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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

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Leptin on Peritoneal Macrophages of Patients with Endometriosis

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  1. 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

  2. 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

  3. 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)

  4. 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 ?

  5. 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 .

  6. Materials and methods

  7. 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

  8. 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

  9. 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%

  10. 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.

  11. Western blotting • COX -1 • COX -2 • IL-1β • IL-1 receptor antagonist (IL-1Ra ) • Signal transducers and activators of transcription (STAT)3 • STAT3 phosphorylation

  12. 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

  13. Results

  14. Peritoneal fluid leptin between the follicular and luteal phases → No significant differences

  15. 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.

  16. 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.

  17. 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

  18. 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. COX-2 expression after different doses of leptin stimulation

  19. 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

  20. 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

  21. Effects of leptin on STAT3 • Total - STAT 3 and STAT 3 phosphorylation → not increased by leptin tx in both control and study group.

  22. Discussion

  23. 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)

  24. 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.

  25. 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 .

  26. 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

  27. 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 .

  28. 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

  29. 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.

  30. Conclusion

  31. 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 .

  32. Thank you !!!

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