Leptin on Peritoneal Macrophages of Patients with Endometriosis

Presenter : Fellow 洪雅珊Superviser : VS 盧道權

Journal of Reproductive Immunology 2010:63:214-221

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 functi

ons 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 st

age 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

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 gr

oup ( 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 co

uld affect the peritoneal macrophages.

Leptin + peritoneal macrophage →evaluated the expression of COX-1 and COX-2 COX-2 : increased after leptin stimulation →decreased 12hr lat

er → 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 endometrios

is. (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 t

he 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 macro

phage → 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 hum

an 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 n

ot 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 !!!