Transcript Leptin on Peritoneal Macrophages of Patients with Endometriosis.

Leptin on Peritoneal Macrophages of
Patients with Endometriosis
Journal of Reproductive Immunology 2010:63:214-221
Presenter : Fellow 洪雅珊
Superviser : VS 盧道權
Introduction
 Endometriosis
 Presence of endometrial tissue outside the uterine cavity
 Common cause of pelvic pain and infertility
 Unclear etiology
 Some kind of immune dysfunction , involving
 Macrophages
 Proinflammatory agents
 Leptin
Literatures review about
immunodysfunction of endometriosis
 No. of macrophages increased in peritoneal fluid of pts
with endometriosis . (Badawy, 1984; Wu MH, 2002)
 Peritoneal macrophages in pts with endometriosis have
higher inflammatory agents producing ability and poor
cytolytic capability. (Steele, 1984; Dmowski,1998; Chuang,2009)
 Hyperactivation of macrophage → PGE2↑→growth of
ectopic endometriotic stromal cell ↑(Wu, 2002; Chuang, 2006)
 Cytotoxic function of peritoneal macrophage is affected
with the stage of endometriosis and PG metabolism.
(Barun,1992)
Literatures about leptin in endometriosis
 Multifunctional hormone : immunoregulatory,
proinflammatory, angiogenic effect
 Significant elevated leptin in ectopic endometriotic cells >
normal eutopic endometrial tissue . (Wu,2002)
 Leptin expression in endometriotic tissue↑→ hypoxia
inducible factor-1a↑→peritoneal fluid leptin level↑(Wu, 2007)
 Macrophage is the target of leptin. (O’Rourke , 2001)
 Effect of leptin on peritoneal macrophage activity ?
Aim of this study
 Investigate whether hyperactivation of macrophages in pts
with endometriosis was mediated by exposure to leptin in
peritoneal fluid.
 Hypothesis of this study
 Leptin has an unantipated role in modulating the
functions of peritoneal macrophages , which might
contribute to the pathophysiological process of
endometriosis .
Materials and methods
Patients
 60 women , aged 20-38 y/o, underwent laparoscopy
 Control : 20 pts without endometriosis
 Study : 40pts with endometriosis ( 20pts stage I/II; 20 pts
stage III/IV)
 Extent of endometriosis : direct vision under laparoscopy
 Stage of endometriosis : revised classification of ASRM
 All pts
 Normal mens cycle
 Not have any endocrine disease or receive any hormone
therapy at least 6 months before
Isolation and culture of monocytes/
Macrophages from peritoneal fluid
 Peritoneal fluid collection : use aspiration needle under
direct vision at the time of laparoscopy.
 Macrophage isolation and purification ( described in
previous report)
 Purified peritoneal mononuclear cells incubated with
various leptin tx.
 Direct lysed the cell and subjected to protein analysis
ELISA
 Commercial leptin kit
 The detection limit of leptin : 1.0ng/ml
 Intra-assay coefficient of variance : 4.6%
 Inter-assay coefficient : 9.6%
 Detected conc of PGF2α in the lab
 Sensitivity (80% bound) of PGF2α assay : 24.7pg/ml
 Intra-assay coefficient : 5.2%
 Inter-assay coefficient : 11.2%
RNA isolation and RT- PCR
 Total RNA was isolated from peritoneal macrophages by
Rneasy mini kit
 RT-PCR → Detect total form ( T-OBR) and long form
(L-OBR) mRNA expression of leptin receptor.
Western blotting
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COX -1
COX -2
IL-1β
IL-1 receptor antagonist (IL-1Ra )
Signal transducers and activators of transcription (STAT)3
STAT3 phosphorylation
Statistical analysis
 ANOVA : differences between and among disease
subgroups or treatment groups
 Duncan’s procedure : post-test multiple comparisons
 Student’s t-test : 2 groups comparison
 P < 0.05
Results
 Peritoneal fluid leptin between the follicular and luteal phases
→ No significant differences
 Age, weight, height, BMI → no significant different
 Peritoneal fluid leptin significantly increased in endometriosis
group ( both early and advanced stages )
 The conc was not different between early and advanced stages.
Effect of leptin on protein expression
in peritoneal macrophages
 Evaluated the presence of mRNA encoding leptin R on
peritoneal macrophages .
 long form R ( use RT-PCR) →Functional →leptin
could affect the peritoneal macrophages.
