine Pgp-l/
Ly24. The high level of CD44 expression on hematopoietic
progenitor cells, especially erythroid progenitors that do not
express LFA-1 or LAM-1, makes CD44 an excellent candidate for a general hematopoietic cell adhesion molecule, as
we have previously3’hypothesized for B cells. In this regard,
Miyake, et aI6’ have recently shown that murine Pgp-1 is
involved in the maintenance of B-cell progenitors in WhitlockWitte cultures and granulocyte-macrophage progenitors in
Dexter cultures. Further studies will be required to delineate
precisely the role of CD44 in these and other adhesive
interactions of human leukocytes.
ACKNOWLEDGMENT
The authors gratefully acknowledge generous gifts of MoAbs
from Alan Krensky, Carol Clayberger, Edgar G. Engleman, James
Griffin, and Becton Dickinson; James Griffin for helpful advice and
discussion; Teresa Duling for expert assistance with flow cytometry;
Doug Padley for providing BM samples; Mari Kucera for expert
secretarial assistance; and M. Snapp for inspiration.
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1990 76: 2483-2492
Expression of the CD11/CD18, leukocyte adhesion molecule 1, and
CD44 adhesion molecules during normal myeloid and erythroid
differentiation in humans
GS Kansas, MJ Muirhead and MO Dailey
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