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Peritoneal Fluid and Plasma Levels of Human Macrophage Colony-Stimulating
Factor in Relation to Peritoneal Fluid Macrophage Content
By J. Brice Weinberg, Arthur F. Haney, F.J. Xu, and S. Ramakrishnan
The peritoneal fluid (PF) of women with infertility (especially
in the presence of endometriosis] contains increased numbers of leukocytes, 90% t o 95% of which are macrophages.
The high numbers of peritoneal macrophages presumably
result from an influx of blood monocytes into the peritoneum, and/or from local proliferation of peritoneal macrophages. Once in the peritoneal cavity, monocytes differentiate into tissue macrophages. Mononuclear phagocyte
proliferation and differentiation are influenced by different
cytokines, including macrophage colony-stimulating factor
(M-CSF). The purpose of this study was to determine the
relationship of M-CSF levels in human PF and plasma t o the
macrophage content, and to the patient diagnoses. Mean
concentrations of PF M-CSF were higher than plasma levels
(2.44 f 0.13 Y 0.95 f 0.06 ng/mL, respectively). The mean
concentrations of plasma M-CSF did not differ in samples
from women of different diagnostic groups (normal, peritoneal adhesions, endometriosis, inactive pelvic inflammatory
disease, uterine fibroids, and idiopathic infertility], but the PF
concentration was slightly higher in normal women. The
absolute (total) amount of PF M-CSF in normal women was
lower than in those of the other diagnostic groups. The total
amount of PF M-CSF in all women correlated closely with the
total number of peritoneal macrophages. The tubal patency
status (open versus closed) did not influence the plasma and
PF concentrations of M-CSF, nor the PF absolute amount of
M-CSF. The PF M-CSF may have come from peritoneal
macrophages, fibroblasts, mesothelial cells, or endothelial
cells. PF M-CSF may play important roles in the proliferation
and/or the differentiation of peritoneal mononuclear phagocytes.
o 7991by The American Society of Hematology.
W
The patients were not taking any medications. Diagnoses were
established by standard means.’,’*Samples from 67 subjects were
studied. There were six normal subjects (those for elective bilateral
tubal ligation [BTL] or tubal reanastomosis), two with idiopathic
peritoneal adhesions, 32 with endometriosis, 16 with inactive pelvic
inflammatory disease (PID), five with uterine fibroids, and six with
infertility. M-CSF was measured with a radioimmunoassay using a
highly specific rabbit anti-M-CSF antibody that does not crossreact with other growth factors6 This assay is capable of detecting
levels 2 0.5 ng/mL.6 Statistical analyses were performed using the
“Statview” program (Abacus Concepts, Berkeley, CA) with a
Apple MacIntosh computer with the Student’s t-test and MannWhitney U test.
OMEN WHO ARE INFERTILE for a variety of
reasons have increased volumes of peritoneal fluid
(PF) and increased numbers of peritoneal macrophage^.'.^
The increased number of peritoneal macrophages results
from the influx of monocytes into the peritoneum, and/or
from local proliferation of peritoneal macrophages. Mononuclear phagocyte proliferation is controlled by macrophage and granulocyte-macrophage colony-stimulating factor (M-CSF and GM-CSF), and interleukin-3 (IL-3).4
M-CSF is the primary growth factor for macrophages. It is
produced by a variety of normal cells (mononuclear phagocytes, fibroblasts, endothelial cells, and some epithelial
cell^)^,^ and by certain tumor cells.6 In addition to the
proliferative effects, M-CSF can cause differentiation or
“activation” of monocytes and ma~rophages.7.~
The proliferative and differentiative effects of M-CSF are mediated by
high-affinity membrane receptor^.‘'^^^'" The purpose of this
study was to determine the relationship of M-CSF levels in
human PF and plasma to the macrophage content, and to
the patient diagnoses. Results demonstrate that PF M-CSF
levels were always higher than plasma levels, and that PF
and plasma M-CSF concentrations did not correlate with
PF volume, PF macrophage concentration or absolute
number, or tubal patency status (open or closed). However,
the absolute (total) amount of PF M-CSF correlated closely
with the absolute number (total) of PF macrophages.
