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Phenotypic Characterization of Macrophages in the Endometrium of the Pregnant Cow Lilian J

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Phenotypic Characterization of Macrophages in the Endometrium of the Pregnant Cow Lilian J ORIGINAL ARTICLE Phenotypic Characterization of Macrophages in the Endometrium of the Pregnant Cow Lilian J. Oliveira, Peter J. Hansen Department of Animal Sciences, University of Florida, Gainesville, FL, USA Keywords Problem Cow, endometrium, macrophage, placentome, Macrophages are recruited in large number to the interplacentomal pregnancy endometrium of the cow during pregnancy. We evaluated whether endometrial macrophages also accumulate in placentomal regions of Correspondence Peter J. Hansen, Department of Animal endometrium during pregnancy and whether endometrial macrophages Sciences, University of Florida, PO Box are regionally differentiated. 110910, Gainesville FL 32611-0910 USA. E-mail: [email protected]fl.edu Method of study Interplacentomal endometrium and placentomes were subjected to Submitted June 18, 2009; dual-color immunofluorescence using CD68 as a pan-macrophage accepted September 8, 2009. marker. Citation Results Oliveira L J, Hansen PJ. Phenotypic CD68+ cells were abundant in stroma of the interplacentomal endome- Characterization of macrophages in the trium and caruncular septa of the placentomes. CD68+ cells were not endometrium of the pregnant cow. Am J Reprod Immunol 2009; 62: 418–426 present in fetal villi of the placentomes or in the interplacentomal cho- rion. Regardless of location, the majority of CD68+ cells also expressed + + doi:10.1111/j.1600-0897.2009.00761.x CD14. In interplacentomal endometrium, CD68 CD11b cells were pres- ent in deeper areas of the stroma but not in shallow endometrial stroma. In caruncular septa of the placentome, CD68+ cells were nega- tive for CD11b. CD68+ cells in the interplacentomal endometrium were negative for MHC class II while most CD68+ cells in caruncular septa were positive for MHC class II. Conclusion CD68+CD14+ macrophages present in the stroma of the interplacentomal endometrium and caruncular septa of the placentome are regionally differentiated with regard to expression of CD11b and MHC class II. in number in caruncular septa after experimental Introduction infection with Neospora caninum in the pregnant Recruitment of macrophages to the endometrium is cow.8 Endometrial macrophages could also be key a characteristic of pregnancy in many species includ- regulators of the immunological interaction between ing the mouse,1 human,2–4 cynomologus and vervet mother and conceptus. Human decidual CD14+ cells monkeys,5 sheep6 and cow.7 One function of endo- express low levels of the costimulatory molecule metrial macrophages may be to serve as sentinels to CD86 when compared with cells in blood2 and prevent pathogens from establishing infection or to spontaneously express alternative activation markers clear dead cells. Endometrial macrophages increase interleukin (IL)-10, stabilin-1 and coagulation factor American Journal of Reproductive Immunology 62 (2009) 418–426 418 ª 2009 John Wiley & Sons A/S ENDOMETRIAL MACROPHAGES IN THE COW XIIIa.2,4 The comparison of the global transcriptome pattern of differentiation than the endometrial of circulating CD14+ cells to decidual CD14+ cells in macrophage in the interplacentomal region. To human revealed that decidual CD14+ have a distinct identify macrophages, we used antibody to CD68, a phenotype and express genes for regulatory proteins lysosomal-associated protein that is expressed on such as DC-SIGN.9 A population of DC-SIGN positive monocytes, macrophages and dendritic cells.19 macrophages is also present near the site of placental Expression of three markers by CD68+ cells in the attachment in early pregnancy in cynomologous and endometrium was evaluated to determine whether vervet monkeys.5 It has also been suggested that regional differentiation of endometrial macrophages endometrial macrophages regulate the concentra- occurs. The first marker examined was CD14, a tions of placental lactogen at the uteroplacental co-receptor for bacterial lipopolysaccharide expressed interface by binding of placental lactogen to macro- on monocytes, macrophages and neutrophils.20,21 phage stabilin-1 and subsequent processing of the CD14 is expressed on most interplacentomal CD68+ protein.10 A role in parturition is also possible cells in the pregnant cow.7 The second was CD11b because macrophages accumulate in the uterus near (b2 integrin) that, with CD18, composes the parturition.11,12 macrophage complex-1 (Mac-1) that is involved in The signal or signals that drive the accumulation leukocyte-endothelial adhesion.22 The third mole- and differentiation of macrophages in the pregnant cule was MHC class II, which is involved in antigen endometrium remains unclear. Using the unilater- presentation, and can be upregulated in activated ally-pregnant sheep as a model, there was evidence macrophages and downregulated in inactivated for both local and systemic signals for macrophage macrophages.23 accumulation in the interplacentomal endometrium during pregnancy.6 There are a variety of cytokines Materials and methods