Journal
of Leukocyte
Biology
50:471-478
(1991)
Murine Eosinophil Granulocytes Bind the Murine Macrophage-Monocyte Specific Monoclonal Antibody F4/80 McGarry
M.P. Departments
of Laboratory
Animal
Resources
(M.P.M.)
and
C.C.
and Flow Cytometry
Stewart (C.C.S.),
Roswell
Park Cancer
Institute,
Buffalo,
New York
Studies designed to identity a panel of monoclonal antibodies useful for the separation of murine eosinophil myeloid progenitors revealed that F4/80, an antigen heretofore thought to be expressed only by murine monocyte/macrophage lineage cells, was
expressed
by eosinophil
granulocytes.
Eosinophils
from several
strains of mice stained
positively with specific antibody for the epitope. A novel pathway for myeloid differentiation is proposed in which neutrophil progenitors exit a common lineage prior to a common progenitor of monocytes and eosinophils. Moreover, the results demonstrate that binding of anti-F4/80 can no longer be viewed as exclusive for mononuclear phagocytes. Key
words:
cell sorting,
differentiation,
hematopoiesis,
INTRODUCTION Over the last decade, monoclonal antibodies (mAb) have been produced to epitopes found on most all hematopoietic cells. It was hoped that these antibodies would be useful to identify the individual cell subsets that make up each lineage within Unfortunately, this approach cessful as antibodies produced immunogen
are
specific specificities
functional represent
example
found
Mac-
the has
to react
with
molecules are now
1 binding
to
hematopoietic system. not been entirely sucusing one cell type as other
cell
types.
The
that these crossreacting being elucidated, as for an
epitope
on
the
C3bi
receptor [10]. Thus, the single purpose of identification of cells is giving way to more fundamental questions concerning the nature and function of molecules identifled by unique antibodies, and possible lineage or functional relationships between cells expressing them. Many hematopoietic cells express common antigens with functional myeloid cells [5] and this property can be exploited to characterize and sort cells. Combinations of antibodies may be used with multiparameter analysis to achieve preferential selection of unique populations. Thus, separate
Lytl and classes
Lyt2 are proteins expressed of peripheral lymphocytes,
on two but their
progenitors express both proteins simultaneously. three populations, LytlLyt2 LytlLyt2 LytFLyt2 can be explicitly resolved when
All and anti-Lytl
,
,
,
and anti-Lyt2, used together
[ 12].
Using this antibody-detected
strategy, we sought epitopes to enable
cytometry ©
labelled
different
fluorochromes,
granulocytes.
Inc.
markers
row and peritoneal exudate, enriched for eosinophils by experimental parasitism, were stained with a panel of antibodies to murine myeloid cells to find candidates that might be useful to resolve marrows were so dominated myeloid fluorescence series have
cells found
series,
high indicated
cells. One an affinity
Previously, against phages,
eosinophils. by cells of
proportions epitopes
of existent
Because the the eosinophil
bound on
antibody eosinophil
such antibody, F4/80, was discovered for eosinophils by this procedure.
this mAb, thioglycollate had demonstrated
of the monocyte/macrophage that the murine eosinophil
lineage
lineage cells
[1 .7]. bound
I [ 19]. eosinophils
We both
When these could be
distinguished from monocyte/macrophage lineage cells because their fluorescence for both Mac- 1 and F4/80 was slightly less intense and because neutrophil granulocytes, while binding Mac-l, did not bind F4/80. Erythroid and lymphoid
cells
did
not
stain
with
either
antibody.
This
demonstration of the F4/80 antigen on eosinophil granulocytes may reveal a closer lineage relationship between monocytes/macrophages and eosinophils. Moreover, results of studies based on a presumed binding specificity of F4/80 for monocytes/macrophages may in some instances require reinterpretation.
are
to define separation
a
set of by flow
Murine
bone
mar-
to
derived from rats immunized broth-elicited murine macrobinding specificity only for
F4/80 and another rat mAb, anti-Mactwo antibodies were used together.
Received
of eosinophil
1991 Wiley-Liss,
with
antigen
November
Reprint
requests:
Animal
Resources,
Streets,
Buffalo,
2.
1990:
Michael Roswell NY
14263.
accepted P.
MeGarry. Park
Cancer
February
14,
1991.
Department Institute,
of Elm
Laboratory and
Carlton
472
McGarry
MATERIALS,
and
Stewart
METHODS,
AND
PROCEDURES
concentration
Ros:(ICR), BALB/cRos, obtained
C57BL/6Ros, AKR/Ros, from
Park
Cancer
iae,
obtained
the
West
Institute from
.
C57BL/ A/StRos
and
Seneca
lORos, female
Mesocestoides
Dr.
