From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 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 From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 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.) From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 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. REFERENCES 1. Haney AF, Muscat0 JJ, Weinberg JB: Peritoneal fluid cell 7. Wing ET, Ampel NM, Waheed A, Shadduck R K Macropopulations in infertility patients. Fertil Steril35:696, 1981 phage colony-stimulating factor (M-CSF) enhances the capacity of 2. Halme J, Becker S, Hammond MG, Raj S: Pelvic macromurine macrophages to secrete oxygen reduction products. J phages in normal and infertile women: The role of patent tubes. Immunol135:2052,1985 Am J Obstet Gynecol142:890,1982 8. Warren MK, Ralph P: Macrophage growth factor CSF-1 3. Olive DL, Weinberg JB, Haney AF: Peritoneal macrophages stimulates human monocyte production of interferon, tumor necroand infertility: The association between cell number and pelvic sis factor, and colony stimulating activity. J Immunol 1372281, pathology. Fertil Steril44:772, 1985 1986 4. Metcalf D: The molecular control of cell division, differentia9. Lyberg T, Stanley ER, Prydz H: Colony-stimulating factor-1 tion, commitment, and maturation in hematopoietic cells. Nature induces thromboplastin activity in murine macrophages and hu339:27, 1989 man monocytes. J Cell Physiol 132:367, 1987 5. Ralph P, Sampson-Johannes A Macrophage growth and 10. Sherr CJ, Rettenmier RW, Sacca R, Roussel MF, Look AT, stimulating factor, M-CSF, in Spivak J, Drohan W, Dooley D (eds): Stanley ER: The c-fms proto-oncogene product is related to the Hematopoietic Growth Factors in Transfusion Medicine. New receptor for the mononuclear phagocyte growth factor, CSF-1. Cell York, NY,Wiley-Liss, 1990, p 43 41:665,1985 6. Ramakrishnan S, Xu FJ, Brandt SJ, Niedel JE, Bast RC Jr: 11. Yam LT, Li CY, Crosby WH: Cytochemical identification of Constitutive production of macrophage colony-stimulating factor monocytes and granulocytes. Am J Clin Pathol55:283,1970 by human ovarian and breast cancer cell lines. J Clin Invest 83:921, 1989 12. Acosta AA, Buttram VC, Besch PK, Malinak LR, Franklin From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 516 RR, Vanderheyden JD: A proposed classification of endometriosis. Obstet Gynecol42:19, 1973 13. Muscato JJ, Haney AF, Weinberg JB: Sperm phagocytosis by human peritoneal macrophages: A possible cause of infertility in endometriosis. Am J Obstet Gynecol 144:503,1982 14. Olive DL, Haney AF, Weinberg JB: The nature of the intraperitoneal exudate associated with infertility: Peritoneal fluid and serum lysozyme activity. Fertil Steril48:802, 1987 15. Haney AF, Misukonis MA, Weinberg JB: Macrophages and infertility: Oviductal macrophages as potential mediators of infertility. Fertil Steril39:310, 1983 16. Halme J: Release of tumor necrosis factor-alpha by human peritoneal macrophages in vivo and in vitro. Am J Obstet Gynecol 161:1718,1989 17. Fakih H, Baggett B, Holtz G , Tsang K-Y, Lee JC, Williamson HO: Interleukin-1: A possible role in the infertility associated with endometriosis. Fertil Steril47:213, 1987 18. Eisermann J, Gast MJ, Pineda J: Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery. Fertil Steril50:573,1988 19. Hill J A The clinical significance of cytokines in female reproductive failure, in Mettler L, Billington WD (eds): Reproductive Immunology 1989. New York, NY,Elsevier Science, 1990, p 299 20. Halme J, Becker S, Haskill S: Altered maturation and function of peritoneal macrophages: Possible role in pathogenesis of endometriosis. Am J Obstet Gynecol 156:783,1987 21. Fibbe WE, van Damme J, Billau A, Duinkerken N, Lurvink E, Ralph P, Altrock BW, Kaushansky K, Willemze R, Falkenburg JHF: Human fibroblasts produce G - , M- and GM-CSF following stimulation by IL-1 and poly IC. Blood 72:860, 1988 22. Fibbe WE, Daha M, Hiemstra PS, Duinkerken N, Lurvink E, Ralph P, Altrock BW, Kaushansky K, Willemze R, Falkenburg J H F IL-1 and poly IC induce production of G-, M- and GM-CSF by human endothelial cells. Exp Hematol 17:229,1989 23. Pollard JW, Bartocci A, Arceci R, Orlofsky A, Ladner MB, WEINBERG ET AL Stanley ER: Apparent role of the macrophage growth factor, CSF-1, in placental development. Nature 330:484, 1987 24. Azuma C, Saji F, Kimura T, Tokugawa Y, Takemura M, Samejima Y, Tanizawa 0: Steroid hormones induce macrophage colony-stimulating factor (MCSF) and MCSF receptor mRNAs in the human endometrium. J Mol Endocrinol5:103, 1990 25. Weinberg JB, Haney A F Spontaneous tumor cell killing by human blood monocytes and human peritoneal macrophages: Lack of alteration by endotoxin or quenchers of reactive oxygen species. J Natl Cancer Inst 70:1005,1983 26. Kurlander RJ, Haney AJ, Gartrell J: Human peritoneal macrophages possess two populations of IgG Fc receptors. Cell Immunol86:479,1984 27. Haskill S, Becker S, Jontell M, Sporn S, Halme J: Normal human peritoneal macrophages are unable to cap and internalize class I1 antigens. Cell Immunol 115:100, 1988 28. Conkling PR, Chua CC, Nadler P, Greenberg CS, Doty E, Misukonis MA, Haney AF, Bast RC Jr, Weinberg JB: Clinical trials with human tumor necrosis factor: In vivo and in vitro effects on human mononuclear phagocyte function. Cancer Res 48:5604, 1988 29. Becker S, Johnson C, Halme J, Haskill S: Interleukin-1 production and antigen presentation by normal human peritoneal macrophages. Cell Immunol98:467,1986 30. Wang JM, Griffin JD, Rambaldi A, Chen ZG, Mantovani A Induction of monocyte migration by recombinant macrophage colony-stimulating factor. J Immunol141:575,1988 31. Gilbert HS, Praloran V, Stanley ER: Increased circulating CSF-1 (M-CSF) in myeloproliferative disease: Association with myeloid metaplasia and peripheral bone marrow extension. Blood 74:1231,1989 32. Kacinski BM, Stanley ER, Carter D, Chambers JT, Chambers SK, Kohorn EI, Schwartz PE: Circulating levels of CSF-1 (M-CSF) a lympho-hematopoietic cytokine may be a useful marker of disease status in patients with malignant ovarian neoplasms. Int J Radiat Oncol Biol Phys 17:159,1989 From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 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 Updated information and services can be found at: http://www.bloodjournal.org/content/78/2/513.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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