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Copenhagen,
Denmark
19 - 22 June 2005
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ER
alpha and beta in macrophages from endometriosic
women
S
Capellino [1], S Ferrero [2], B Villaggio
[3]
V Remorgida [2], P Montagna [1], N Ragni [2],
M Cutolo [1]
[1] University of Genoa
Division of Rheumatology DIMI
Genoa, Italy
[2] San Martino Hospital and University of
Genoa Department of Obstetrics and Gynaecology
Genoa, Italy
[3] University of Genoa
Division of Nephrology DIMI
Genoa, Italy
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Introduction
This study aims to determine the expression and
distribution of a and b isoforms of estrogen receptor
(ER) in peritoneal fluid macrophages obtained from
women affected or not by endometriosis.
Materials and methods
Peritoneal fluid samples were obtained from 30 women
with endometriosis and 22 controls (infertility,
n=12; pelvic pain, n=10). Only women of reproductive
age with a menstrual cycle length between 21 and
35 days were included in the study. Exclusion criteria
for the study were: menses at the time of laparoscopy,
signs of pelvic infection, pregnancy or lactation
in the 6 months prior to laparoscopy, previous treatment
with GnRH analogues, and other hormonal therapies
(i.e. oral contraception, ovarian stimulation) in
the 3 months prior to surgery.
Peritoneal fluids were centrifuged at 300 g for
10 min and the contaminant erythrocytes were eliminated
from the cellular pellet by osmotic lysis. Vitality
of freshly isolated macrophages was determined by
trypan blue dye exclusion. The macrophages were
resuspended
in DBPS, placed on glass slides, incubated for 40
min at 4°C, fixed in cold acetone and stored
at -20°C until analysis. Immunohistochemical
localization of two ER subtypes, ER-a and ER-b,
was performed by using respectively a mouse-monoclonal
and a rabbit-polyclonal antibody (Affinity Bioreagents,
CO, USA). In addition, the expression of CD68, NCL-MACRO,
HAM56 (markers of macrophage differentiation) and
of IL-1b, TNF-a, IL-6 was evaluated.
Immunohistochemical staining was performed with
the biotinstreptavidin-peroxidase method. Image
analysis was performed byusing the Leica Q500 MC
image analysis system (Leica, Cambridge, UK); for
the digital image analysis, about 100 cells were
evaluated for each case.
Results
ER-a and ER-b were expressed by macrophages of women,
affected or not, by endometriosis. However, ER-a
was found significantly more expressed than ER-b.
Both ER-a and ER-b were more expressed in macrophages
of women with endometriosis than in controls. When
comparing women with and without endometriosis,
ER-b was found more activated than ER-a, in fact
the ratio (ER-a in macrophages of women with endometriosis/ER-a
in macrophages of controls) was lower than the ratio
(ER-b in macrophages of women with endometriosis/ER-b
in macrophages of controls).
An interesting difference was observed in the distribution
pattern of the two ER isoforms within the macrophages:
ER-a was more localized near the nucleus, whereas
ER-b had a widespread distribution. Macrophages
from both groups expressed differentiation markers
(CD68, NCL-MACRO, and HAM56), but their expression
was significantly higher in macrophages obtained
from women with endometriosis.
A correlation was detected between the expression
of CD68, NCL-MACRO, HAM56 and ER in the macrophages
of women with endometriosis. Peritoneal macrophages
also expressed higher levels of IL-1b, TNF-a, IL-6
in women with endometriosis.
Conclusions
An overexpression of both ER isoforms (a and b)
was observed on macrophages of women with endometriosis.
ER-b seems to have a stronger activation than ER-a
in endometriosis, therefore ER-b might be more relevant
to the pathogenesis of endometriosis.
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