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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1423-1431
By
From the Inflammation Research Centre, University of Melbourne,
Department of Medicine, The Royal Melbourne Hospital, Parkville,
Victoria, Australia; the Department of Immunology, Erasmus University,
Rotterdam, The Netherlands; the Peter MacCallum Cancer Institute, East
Melbourne, Victoria, Australia; and the Department of Molecular
Genetics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto,
Japan.
Macrophage populations resident in tissues and at sites of
inflammation are heterogeneous and with local proliferation sometimes evident. Using the convenient murine peritoneal cavity as an
inflammation model, the appearance of macrophage lineage cells was
followed with time in both thioglycollate- and sodium periodate-induced exudates. The cells were characterized by their proliferative response
in vitro in response to colony-stimulating factor-1 (CSF-1) (or
macrophage colony-stimulating factor [M-CSF]), particularly by their
ability to form colonies in agar, in combination with flow cytometry
(surface marker expression and forward and side scatter
characteristics). We propose that c-Fms (CSF-1 receptor), unlike other
markers, is a uniformly expressed and specific marker suitable for the
detection of macrophage-lineage cells in tissues, both in the steady
state and after the initiation of an inflammatory reaction. It was
shown that the bone marrow myeloid precursor markers, ER-MP58 and
ER-MP20 (Ly-6C), but not ER-MP12 (PECAM-1), are expressed by a high
proportion of macrophage-lineage cells in the inflamed peritoneum. The
macrophage colony-forming cells (M-CFCs) in a 16-hour
thioglycollate-induced exudate were phenotyped as
c-Fms+ERMP12 © 1998 by The American Society of Hematology.
MACROPHAGES IN various tissues during the
steady state and at sites of inflammation are heterogeneous with
respect to phenotype as well as function.1 The murine
peritoneum is often used as a convenient sterile site to explore the
characteristics and the involvement of macrophage-lineage cells in the
development of an inflammatory reaction. During the steady state,
resident peritoneal macrophages can be derived locally in the
peritoneum2,3 and, from the op/op4 (colony
stimulating factor-1 [CSF-1]-deficient) mouse, this production is
dependent on the presence of CSF-1 (or macrophage-CSF [M-CSF]). Soon
after injection of an irritant, such as thioglycollate medium (TM),
resident peritoneal macrophages disappear by adhering to the peritoneal
lining wall, with a concomitant influx of neutrophils and a subsequent
appearance of macrophages.5,6 Many of the monoclonal
antibodies (MoAbs) reacting with peritoneal macrophages recognize
elicited or activated cells or functionally defined
subpopulations.1 However, some that react with surface antigens expressed on all or nearly all resident peritoneal macrophages have also been described, eg, F4/807 and
MUM-4.8 These markers, which are not detected on
all peritoneal macrophage populations, in particular inflammatory
macrophages, provide therefore an underestimate of macrophage numbers;
the same conclusion holds for other tissues.9 In addition,
macrophages in different tissues show different patterns of expression
of the currently used markers.9 One other problem is that
most of the markers used to detect macrophages are also expressed by other cell types.
Macrophages at many different sites of inflammation have been shown to
proliferate, and this local proliferation may contribute to the
increased numbers of macrophages, particularly in chronic lesions (see,
eg, Bitterman et al10 and Jutila and Banks11). It has been shown that murine elicited peritoneal macrophages can
proliferate in vitro in response to CSF-112-14; also
evident in the inflamed peritoneum (and other tissues) is a
subpopulation of immature cells of the macrophage lineage, the
so-called macrophage colony-forming cells (M-CFCs or CFU-M), which form colonies in response to CSF-1,12,14-19 again
indicating substantial heterogeneity. These M-CFCs could contribute to
the local development of more mature inflammatory macrophage
populations. The surface marker expression of the peritoneal M-CFCs is
unknown, as is their relationship to M-CFCs in hematopoietic organs
such as bone marrow and spleen.