DAB/2Ros, mice were
Laboratory
David
of
corti
Warren
tubes
Sloan
Kettering Cancer Institute, New York, NY), were maintamed by serial intraperitoneal passage in mice. Schistosoma mansoni-infected CD1 female mice were obtained
ceinated
and
Antibody
were
chronically
eosinophilic
An initial panel of mAbs was selected based on reported binding specificities for cells of various lymphohematopoietic lineages (Table 1). For detection of bound mAb by indirect immunofluorescence, marrow cells were obtained by a cold (4#{176}C) phosphate buffered saline (PBS) flush of the marrow cavity of both excised femurs and one tibia from donor mice. An aliquot was taken for cytospin and stained for differential. A direct
trifuged
at 800g
and
resuspended
RA34B62”
Mac 3 Mac 2 Mac 1
was
IgG2a
30G
I
-
1.2)
IgG2b IgG2b -
at unit free
in part
Baxter
Corp., CHK
1.4
Chicago, dF4/80:
from
was
bRA34B62
was IL
Walter
et al.,
a personal personal
gift
cell-rich
Park, from
I
cells
for Use on on normal
tissuesa
Marrow
B cells
cells pos.)
-
-
-
-
Thymus
? 0 0
? 0 0 50
0
-
-
1111 1111
+ +
-?
49
>96
1111
+
+
71
>85
+
+
65
-
-
+
+
Dr. J. Bender,
Round
40 9 >90 97
98 90% recovery.
and
of the relative intensi1 . Sorted cells were
collected into slightly hypotonic agamma with distilled water). Cytocentrifuged sorted populations were Giemsa stained Computations sorted that
FACSCAN
antiserum was Mean eosinophil
preps of final suspensions
Siemens
instrumentation (Becton-Dickinson ImmuSystems, San Mateo, CA). Forward right angle light scatter, and fluorescein and fluorescence were measured using an operating at 488 nM. Subpopulations of cells
were defined based ties of the FL-F4/80
FGAR mAbs.
30G2, and Surprisingly,
73. 1 + marrow suspension.
I .5 smear
to determine characteristics
which, of each
in
Table 1 and the 1 . Each mAb was of a murine marrow
eosinophils and
cytospin
by differprepara-
with defined specificities for neutrophils or exhibited little or no binding to the eosinomarrows. pan-myeloid 30Gl2 one
bound mAb,
Antibodies affinities, to a high anti-F4/80,
that had previously such as DNL 4.4, proportion reported
less than 12% of cells in normal BM , stained cells. In a suspension with 73% eosinophils, only be interpreted as evidence that some
ofthe cells. [1] to stain 68% of the this could cells in the
474
McGarry
and Stewart
eosinophil myelocyte series F4/80. Normal hematopoietic
expressed marrow
than 5% of cells morphologically myelocyte series. It is of interest isotype rat mAbs did not consistently levels (Table 4.9% (3OH12) Mac- I ). Only amount equal tion,
for less
sisted
in to
evidencing
against
a role
for
the eosinophil note that IgG21, bind at equivalent
used
non-specific, in
the
cells that lymphoid suspension
To
screen,
phils,
we
stained
PEC
and
that
also expressed F4/80 bound
BM
mice with FL-F4/80 and PE-Mac1 was included so neutrophils, phages could be distinguished populations prior to resolution fluorescence. Results for PEC
cells
from
from phils, tical
it. to eosinoMacmacro-
FL 99% with
(F4/80)
determined with both
fluorescence
macrophages. somewhat
(see
microscopically intense PE (MacFigure
2A)
any F4/80 granulocytes
did not stain with either (see Fig. 2A). Greater was
recovered,
and
eosinophil
stained
gran-
for PE (Mac-l)
associated (see
Fig.
fluorescence 2A). Other
antibody were primarily than 95% of the initial there
was
a marrow suspension 84% were eosinophils. to the binding profile
no
evidence
containing 24.5% eosinoThis was essentially idenfor peritoneal eosinophils.
cells sorted from the eosinophil region identity of the F4/80, Mac- 1 subpopulation
confirmed the as eosino-
phil-series cells (Fig. 3). Several strains of mice were tested for the presence of the F4/80 antigen. M. corti antigen-primed mice of selected strains were made eosinophilic by peritoneal
The 1 ) and .
consisted
In contrast, a subpopulation less intense combined fluorescence
of mature that
fluorescent for Mac- 1 than those from the peritoneal cavity. This was a consistent finding. Lymphocytes, erythroblasts. and some other blasts were negative for both mAbs. The internal lysosomal granule crystalloid structure in
from other cells in both on the basis of FL-F4/80 are shown in Figure 2A.