 Leptin + peritoneal macrophage →evaluated the expression of
COX-1 and COX-2
 COX-2 : increased after leptin stimulation →decreased
12hr later → more obvious 24hr later
 COX-1 : continuous expression and increased after 24 hr
treated with leptin
COX-2 expression after
different doses of leptin stimulation
 Expression of protein ratio was
not different between normal
and endometriotic group after
12hr leptin tx.
 COX-2 expression
significantly increased in
endometriotic group only after
high-dose ( 1000ng/ml) leptin
stimulation.
 COX-2 was not significantly
increased in normal control
→may be attributable to wide
variation in the values after
1000ng/ml leptin tx.
Effect of Leptin on PGF2α
 Evaluate the association with
leptin and pelvic pain
 Endometriotic group : PGF2α in
peritoneal marcophages were
markedly induced by leptin .
 Early & advanced stage : no
difference in conc of PGF2α
 Control : no effect
Effects of leptin on COX-1, IL-1
 COX-1 : no dose-dependent changes after leptin tx
 COX-2 : expressed obviously in high does of leptin tx
 IL-1β and IL-1Ra : not affected by leptin tx in both control and
study group
Effects of leptin on STAT3
 Total - STAT 3 and STAT 3 phosphorylation
→ not increased by leptin tx in both control and study
group.
Discussion
Leptin in endometriosis
 In animal study (Styer, 2008)
 Involved lesion proliferation and survival , early vascular
recruitment and neoangiogenesis.
 In human studies
 Leptin increase in peritoneal fluid of pts with
endometriosis. (Matarese G, 2000)
 Peritoneal endometriosis (+) →influences leptin conc in
peritoneal fluid . (De Placido , 2001)
 Peritoneal implants + ov endometrioma →peritoneal fluid
leptin positively correlated with the stage of
endometriosis (Bedaiwy, 2006)
Leptin in endometriosis
 In this study
 Peritoneal fluid leptin was increased in women with
endometriosis .
 A majority of pts with advanced stage associated with
peritoneal lesions
→agreed with the above reports .
 Leptin receptor expressed in peritoneal macrophages in
pts with or without endometriosis
→ peritoneal leptin may be involved in the immune
function of peritoneal macrophages.
Leptin vs PGF2α
 PGF2α : smooth muscle contractile agent, etiology of
dysmenorrhea
 Increase peritoneal fluid leptin associated with pelvic pain
in endometriotic women. ( Bedaiwy, 2006)
 This study : PGF2α increased after leptin tx .
 Previous study : peritoneal PG increased in endometriotic
women
→ PGF2α continued to elevate after leptin tx in peritoneal
macrophages especially in endometriotic pts , which was
different from PG producing after IL-1β and TNF - α
stimulation in previous report .
Leptin vs COX-1 and COX-2
 Increased COX-2 in peritoneal endometriosis is highly
correlated with chronic pelvic pain . (Buchweitz, 2006)
 In this study
 COX-2 in peritoneal macrophage was increased with
the highest expression at 4hr that may be transient after
leptin tx
→ leptin did not induce COX-2 continuously
 COX-1 was expressed continuously →may play a role
in the progressive increase in PGF2α
Leptin vs COX-1 and COX-2
 Leptin induced COX-2 and/or COX-1 on peritoneal
macrophage → in turn , PGF2α production
 COX-2 increased only in high dose of leptin , COX-1 kept
expression in different dose of leptin
→may more cytokines or growth factor leading to
increased COX in the pathogenesis of endometriosis .
 Leptin was one of the factors that regulates the peritoneal
macrophage in endometriosis .
 Leptin tx → COX-2 and PG↑ →may play an
immunomodulatory role on peritoneal activated
macrophages→ affect the secretion of proinflammatory
cytokines or upregulate of activation markers
Leptin vs IL-1
 Leptin induces the IL-1Ra , without effect on IL-1, in
human monocytes via functional leptin receptor . (Gabay, 2001)
 In this study
 IL-1b and IL-1Ra were not affected by leptin .
 STAT 3 was not affected with dose or time
 Functional leptin receptor on peritoneal macrophages did
not affect COX-2 expression .
→Peritoneal fluid leptin did not regulate macrophage
of secretion of proinflammatory cytokines or up
regulate activation markers .
→Leptin-stimulated PGF2α was not through the
induction of IL-1 signal pathway.
Conclusion
 The mechanism in the involvement of other factors in
mediating leptin action on PG producing capacity →
need more studies
 This study confirmed
→leptin had a significant and direct effect on COX-2
and PGF production in peritoneal macrophages
→Leptin may play an immunomodulatory role in the
local inflammatory function in the pathogenesis of
endometriosis .
Thank you !!!