MATERIALS AND METHODS
After appropriate informed consent, matched sets of P F and
blood were obtained from women undergoing laparoscopy for
elective sterilization or investigationitreatment for infertility. The
samples were anticoagulated with 10 UImL of heparin, 0.3%
EDTA, and 0.2 moliL epsilon amino caproic acid. P F was obtained
via a laparoscope as described before.’ The patient was positioned
so that the fluid pooled in the dependent aspect of the peritoneal
cavity and all of the free PF was collected; no rinsing of the
peritoneal cavity was performed. Care was taken to avoid contaminating the PF with blood. Greater than 90% of the peritoneal cells
were macrophages (nonspecific esterase [alpha naphthyl butyrate
esterasel-positive)” and all were viable (trypan blue exclusion).
Samples were taken between days 10 and 20 of the menstrual cycle.
Blood, Vol 78, No 2 (July 15). 1991: pp 513-516
RESULTS
In all sets of PF and plasma examined, the mean PF
M-CSF level was higher than the mean plasma level
(2.44 ~fr0.13 [mean ? SEMI v 0.95 ? 0.06 ng/mL for PF
and plasma, respectively [n = 671; P < .0005), with the PF
levels higher than the corresponding plasma levels in all
individual cases. The concentrations of M-CSF in plasma
did not differ among the various diagnostic groups, but the
PF concentrations were slightly higher in normal women
undergoing bilateral tubal ligation (Fig 1A and B). PF
volumes varied considerably among the groups, with the
From the Departments of Medicine and Obstetrics and Gynecology,
VA and Duke University Medical Centers, Durham, NC; and the
Department of Pharmacology, University of Minnesota Medical Center, Minneapolis.
Submitted October I , 1990; accepted March 19, 1991.
Supported in part by the VA Research Service, the James Swiger
Hematology Research Fund, and Grants No. CA 48068, POI AI23308,
POI 32682, and P50 AR39162from the National Institutes of Health.
Address reprint requests to J. Brice Weinberg, MD, VA and Duke
University Medical Centers, 151G, 508 Fulton St, Durham, NC 27705.
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 I8 U.S.C.section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971I91/7802-0001$3.00iO
513
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514
WEINBERG ET AL
Ij T
10
n.16
T
09
=
.
m
E
-
08
M
07
06
LL
P
05
04
03
02
a1
n.n.
Adhesons
BTL
Endomelrlosas
Ffbroids
PID
Diagnosis
n=4
T
Fig 2. Total PF M-CSF levels (concentration times volume) in
women with different diagnoses. The bars show the means f SEM.
Statistical comparisons among the different groups showed significant differences between values for BTL versus endometriosis
(P < .05), and BTL and reanastomosisversus endometriosis (P < .025).
Otherwise, there were no significant differences.
-E
.
m
c
1
there were no differences in the concentrations of PF or
plasma M-CSF nor the total amount of PF M-CSF with
regard to the tubal patency status. In plasma, the concentrations were 0.90 ? 0.16 and 1.00 ? 0.07 ng/mL
(mean ? SEM) for women with closed and open tubes,
respectively; in PF, the concentrations were 2.60 2 0.31 and
2.50 f 0.14 for women with closed and open tubes,
respectively; and in PF, the total amounts of M-CSF were
Adhesions
BTL
Endometriosis
Fibrolds
PID
Diagnosis
Fig 1. Plasma (A) and PF (6)M-CSF concentrations in women with
different diagnoses. The bars show the means +- SEM. Statistical
comparisons among the different groups showed significant differences between values for BTL versus endometriosis (P < ,005). BTL
versus fibroids (P < .02), and BTL versus PID (P < .005).
volumes being lowest in women undergoing BTL or tubal
reanastomosis and highest in women with adhesions, endometriosis, fibroids, and PID. The absolute (total) amount
(concentration times volume) of PF M-CSF was significantly lower in normal women (those undergoing a BTL
and/or reanastomosis) (Fig 2 ) . The absolute (total) amount
of PF M-CSF correlated very closely with the absolute
(total) number of peritoneal macrophages (Fig 3).
Because the peritoneal cavity in women communicates
through the oviducts to the uterus, vagina, and the environment, patent oviducts represent an avenue through which
exogenous materials (eg, microorganisms or their constituents [such as endotoxin]) could gain contact with normal
peritoneal macrophages and modulate their function (including the stimulation of M-CSF elaborati~n).~.~
However,
-
m
C
Total Number of Peritoneal Cells (millions)
Fig 3. Total number of PF cells (macrophages] as a function of the
absolute (total) PF M-CSF. The bars show the means f SEM. There is
a statistically significant correlation between the t w o parameters
(P < ,0001). y = 9.95 + 0.59~.(r = ,637; P < ,0001.)