Data in Table 2 present differentials for initial (unsorted) and variously sorted populations. The PEC contained 12% eosinophil granulocytes. Four subpopulations of cells were defined and differentials on Giemsa stained cytospin preparations subpopulation of PEC
express neutrophil
Cells
Cells positive for Mac-l but negative for F4/80 were neutrophils and some undifferentiated blasts. Note that the neutrophils from bone marrow appear to be more
eosinophilic
1 simultaneously. eosinophils, and
(97%)
2A).
marrow does not contain a high proportion of reactive macrophages, defined here as brightly F4/80 Mac- 1 cells. When cells in the region of moderate FL-F4/80 and PE-Mac-l fluorescence (Fig. 2B Table 2) were sorted
intensity indicated the eosinophilic marrow cells that had a moderate to high expression of
at least some eosinophils confirm the finding
entirely Fig.
for a selective loss of any subpopulation due to the procedures. A similar profile of mAb binding, shown in Figure 2B, was found in eosinophil-rich marrow, except that such
the epitope for Mac- 1 , a mAb specific for the C3bi receptor on neutrophils and macrophages. It was detected on 53.4% of cells in the marrow suspension, indicating that
(see
but did not were mostly
isotype
initial
almost
ulocytes
1 ). Binding ranged from 3.6% (3OE2) and through 24.6% (3OH1 1) to 53.4% (antianti-F4/80 was bound in the approximate to the eosinophils present in the prepara-
related Fc receptor binding. Among the other mAbs fluorescence contained
the epitope usually has
of
of PEC con-
challenge with 50 i.Lg of the M. corti antigen preparation. Three days later PEC and BM cell suspensions were
B
A A
FL-F4/80
Fig. 2. Resolution of eosinophil granulocytes.
PEC (A) or BM
cells (B) from CD1 mice infected with S. mansoni cells (C) were stained with FL-F4/80 and PE-Maci by flow cytometry. Cells were sorted, stained
or normal BM and analyzed with Wright’s
blood stain, and identified using light microscopy to contain neutrophils (+), macrophages ( ), eosinophils (0), and other cells (0) consisting mainly of erythroid and lymphoid elements. Groups roughly correspond to the four sorted groups in Table 2.
Murine TABLE
2. Differential
Peritoneal
Cellular
Composition
Exudate and Femoral Marrow
Eosinophilopoietic
(Percent)
of Initial
Suspensions
from
PeritoneaL exudate Unsorted Subpopulations
and Sorteda
M. Cortl-Infected,
Macrophages
Eosinophils
78
12
99 16 2.5 1
0 1 97 0
1 42 0 0
24.5
29.5
46
1 0
17 87
82 13
7.5 0 41 0.5
+ 0
99
Neutrophils
Other
1.5
marrow
Unsorted Subpopulations + 0
84
0
16
0
22
0
78
aSorted
populations
fluorescence bwright
obtained
stained
cytocentrifuged selected
phycoerythrin
dCells
using
for fluorescein
C5ubpopulations
this
are F4/80 Positive
Mlcebc Lymphocytes
Femoral
Eosinophils
on
(PE-Mac-l).
FACSTAR
(F4/80)
cell preparations.
the
basis
Symbols
of the erythroid,
flow
and phycoerythrin of
relative
comparable
lymphoid,
to lineages.
monocyte
cytometer
(Mac-I) fluorescence
and
based
on
relative
intensity
of
(See Fig. 2A, B). for
fluorescein
Fig. 2A, B. and all undifferentiated
(FL-F4/80) blasts
were
and placed
in
category. ;
.
.
-r---(
‘
.
I-
‘
-. I
I
-
-
.,
-.
-
.
:
1
-
:
‘-
I:
(‘
:: Fig. 3. intensity structure
Electron micrograph of marrow eosinophil from sorted of both FL-F4/80 and PE-Mac-1 associated with eosinophils. is uniquely
characteristic
of eosinophils.
x 18,000.
population moderate for Intragranular crystalloid
475
476
McGarry
and Stewart
BONE
MARROW
CELLS C57BL1O
C57BL6
E
DBA/2
BALB/c
E
Cl)
A/St (U)
E
E
0
CELLS C57BL1O
C57BL6 E M
9
M
CD1
M
LOG Fig. 4.
Binding
PEC enriched
of fluoresceinated
for eosinophils
anti-F4/80
by challenge
to murine
FLUORESCENCE BM or
with 50 ig M. corti
soluble antigen. Filled histogram shows control cells while unfilled histogram shows F4/80 cells. For BM, “E” arrows point to region of eosinophils. Percent eosinophils in BM: C57BLJ
6Ros, 20%; C57BL/lORos, 12%; AKR/Ros,
15%; DBAJ2Ros, 17%; BALB/cRos, 18%; AIStRos, 5%. For PEC, “E” arrows point to
eosinophils eosinophils date:
and “M” arrows and macrophages,
C57BLJ6Ros,
25%,
16%;
point
autofluorescence
30%,
are distinctly
in C57BL/lORos
Percent
in peritoneal
C57BLJ1ORos,
27%, 16%. Note that macrophages their cells.
to macrophages.
respectively,
and
CD1
20%;
exuCD1,
resolved peritoneal
by
Murine examined
for
evident tested.