ER-MP12, ER-MP20, and ER-MP58 have been used for the identification of
myeloid-committed progenitors and used as markers of murine macrophage
development in murine bone marrow.20-24 ER-MP12 has been
recently identified as the adhesion molecule, PECAM-1 (CD-31),25 and ER-MP20 as Ly-6C.26 The earliest
CSF-1-responsive cells in murine bone marrow have the
ER-MP12hi20 What is needed for the identification of macrophage populations in
tissues are specific markers that are expressed on all macrophage
populations, as well as suitable markers to delineate the relationships
between them. Seeing that CSF-1 is likely to be playing a role in the
survival, proliferation, and perhaps development of macrophage-lineage
cells at such sites4 and that its receptor (c-Fms) is not
expressed on committed cells of other myeloid or lymphoid
lineages,28 it was reasoned that c-Fms may be a useful
marker for identifying macrophage-lineage cells in tissues. It was also
reasoned that, in combination with surface marker expression, the
degree of the proliferative response to CSF-1, reflecting relative
cellular immaturity, may be a useful functional criterion to explore
the developmental relationships between macrophage subpopulations. From
the studies below we suggest, on the basis of the inflamed murine
peritoneum as a model and using flow cytometry, that c-Fms should be
considered as a convenient uniform marker for macrophage-lineage cells
in tissues; we also report that inflammatory macrophage-lineage cells
express ER-MP58 and/or ER-MP20 and that, in a 16-hour TM
exudate, the peritoneal M-CFC are
c-Fms+ER-MP12 Mice
Reagents
Peritoneal Cells Peritoneal cells were washed from the peritoneal cavity of mice by lavage with 5 mL of ice-cold, sterile phosphate-buffered saline (PBS; Trace BioSciences Pty Ltd, New South Wales, Australia). Peritoneal exudate cells were elicited by intraperitoneal injection of either 1 mL Brewer's Thioglycollate medium (Difco Laboratories, Detroit, MI) or 1 mL of 5.6% sodium periodate (Sigma, St Louis, MO). Cells were harvested at various time points after injection. In each experiment, the peritoneal cells were harvested from 8 mice, a cell count was obtained for each individual mouse, and the cells were pooled for experiments.Autoradiography Autoradiography was performed to determine the percentage of adherent cells synthesizing DNA (S-phase). Resident or exudate cells (1.2 × 105/well) were allowed to adhere to a 12-well plate (Costar, Cambridge, MA) containing a circular (16-mm diameter) glass coverslip in 1 mL of -minimum essential medium
(-MEM; Trace BioScience) plus 10% fetal calf serum
(FCS; CSL, Parkville, Australia) for 2 hours at 37°C.
Three washes with PBS were performed to remove nonadherent cells. Each
well was replenished with 1.5 mL of -MEM plus 10% FCS in the
presence or absence of 3,000 U/mL of recombinant human CSF-1 (Chiron
Co, Emeryville, CA) or 10,000 U/mL of recombinant murine
granulocyte-macrophage colony-stimulating factor (GM-CSF; Sandoz, Vienna, Austria). 3H-thymidine (3H-TdR;
Amersham Life Science, New South Wales, Australia; specific activity,
82.0 Ci/mmol) was added to each culture well at 2.5 µCi/mL at t = 0. After 4 days, cells were fixed in EtOH for 10 minutes on ice and 30 minutes at room temperature and then allowed to air dry. A long
labeling protocol was used because of the lag period before the cells
enter S phase.12-14 Coverslips were adhered to glass slides
and dipped in liquid emulsion (Ilford Scientific Product, K-2 Emulsion
in gel form; Mobberley, Cheshire, UK), exposed for 6 days at room
temperature, developed in Kodak D-19 developer (Eastman Kodak,
Rochester, NY) for 3 minutes, fixed, and counterstained with hematoxylin. The percentage of labeled cells was determined by
counting a total of 300 cells per coverslip.
Colony Assay The double-layer nutrient agar culture,30 consisting of 1 mL underlayer of 0.5% agar (Bacto Agar; Difco) plus 800 U/dish of CSF-1 or 600 U/dish of GM-CSF and 0.5 mL overlay of 0.33% agar plus 1,000 or 20,000 peritoneal cells in a 35-mm Petri dish for M-CFC or GM-CFC detection, respectively, was used throughout.31-MEM was used, supplemented with vitamins (×2; ICN,
Biomedicals Inc, Costa Mesa, CA), L-glutamine, and 20% bovine calf
serum (Hyclone Laboratories Inc, Logan, UT). Cultures were performed in
triplicate. The culture dishes were incubated in 10% CO2
for 14 days and colonies were scored as greater than 50 cells; clusters
were scored as less than 50 cells. No colonies were found in the
absence of growth factor. Unfractionated bone marrow cells were
included in every experiment as an internal control for the cloning
efficiency of a particular experiment. We routinely detected an average
of 60 M-CFCs per 2,500 cells plated in response to CSF-1.