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PERITONEAL FLUID M-CSF
21.60 f 6.70 and 26.40 2 4.00 ng for women with closed and
open tubes, respectively.
DISCUSSION
Infertile women are known to have increased numbers of
peritoneal leukocytes, 90% to 95% of which are macrophages. The highest numbers are found in women with
endometri~sis.”~
We and others have hypothesized that
macrophages (or their products) may adversely affect fertilization by injuring gametes or the embry0.‘.~3’~-~~
Conditions
characterized by high numbers of peritoneal macrophages
(eg, endometriosis) are often accompanied by “idiopathic”
infertility. 1,3,13,14.20 Importantly, peritoneal macrophage number correlates with oviductal macrophage n ~ m b e r , docu’~
menting that the peritoneal macrophages have access to
oviducts, the site of fertilization. Information from studies
of women with open or closed oviducts suggest that oviductal macrophages derive from peritoneal macrophages that
emigrate through the open fimbriae into the oviducts.”
While it has been assumed that the increased numbers of
peritoneal macrophages in infertile women result from an
influx of blood monocytes into the peritoneal cavity in
response to some inflammatory stimulus, it is also possible
that some of the increase is caused by local proliferation of
macrophages in response to high levels of growth factors. In
this study, we demonstrate that PF levels of M-CSF are
significantly higher than plasma levels, and that the absolute (total) amount of PF M-CSF correlates closely with the
absolute (total) number of peritoneal macrophages present.
The source of the PF M-CSF is not known. Macrophages in
the peritoneal cavity could elaborate the factor and cause
the noted elevations. Alternately, the M-CSF could in part
(or whole) come from peritoneal fibroblasts or mesothelial
cells stimulated to elaborate M-CSF by products of macrophages.2L,22
Also, uterine epithelial (endometrial) cells can
elaborate M-CSF.232”
Because PF levels of M-CSF were the
same in women with open and closed fallopian tubes, it is
unlikely that reflux of uterine cavity M-CSF (derived from
endometrial cells) contributed substantially to the PF
levels.
515
As a blood monocyte enters extravascular tissues, certain
micro-environmental factors cause the cell to differentiate
into a phenotypically different cell, the tissue macrophage.
The factors causing this in vivo differentiation are not
known. Peritoneal macrophages are phenotypically different than blood monocytes in several respects: they are
larger and have more vacuolated cytoplasm^,'"^ have increased ability to phagocytize normal sperm,13 have increased ability to mediate spontaneous tumor cell killing in
have altered expression of Ig Fc receptors,26 are
unable to cap and internalize major histocompatibility
complex class I1 antigen^,'^ have enhanced tissue factor
(thromboplastin) expression,zs and have diminished ability
to express/elaborate IL-1 activity and present antigenz9
(Weinberg JB, unpublished). Clearly, peritoneal macrophages are “differentiated” mononuclear phagocytes as
compared with blood monocytes. PF M-CSF could play a
role in the differentiation process; this factor causes “differentiation” or “activation” of monocytes and macrophages
as judged by several different
Because M-CSF
can serve as a chemotactic factor for monocytes,% PF
M-CSF could attract blood monocytes into the peritoneal
cavity, and then M-CSF (alone or in combination with other
PF factors) could cause differentiation of the blood monocytes into the phenotypically different peritoneal macrophages.
The controls of partitioning of M-CSF between plasma
and tissue fluids are not known. High serum levels of
M-CSF have been reported in patients with myeloproliferative disease31 and in plasma of patients with ovarian
cancer.” The greater than 2.5-fold higher PF M-CSF levels
(compared with plasma levels) that we observed here
suggest a high rate of production of M-CSF in the peritoneal cavity, and/or a selective retention of, and/or a concentrating mechanism for M-CSF in PF. Based on our findings
of compartmentally regulated levels of M-CSF, we can
assume that plasma or serum levels will not always accurately reflect levels in tissue fluids. This finding is important
in evaluating M-CSF levels in various pathologic states.
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1991 78: 513-516
Peritoneal fluid and plasma levels of human macrophage colonystimulating factor in relation to peritoneal fluid macrophage content
JB Weinberg, AF Haney, FJ Xu and S Ramakrishnan
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