F4/80
in Figure
binding
and
4, eosinophils
percent were
eosinophils.
F4/80
As
in all strains
In this obtained
rately
study, from panel
shown that investigation of the
we characterized the intentionally parasitized of
eosinophils has shown
mononuclear
to eosinophils istry studies, granulocyte
mAbs.
bind the F4/80 that this antibody
phagocyte
was not discovered. tissues examined content and F4/80
fied as macrophages others using this greatly enhanced the background to bind F4/80
suggest
a need
have
Since
tissues
small proportions of surprising that binding In immunocytochemwere low in eosinophil cells were only identi-
percentage of marrow is consistent with present
We
mAb. Previous bound to cells
[71 .
series
[6,8]. Our study antibody because we numbers of eosinophils
granulocytes studies
eosinophilic BM mice using a
myeloid-associated
rich in macrophages with only eosinophils were studied, it is not
eosinophil marrow. These
using F4/80 of macrophages.
(3-7%) for
cells reported the percentage in normal reevaluation
[ 1]
with eosinophils. activities may
to macrophages Especially quantitation
that
inferences phils from
about these
have in fact
the differentiation findings. It is our arise
cell lineages differentiate acquire new functional identified by Mac-l and 1 .2 or La [6].
the
differentiation
are functions
during progenitor
to make
the
differentiation of cells. As specific
from these progenitors, they cell surface proteins like those F4/80, and they may lose others,
We believe
cells
that
pathways
from from These
acquire
associated
with
murine
lineage. anti-Mac-
of data
with C3bi
that there is an epitope on eosinophils, neutrophils,
the demonstration receptor present
1 binding
MACROPHAGE
MACROPHAGEEOSINOPHIL precursor cell
EOSINOPHIL common myelold progenitor cell
NEUTROPHIL
Fig. 5. Proposed differentiation pathway for eosinophils epitopes. Data indicate a closer association between between eosinophils and neutrophils.
based on expression of mAb eosinophils and macrophages
defined than
a new
oligopotent hematopoiedivergences
particular hematopoietic Our results with the
of
some
pathway of eosinobelief that epitopes
epitope, like F4/80, diverge together progenitors along a pathway different tic cells that don’t acquire the epitope. define
Moreover, it is been maccu-
vulnerable are studies in which is accomplished through F4/80
found on mature cells oligopotent hematopoietic
Thy
477
the presence and that some of these
binding measured biochemically [13,14]. As shown in Figure 5, it is possible
like
differs from all used mice with . Interestingly,
attributed
ofeosinophils. macrophage
are F4/80 Positive
binding to assess It is possible
functions may be shared also possible that some
DISCUSSION
selected
obtained function
Eosinophils
any
are consistent on
the and
478
McGarry
macrophages. myeloid
and
This
result
progenitor
rophages, acquire
Stewart is consistent
for
these
three
and now eosinophils, F4/80 antigen as they
the
with lineages.
but mature,
not
a common
macrophage-specific
Since
mouse
mac-
neutrophils it seems quite
possible that eosinophils and macrophages initially branch together away from neutrophil differentiation as shown in Figure 5 . Committed neutrophil progenitors then would independently differentiate and not acquire the F4/80 antigen. At some later stage in the differentiation scheme, eosinophils appear to branch monocyte/macrophage series, but retain, albeit level of expression, the F4/80 epitope. Colonies rophage shown
derived
colony to contain
from
forming many
that these cells stage [20]. F4/80 should be present
the
murine
responsiveness
(CSF),
similarly
to GM-colony
Hume,
in their
expression
cells
express
antigen
F4/80
monocyte/macrophage
by BM cannot
[3 ,2 1]
Mesocestoides
acknowledge
the
expert
iA. cell
,
MeGarry,
directed 11,805. 2.
Hapel.
3.
Hirsch.
against
the
macrophage.
H.S.
and
Hume,
specifically
and
F4/80,
a monoclonal Eur.
antibody J.
Exp.
162,60,
Med.
factors
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FDC-Pl S.
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The
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Allergy
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differentiation
gen
L.A.
GM-
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stimulating
differ
FDC-PI
binding, to various
6.
cells
subpopulation-speeifie
surface
granulocyte-mac-
cell (GM-CFC) have not been eosinophils leading to the belief
and
.
Holmes,
may branch earlier than the GM-CFC expression would infer that eosinophils if macrophages are differentiating from
in their
M.-K.
elonal 5.