Immunofluorescence Labeling, Flow Cytometric Analysis, and Cell Sorting For phenotypic analyses, 0.5 to 1 × 106 freshly isolated peritoneal cells/well were aliquotted into 96-microwell plates (V-bottom; Nunc, Roskilde, Denmark) and labeled with the appropriate antibodies at the predetermined dilutions. All incubations were performed on ice for 20 minutes and were followed by three washes with PBS. For single-color analysis, cells were incubated first with hybridoma supernatant, washed, and then incubated with donkey-antirat-PE. For two-color analysis, cells were incubated first with hybridoma supernatant, washed, and then treated with antirat-FITC (Silenus), followed by biotinylated MoAb and streptavidin-PE (Dako, Carpinteria, CA). The same batch of rat isotype Igs (Silenus) was used as the control. The percentage of positive cells for any particular marker was determined by subtracting the background irrespective of the number of positive peaks. Phenotypic analyses were performed with a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA). The identification of cell populations in forward and side scatter profiles for the different MoAbs used was performed by back-gating positive cell populations in histogram plots using CellQuest version 3.0.1. software (Becton Dickinson).
Appearance of Proliferative Macrophage-Lineage Cells in the Inflamed Peritoneum Adherent cells and DNA synthesis. Injection of TM is known to induce a population of macrophages of which a high proportion is capable of proliferating in vitro in response to CSF-1.12-14 We confirm these observations in Fig 1A, in which we plot the total number of peritoneal cells appearing over a 96-hour period after TM injection and the percentage of the adherent cells in the populations capable of undergoing DNA synthesis in vitro in response to CSF-1. When cells were cultured in the absence of CSF-1, less than 2% of both resident and elicited cells entered S-phase. Only 5% to 10% of the adherent cells incorporated 3H-TdR in the presence of murine GM-CSF over a 4-day period in vitro (data not shown).
Appearance of M-CFCs. We also compared the kinetics of appearance into the cavity of cells capable of forming colonies in agar in response to CSF-1 or GM-CSF, ie, in assays that allow for cells with high proliferative capacity to be measured. There were no colony-forming cells detected in the unstimulated cavity with either CSF-112,17 or GM-CSF13 (data not shown). When GM-CSF was used in vitro as growth factor, no GM-CFCs were found in peritoneal cells from either stimuli at any of the time points tested above, although a few clusters were detected (data not shown). M-CFCs were observed clearly within 5 hours after TM injection and increased over the 96-hour exudate period when calculated both as a percentage and absolute number of cells (Fig 1B). In the sodium periodate-induced exudate, a low percentage (0.3%; P < .05) of M-CFCs could be detected within 24 hours, whereas the percentages and numbers increased to a maximum at 16 hours, followed by a slow decrease (data not shown). Many clusters (<50 cells) were also observed in the colony assay with both in vivo stimuli. c-Fms as a Marker for Macrophage-Lineage Cells Flow cytometric analysis was then used to define the macrophage-lineage cells at different times after the injection of the irritants. In the first instance, we chose to examine whether flow cytometry could be used to monitor macrophage-lineage cells by c-Fms expression in the peritoneal cavity and whether it would therefore be a useful marker for such cells, given its specificity,28 both in the steady state and during an inflammatory reaction. In Fig 2, it can be seen that c-Fms+ cells can be detected in the unstimulated cavity and that the initial decline in c-Fms+ cells is presumably due to the so-called macrophage disappearance reaction described previously.5,6 After this initial decrease, the number of detectable c-Fms+ cells gradually increases with time. In contrast, Mac-1+ cells increase to a maximum number at 16 hours, presumably due to the influx of neutrophils; at later times, the total number remains constant presumably due to the appearance of macrophage-lineage cells (see below) at the expense of the neutrophils.