produced by the uterus or placenta that can regu- late monocyte or macrophage differentiation. In the Materials cow, these include transforming growth factor-b13, IL-10, IL-12, IL-18, tumor necrosis factor-a and Mouse anti-human CD68 (clone EBM11; ascites, interferon-c.8 2.3 lg ⁄ mL) was obtained from Dako (Carpinteria, In the ruminant, the nature of the relationship CA, USA)6, mouse anti-bovine CD14 (clone between mother and fetus varies regionally within MM61A, ascites, 10 lg ⁄ mL),24 mouse anti-bovine the placenta. Most gas and nutrient exchange occurs MHC class II [a mixture of and equal mass of anti- at specialized structures called placentomes where DR alpha (clone TH14B) and an anti-DQ (clone fetal villi are interdigitated with maternal caruncular TH81A5); ascites, 1 mg ⁄ mL]25 and mouse anti- septa. In the intervening interplacentomal areas, the human CD11b (clone MM12A; ascites, 1 mg ⁄ mL)26 chorioallantois is apposed to the luminal epithelium. were from VMRD (Pullman, WA, USA). IgG controls A synctium forms in the endometrial epithelium as a matching the isotype of each antibody used were result of migration of fetal binucleated cells and their obtained as control mouse ascites fluid (Sigma- fusion with maternal epithelium.14 The immuno- Aldrich, St Louis, MO, USA). The Zenon Alexa Fluor genicity of the placenta differs between placentomal labeling kit and the ProlongÒ Antifade mounting and interplacentomal sites. Chorionic tissue in the medium were obtained from Invitrogen (Eugene, placentome is negative for MHC class I expression OR, USA). throughout pregnancy while chorionic tissue in the The antibodies, including the IgG control, were interplacentomal regions can express MHC class I in tagged with fluorescently-labeled Fab fragments the third trimester of pregnancy.15,16 MHC class I against mouse IgG conjugated using the ZenonÒ proteins on the chorion are predominately encoded Mouse Labeling IgG kits as per manufacturer’s by non-classical MHC class I genes.17 These non- instructions. The anti-CD68 was labeled with Alexa classical MHC class I proteins are possibly analogous Fluor 488, the anti-CD14 was labeled with Alexa to human leukocyte antigen (HLA)-G in humans Fluor 488 or Alexa Fluor 594 for immunofluores- that can regulate natural killer cell and macrophage cence and with Alexa Fluor R-Phycoerythrin for function.18 flow cytometry, and anti-bovine MHC class II and In this study, we hypothesized that the endome- anti-CD11b were labeled with Alexa Fluor 594. trial macrophage in the placentome has a different American Journal of Reproductive Immunology 62 (2009) 418–426 ª 2009 John Wiley & Sons A/S 419 OLIVEIRA AND HANSEN Normal goat serum and fetal bovine serum were washed an additional three times for 5 min each, purchased from Sigma-Aldrich or Pel-Freez Biologi- cover slips were mounted using ProlongÒ Antifade cals (Rogers, AR, USA). The tissue freezing medium reagent and slides were examined using a Zeiss Axi- (Tissue-Tek OCT) was purchased from Sakura Fine- oplan 2 epifluorescence microscope (Zeiss, Gottin- tek USA, Inc. (Torrance, CA, USA). gen, Germany) with a 40· objective and using Zeiss Tissue Culture Medium-199 (TCM-199), bovine filter set 02 (DAPI filter), Zeiss filter set 03 (FITC fil- serum albumin (BSA) Fraction-V, Dulbecco’s phos- ter) and Zeiss filter set 15 (rhodamine filter). Digital phate buffered saline (DPBS), collagenase type I and images were acquired using AxioVision software Hoescht 33342 were purchased from Sigma-Aldrich (Zeiss) and a high-resolution black and white Zeiss (St. Louis, MO, USA). AxioCam MRm digital camera. Tissues Dual-Color Flow Cytometric Analysis for CD68 and CD14 in Dispersed Endometrial Cells Uteri were obtained from pregnant cows of various breeds at a local abattoir. The uterus was transported Two-color immunofluorescence was performed using to the lab on ice within 1.5 hr after slaughter. Fetal preparations of dispersed endometrial cells from the crown-rump length was measured to estimate fetal interplacentomal endometrium of a pregnant cow at age.27 Tissues from a total of 20 pregnant cows an estimated Day 166 of pregnancy. The uterus was (estimated fetal age 137–242 days; mean ± S.D. = obtained at a local abattoir and transported to the lab 182.0 ± 37 days) were collected. Samples of inter- on ice. Interplacentomal endometrium was obtained placentomal endometrium and placentome ipsilateral by dissection. Tissue fragments were collected into a to the corpus luteum were snap-frozen in Tissue-Tek 50 mL sterile culture tube containing 30 mL Tissue OCT embedding compound (see Noden and de Culture Medium (TCM)-199 supplemented with type Lahunta27 and Davies et al.28 for representation of I collagenase at 150 U ⁄ mL and 10% (v ⁄ v) fetal bovine the anatomy of the bovine placenta highlighting fea- serum. Cells were incubated at 37°C for 1 hr under tures of interplacentomal and placentomal endo- gentle rotation. Dispersed cells were then filtered metrium). Samples of lymph node, thymus and through a sterile 200 lm cell strainer into 50 mL ster- spleen were also collected from cows at Day 17 of ile culture tubes and centrifuged at 110 · g for 5 min. pregnancy for use as positive controls.
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