8.
eosinophils may not have arisen in these colonies in sufficient number to attract attention. Interestingly, two IL-3 dependent cell lines, differing
CSF receptors, GM-CSF and
Ho,
antigen marrow
maerophage
from the at a lower
a progenitor also yielding cells of the neutrophil cyte series. One possibility is that growth conditions GM-CFC may not have favored eosinophilopoiesis, perhaps to a lack of interleukin-5 (IL-5) [17,22].
epitope.
4.
bone
37,121.
1985.
among
interleukin of eosinophils
Immunol.
140,94.
of Leukocyte
Biology
50:471-478
(1991)
Murine Eosinophil Granulocytes Bind the Murine Macrophage-Monocyte Specific Monoclonal Antibody F4/80 McGarry
M.P. Departments
of Laboratory
Animal
Resources
(M.P.M.)
and
C.C.
and Flow Cytometry
Stewart (C.C.S.),
Roswell
Park Cancer
Institute,
Buffalo,
New York
Studies designed to identity a panel of monoclonal antibodies useful for the separation of murine eosinophil myeloid progenitors revealed that F4/80, an antigen heretofore thought to be expressed only by murine monocyte/macrophage lineage cells, was
expressed
by eosinophil
granulocytes.
Eosinophils
from several
strains of mice stained
positively with specific antibody for the epitope. A novel pathway for myeloid differentiation is proposed in which neutrophil progenitors exit a common lineage prior to a common progenitor of monocytes and eosinophils. Moreover, the results demonstrate that binding of anti-F4/80 can no longer be viewed as exclusive for mononuclear phagocytes. Key
words:
cell sorting,
differentiation,
hematopoiesis,
INTRODUCTION Over the last decade, monoclonal antibodies (mAb) have been produced to epitopes found on most all hematopoietic cells. It was hoped that these antibodies would be useful to identify the individual cell subsets that make up each lineage within Unfortunately, this approach cessful as antibodies produced immunogen
are
specific specificities
functional represent
example
found
Mac-
the has
to react
with
molecules are now
1 binding
to
hematopoietic system. not been entirely sucusing one cell type as other
cell
types.
The
that these crossreacting being elucidated, as for an
epitope
on
the
C3bi
receptor [10]. Thus, the single purpose of identification of cells is giving way to more fundamental questions concerning the nature and function of molecules identifled by unique antibodies, and possible lineage or functional relationships between cells expressing them. Many hematopoietic cells express common antigens with functional myeloid cells [5] and this property can be exploited to characterize and sort cells. Combinations of antibodies may be used with multiparameter analysis to achieve preferential selection of unique populations. Thus, separate
Lytl and classes
Lyt2 are proteins expressed of peripheral lymphocytes,
on two but their
progenitors express both proteins simultaneously. three populations, LytlLyt2 LytlLyt2 LytFLyt2 can be explicitly resolved when
All and anti-Lytl
,
,
,
and anti-Lyt2, used together
[ 12].
Using this antibody-detected
strategy, we sought epitopes to enable
cytometry ©
labelled
different
fluorochromes,
granulocytes.
Inc.
markers
row and peritoneal exudate, enriched for eosinophils by experimental parasitism, were stained with a panel of antibodies to murine myeloid cells to find candidates that might be useful to resolve marrows were so dominated myeloid fluorescence series have
cells found
series,
high indicated
cells. One an affinity
Previously, against phages,
eosinophils. by cells of
proportions epitopes
of existent
Because the the eosinophil
bound on
antibody eosinophil
such antibody, F4/80, was discovered for eosinophils by this procedure.
this mAb, thioglycollate had demonstrated
of the monocyte/macrophage that the murine eosinophil
lineage
lineage cells
[1 .7]. bound
I [ 19]. eosinophils
We both
When these could be
distinguished from monocyte/macrophage lineage cells because their fluorescence for both Mac- 1 and F4/80 was slightly less intense and because neutrophil granulocytes, while binding Mac-l, did not bind F4/80. Erythroid and lymphoid
cells
did
not
stain
with
either
antibody.
This
demonstration of the F4/80 antigen on eosinophil granulocytes may reveal a closer lineage relationship between monocytes/macrophages and eosinophils. Moreover, results of studies based on a presumed binding specificity of F4/80 for monocytes/macrophages may in some instances require reinterpretation.
are
to define separation
a
set of by flow
Murine
bone
mar-
to
derived from rats immunized broth-elicited murine macrobinding specificity only for
F4/80 and another rat mAb, anti-Mactwo antibodies were used together.
Received
of eosinophil
1991 Wiley-Liss,
with
antigen
November
Reprint
requests:
Animal
Resources,
Streets,
Buffalo,
2.
1990:
Michael Roswell NY
14263.
accepted P.
MeGarry. Park
Cancer
February
14,
1991.