Definition of Macrophage-Lineage Cells in the Inflamed Peritoneum We next decided to combine forward and side scatter properties with surface marker expression to try to define populations rich in macrophage-lineage cells in the peritoneal cavity. The forward and side scatter profiles for the steady state peritoneal cells are depicted in Fig 3A. For the unstimulated cavity, a macrophage-rich region was defined (region 1) on the basis of c-Fms, F4/80, MUM-4, and Mac-1 (Table 1). Region 2, one of lower forward and side scatter, is positive for T- and B-lymphocyte markers and class II antigen, but negative for the other macrophage markers. Neutrophils were not found in significant numbers in the resident peritoneal cavity.32 Region 3 was defined as a neutrophil-rich region in the TM-induced 16-hour exudate (Fig 3B) by their initial absence then appearance, by their relatively discrete size and granularity, and by their expression of Mac-1 antigen (16 hours: 90% ± 3%; n = 11) but not of the other surface markers (eg, F4/80: <3%; n = 11; c-Fms: <3%; n = 9).
Peritoneal M-CFCs Are
c-Fms+ER-MP12
We used the murine peritoneal cavity as a suitable site to follow the
appearance of macrophage-lineage cells during an inflammatory reaction.
There have been reports that macrophages, presumably of a relatively
immature phenotype, can undergo DNA synthesis at sites of inflammation
(see, eg, Bitterman et al10 and Jutila and
Banks11), possibly in response to CSF-1, which can be found at elevated levels at such sites.34 We therefore used the
proliferative response to CSF-1, as well as cell surface markers found
on immature myeloid bone marrow cells, including cells belonging to the
macrophage lineage, to help define subpopulations of the macrophage
lineage in the inflamed peritoneal cavity. Two stimuli that have no
obvious common properties were used as an indication of the generality of any conclusion, the logic being that, if surface marker patterns were similar in the two exudates, then they might reflect lineage maturation rather than have resulted from cellular activation by a
particular inflammatory insult. The data in this report show that, in
general, the two stimuli result in a similar response. As reported
elsewhere,12,14,15,17 we could not find significant numbers
of M-CFCs in the unstimulated cavity.
Submitted November 12, 1997;
accepted April 20, 1998.
Helpful discussions were held with S. Moss and I. Campbell. R. Sallay
and J. Scopes are thanked for typing the manuscript. We also thank R. Rossi and B. Williams (Peter MacCallum Institute) for assistance with
cell sorting and colony assays, respectively.
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Abstract
Introduction
20+58+,
properties consistent with their being more mature than bone marrow
M-CFCs. It is proposed that ER-MP58, as well as ER-MP20, may be a
useful marker for distinguishing inflammatory macrophage-lineage cells
from the majority of those residing normally in tissues.
Abstract
)2 fragment (Jackson Immuno Research Lab Inc, PA) and FITC-conjugated antirat IgG (Silenus,
FL) were used as secondary antibodies.
; see the Materials and
Methods). Data are the mean values ± SEM and are pooled from 5 experiments, each with 8 mice. Also, at various times after intraperitoneal injection of TM, adherent cells were treated in vitro
for 4 days with CSF-1 and [3H]-TdR (
; see the
Materials and Methods). Autoradiography was used to measure the number
of S-phase cells (see the Materials and Methods). Data are the mean
values ± SEM from triplicate cultures and are from a representative
experiment that was repeated a total of 3 times. (B) Kinetics of
appearance of peritoneal M-CFCs. At various times after intraperitoneal
injection of TM, M-CFCs were measured as agar colonies (1,000 cells
plated; see the Materials and Methods). Data are plotted as the number
of M-CFCs in the peritoneal cavity (
) using the total cell numbers
presented in (A) and as a percentage of the peritoneal cells (
) expression by flow cytometry (see the Materials and Methods).
Data are the mean values ± SEM from 3 experiments (8 mice/time
point/per experiment). A partial kinetic analysis was also performed
another 8 times. When error bars are not presented, the errors are
smaller than the size of the symbols.
90% of the cells in region 1 are
c-Fms+ (the corresponding figure for F4/80 is 8%; see
Table 
) and region 2 (
; see Fig 
One mechanism for the expansion of the mononuclear phagocyte population in the chronically inflamed lung.
J Clin Invest
74:460,
1984 