Department Institute,
of Elm
Laboratory and
Carlton
472
McGarry
MATERIALS,
and
Stewart
METHODS,
AND
PROCEDURES
concentration
Ros:(ICR), BALB/cRos, obtained
C57BL/6Ros, AKR/Ros, from
Park
Cancer
iae,
obtained
the
West
Institute from
.
C57BL/ A/StRos
and
Seneca
lORos, female
Mesocestoides
Dr.
DAB/2Ros, mice were
Laboratory
David
of
corti
Warren
tubes
Sloan
Kettering Cancer Institute, New York, NY), were maintamed by serial intraperitoneal passage in mice. Schistosoma mansoni-infected CD1 female mice were obtained
ceinated
and
Antibody
were
chronically
eosinophilic
An initial panel of mAbs was selected based on reported binding specificities for cells of various lymphohematopoietic lineages (Table 1). For detection of bound mAb by indirect immunofluorescence, marrow cells were obtained by a cold (4#{176}C) phosphate buffered saline (PBS) flush of the marrow cavity of both excised femurs and one tibia from donor mice. An aliquot was taken for cytospin and stained for differential. A direct
trifuged
at 800g
and
resuspended
RA34B62”
Mac 3 Mac 2 Mac 1
was
IgG2a
30G
I
-
1.2)
IgG2b IgG2b -
at unit free
in part
Baxter
Corp., CHK
1.4
Chicago, dF4/80:
from
was
bRA34B62
was IL
Walter
et al.,
a personal personal
gift
cell-rich
Park, from
I
cells
for Use on on normal
tissuesa
Marrow
B cells
cells pos.)
-
-
-
-
Thymus
? 0 0
? 0 0 50
0
-
-
1111 1111
+ +
-?
49
>96
1111
+
+
71
>85
+
+
65
-
-
+
+
Dr. J. Bender,
Round
40 9 >90 97
98 90% recovery.
and
of the relative intensi1 . Sorted cells were
collected into slightly hypotonic agamma with distilled water). Cytocentrifuged sorted populations were Giemsa stained Computations sorted that
FACSCAN
antiserum was Mean eosinophil
preps of final suspensions
Siemens
instrumentation (Becton-Dickinson ImmuSystems, San Mateo, CA). Forward right angle light scatter, and fluorescein and fluorescence were measured using an operating at 488 nM. Subpopulations of cells
were defined based ties of the FL-F4/80
FGAR mAbs.
30G2, and Surprisingly,
73. 1 + marrow suspension.
I .5 smear
to determine characteristics
which, of each
in
Table 1 and the 1 . Each mAb was of a murine marrow
eosinophils and
cytospin
by differprepara-
with defined specificities for neutrophils or exhibited little or no binding to the eosinomarrows. pan-myeloid 30Gl2 one
bound mAb,
Antibodies affinities, to a high anti-F4/80,
that had previously such as DNL 4.4, proportion reported
less than 12% of cells in normal BM , stained cells. In a suspension with 73% eosinophils, only be interpreted as evidence that some
ofthe cells. [1] to stain 68% of the this could cells in the
474
McGarry
and Stewart
eosinophil myelocyte series F4/80. Normal hematopoietic
expressed marrow
than 5% of cells morphologically myelocyte series. It is of interest isotype rat mAbs did not consistently levels (Table 4.9% (3OH12) Mac- I ). Only amount equal tion,
for less
sisted
in to
evidencing
against
a role
for
the eosinophil note that IgG21, bind at equivalent
used
non-specific, in
the
cells that lymphoid suspension
To
screen,
phils,
we
stained
PEC
and
that
also expressed F4/80 bound
BM
mice with FL-F4/80 and PE-Mac1 was included so neutrophils, phages could be distinguished populations prior to resolution fluorescence. Results for PEC
cells
from
from phils, tical
it. to eosinoMacmacro-
FL 99% with
(F4/80)
determined with both
fluorescence
macrophages. somewhat
(see
microscopically intense PE (MacFigure
2A)
any F4/80 granulocytes
did not stain with either (see Fig. 2A). Greater was
recovered,
and
eosinophil
stained
gran-
for PE (Mac-l)
associated (see
Fig.
fluorescence 2A). Other
antibody were primarily than 95% of the initial there
was
a marrow suspension 84% were eosinophils. to the binding profile
no
evidence
containing 24.5% eosinoThis was essentially idenfor peritoneal eosinophils.
cells sorted from the eosinophil region identity of the F4/80, Mac- 1 subpopulation
confirmed the as eosino-
phil-series cells (Fig. 3). Several strains of mice were tested for the presence of the F4/80 antigen. M. corti antigen-primed mice of selected strains were made eosinophilic by peritoneal
The 1 ) and .
consisted
In contrast, a subpopulation less intense combined fluorescence
of mature that
fluorescent for Mac- 1 than those from the peritoneal cavity. This was a consistent finding. Lymphocytes, erythroblasts. and some other blasts were negative for both mAbs. The internal lysosomal granule crystalloid structure in
from other cells in both on the basis of FL-F4/80 are shown in Figure 2A.
Data in Table 2 present differentials for initial (unsorted) and variously sorted populations. The PEC contained 12% eosinophil granulocytes. Four subpopulations of cells were defined and differentials on Giemsa stained cytospin preparations subpopulation of PEC
express neutrophil
Cells
Cells positive for Mac-l but negative for F4/80 were neutrophils and some undifferentiated blasts. Note that the neutrophils from bone marrow appear to be more
eosinophilic
1 simultaneously. eosinophils, and
(97%)
2A).
marrow does not contain a high proportion of reactive macrophages, defined here as brightly F4/80 Mac- 1 cells. When cells in the region of moderate FL-F4/80 and PE-Mac-l fluorescence (Fig. 2B Table 2) were sorted
intensity indicated the eosinophilic marrow cells that had a moderate to high expression of
at least some eosinophils confirm the finding
entirely Fig.
for a selective loss of any subpopulation due to the procedures. A similar profile of mAb binding, shown in Figure 2B, was found in eosinophil-rich marrow, except that such
the epitope for Mac- 1 , a mAb specific for the C3bi receptor on neutrophils and macrophages. It was detected on 53.4% of cells in the marrow suspension, indicating that
(see
but did not were mostly
isotype
initial
almost
ulocytes
1 ). Binding ranged from 3.6% (3OE2) and through 24.6% (3OH1 1) to 53.4% (antianti-F4/80 was bound in the approximate to the eosinophils present in the prepara-
related Fc receptor binding. Among the other mAbs fluorescence contained
the epitope usually has
of
of PEC con-
challenge with 50 i.Lg of the M. corti antigen preparation. Three days later PEC and BM cell suspensions were
B
A A
FL-F4/80
Fig. 2. Resolution of eosinophil granulocytes.
PEC (A) or BM
cells (B) from CD1 mice infected with S. mansoni cells (C) were stained with FL-F4/80 and PE-Maci by flow cytometry. Cells were sorted, stained
or normal BM and analyzed with Wright’s
blood stain, and identified using light microscopy to contain neutrophils (+), macrophages ( ), eosinophils (0), and other cells (0) consisting mainly of erythroid and lymphoid elements. Groups roughly correspond to the four sorted groups in Table 2.
Murine TABLE
2. Differential
Peritoneal
Cellular
Composition
Exudate and Femoral Marrow
Eosinophilopoietic
(Percent)
of Initial
Suspensions
from
PeritoneaL exudate Unsorted Subpopulations
and Sorteda
M. Cortl-Infected,
Macrophages
Eosinophils
78
12
99 16 2.5 1
0 1 97 0
1 42 0 0
24.5
29.5
46
1 0
17 87
82 13
7.5 0 41 0.5
+ 0
99
Neutrophils
Other
1.5
marrow
Unsorted Subpopulations + 0
84
0
16
0
22
0
78
aSorted
populations
fluorescence bwright
obtained
stained
cytocentrifuged selected
phycoerythrin
dCells
using
for fluorescein
C5ubpopulations
this
are F4/80 Positive
Mlcebc Lymphocytes
Femoral
Eosinophils
on
(PE-Mac-l).
FACSTAR
(F4/80)
cell preparations.
the
basis
Symbols
of the erythroid,
flow
and phycoerythrin of
relative
comparable
lymphoid,
to lineages.
monocyte
cytometer
(Mac-I) fluorescence
and
based
on
relative
intensity
of
(See Fig. 2A, B). for
fluorescein
Fig. 2A, B. and all undifferentiated
(FL-F4/80) blasts
were
and placed
in
category. ;
.
.
-r---(
‘
.
I-
‘
-. I
I
-
-
.,
-.
-
.
:
1
-
:
‘-
I:
(‘
:: Fig. 3. intensity structure
Electron micrograph of marrow eosinophil from sorted of both FL-F4/80 and PE-Mac-1 associated with eosinophils. is uniquely
characteristic
of eosinophils.
x 18,000.
population moderate for Intragranular crystalloid
475
476
McGarry
and Stewart
BONE
MARROW
CELLS C57BL1O
C57BL6
E
DBA/2
BALB/c
E
Cl)
A/St (U)
E
E
0
CELLS C57BL1O
C57BL6 E M
9
M
CD1
M
LOG Fig. 4.
Binding
PEC enriched
of fluoresceinated
for eosinophils
anti-F4/80
by challenge
to murine
FLUORESCENCE BM or
with 50 ig M. corti
soluble antigen. Filled histogram shows control cells while unfilled histogram shows F4/80 cells. For BM, “E” arrows point to region of eosinophils. Percent eosinophils in BM: C57BLJ
6Ros, 20%; C57BL/lORos, 12%; AKR/Ros,
15%; DBAJ2Ros, 17%; BALB/cRos, 18%; AIStRos, 5%. For PEC, “E” arrows point to
eosinophils eosinophils date:
and “M” arrows and macrophages,
C57BLJ6Ros,
25%,
16%;
point
autofluorescence
30%,
are distinctly
in C57BL/lORos
Percent
in peritoneal
C57BLJ1ORos,
27%, 16%. Note that macrophages their cells.
to macrophages.
respectively,
and
CD1
20%;
exuCD1,
resolved peritoneal
by
Murine examined
for
evident tested.
F4/80
in Figure
binding
and
4, eosinophils
percent were
eosinophils.
F4/80
As
in all strains
In this obtained
rately
study, from panel
shown that investigation of the
we characterized the intentionally parasitized of
eosinophils has shown
mononuclear
to eosinophils istry studies, granulocyte
mAbs.
bind the F4/80 that this antibody
phagocyte
was not discovered. tissues examined content and F4/80
fied as macrophages others using this greatly enhanced the background to bind F4/80
suggest
a need
have
Since
tissues
small proportions of surprising that binding In immunocytochemwere low in eosinophil cells were only identi-
percentage of marrow is consistent with present
We
mAb. Previous bound to cells
[71 .
series
[6,8]. Our study antibody because we numbers of eosinophils
granulocytes studies
eosinophilic BM mice using a
myeloid-associated
rich in macrophages with only eosinophils were studied, it is not
eosinophil marrow. These
using F4/80 of macrophages.
(3-7%) for
cells reported the percentage in normal reevaluation
[ 1]
with eosinophils. activities may
to macrophages Especially quantitation
that
inferences phils from
about these
have in fact
the differentiation findings. It is our arise
cell lineages differentiate acquire new functional identified by Mac-l and 1 .2 or La [6].
the
differentiation
are functions
during progenitor
to make
the
differentiation of cells. As specific
from these progenitors, they cell surface proteins like those F4/80, and they may lose others,
We believe
cells
that
pathways
from from These
acquire
associated
with
murine
lineage. anti-Mac-
of data
with C3bi
that there is an epitope on eosinophils, neutrophils,
the demonstration receptor present
1 binding
MACROPHAGE
MACROPHAGEEOSINOPHIL precursor cell
EOSINOPHIL common myelold progenitor cell
NEUTROPHIL
Fig. 5. Proposed differentiation pathway for eosinophils epitopes. Data indicate a closer association between between eosinophils and neutrophils.
based on expression of mAb eosinophils and macrophages
defined than
a new
oligopotent hematopoiedivergences
particular hematopoietic Our results with the
of
some
pathway of eosinobelief that epitopes
epitope, like F4/80, diverge together progenitors along a pathway different tic cells that don’t acquire the epitope. define
Moreover, it is been maccu-
vulnerable are studies in which is accomplished through F4/80
found on mature cells oligopotent hematopoietic
Thy
477
the presence and that some of these
binding measured biochemically [13,14]. As shown in Figure 5, it is possible
like
differs from all used mice with . Interestingly,
attributed
ofeosinophils. macrophage
are F4/80 Positive
binding to assess It is possible
functions may be shared also possible that some
DISCUSSION
selected
obtained function
Eosinophils
any
are consistent on
the and
478
McGarry
macrophages. myeloid
and
This
result
progenitor
rophages, acquire
Stewart is consistent
for
these
three
and now eosinophils, F4/80 antigen as they
the
with lineages.
but mature,
not
a common
macrophage-specific
Since
mouse
mac-
neutrophils it seems quite
possible that eosinophils and macrophages initially branch together away from neutrophil differentiation as shown in Figure 5 . Committed neutrophil progenitors then would independently differentiate and not acquire the F4/80 antigen. At some later stage in the differentiation scheme, eosinophils appear to branch monocyte/macrophage series, but retain, albeit level of expression, the F4/80 epitope. Colonies rophage shown
derived
colony to contain
from
forming many
that these cells stage [20]. F4/80 should be present
the
murine
responsiveness
(CSF),
similarly
to GM-colony
Hume,
in their
expression
cells
express
antigen
F4/80
monocyte/macrophage
by BM cannot
[3 ,2 1]
Mesocestoides
acknowledge
the
expert
iA. cell
,
MeGarry,
directed 11,805. 2.
Hapel.
3.
Hirsch.
against
the
macrophage.
H.S.
and
Hume,
specifically
and
F4/80,
a monoclonal Eur.
antibody J.
Exp.
162,60,
Med.
factors
lines
FDC-Pl S.
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Austyn.
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32D J.M.
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GM-
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