- Email: [email protected]
Ectopic, autologous eutopic and normal endometrial stromal cells have altered expression and chemotactic activity of RANTES
European Journal of Obstetrics & Gynecology and Reproductive Biology 143 (2009) 55–60
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Ectopic, autologous eutopic and normal endometrial stromal cells have altered expression and chemotactic activity of RANTES Fang Chun-Li, Han Su-Ping, Fu Shi-Long, Wang Wei, Kong Na, Wang Xiu-Li* Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, 99 Ding Huai Men Street, 210036 Nanjing, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 March 2008 Received in revised form 28 September 2008 Accepted 1 December 2008
Objective: To evaluate if the expression and chemotactic activity of RANTES are different in IL-1b treated autologous eutopic endometrial stromal cells compared to ectopic and normal endometrium. Study design: Conditioned media from IL-1b-treated ectopic, autologous eutopic and normal endometrial stromal cells were analyzed with a specific sandwich ELISA to quantify RANTES. The monocyte chemotactic activity of RANTES was assayed in a Boyden Chamber. Results: RANTES expression in IL-1b-treated autologous eutopic and normal endometrial stromal cells was significantly lower than ectopic endometrium. Autologous eutopic endometrial stromal cells showed a significant increase in RANTES expression compared to normal endometrium after IL-1b stimulation for 60 h. The monocyte chemotactic activities of these conditioned media were highly correlated with the immunoreactive RANTES concentration. We observed significantly increased monocyte chemotactic activity in conditioned media of ectopic stromal cells compared to autologous eutopic and normal endometrium. The different chemotactic activity of RANTES between the autologous eutopic and normal endometrial stromal cells was also statistically significant. RANTES accounts for the majority (62%) of the monocyte chemotactic activity in ectopic endometrial stromal cells conditioned media and 55% of that activity in autologous eutopic endometrium. Conclusions: Although the eutopic endometric of women with and without endometriosis are histologically similar, our findings confirm that different expression and chemotactic activity of RANTES exist between autologous eutopic and normal endometrium. The altered expression of RANTES and monocyte chemotactic activity observed in ectopic, autologous eutopic and normal endometrium suggest the autologous eutopic endometrium may contribute to the pathogenesis of endometriosis. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: RANTES Ectopic endometrium Eutopic endometrium Macrophage
1. Introduction The inflammatory milieu in the peritoneal cavity of women with endometriosis has been extensively characterized. It was noted that the macrophage was the most abundant leukocyte in the pelvic fluid of women with endometriosis and plays a central role in the immunobiology of endometriosis [1]. Peritoneal fluid specimens from women with endometriosis have elevated levels of a variety of cytokines, including IL-1b, tumor necrosis factor a (TNF-a), IL-6, IL-8, and RANTES (regulated on activation, normal T cell expressed and secreted), compared to normal controls [2]. RANTES is an 8-kDa secreted protein with chemoattractant actions on monocytes, natural killer cells, T cells, and eosinophils. Hornung et al. reported that the concentration of RANTES is elevated in the peritoneal cavity of women with endometriosis and correlates
with the severity of the disease [3]. Hornung et al. have shown that a predominant activated macrophage product, IL-1b, induces RANTES gene expression in ectopic endometrial stromal cells [4]. Although the endometria of women with and without endometriosis are histologically similar, studies revealed that there are many differences between these two tissues [5]. In the current study, we examined the expression and chemotactic activity of RANTES in IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells. Our results indicated that there were significant differences of RANTES expression and chemotactic activity among ectopic, autologous eutopic and normal endometrium. 2. Materials and methods 2.1. Sources of tissues
* Corresponding author. Tel.: +86 13813382652; fax: +86 25 86227420. E-mail address: [email protected] (X.-L. Wang). 0301-2115/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2008.12.001
Ovulatory women, who had not received hormones or GnRH agonist therapy for at least 3 months before surgery, were
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W. Wang, N. Kong / European Journal of Obstetrics & Gynecology and Reproductive Biology 143 (2009) 55–60
recruited after they had provided written informed consent under a study protocol approved by the First Affiliated Hospital of Nanjing Medical University. Ectopic and autologous eutopic endometrium were obtained from women with ovarian endometriomata (n = 11, stage III or IV) who have been identified pathologically and staged according to a modification of the revised American Fertility Society system. Control endometrial tissue was obtained from five healthy women undergoing removal of intrauterine device. All of the tissues were acquired during the midproliferative phase of the menstrual cycle. Although most of the endometrial biopsies immediately after IUD removal showed inflammatory changes and edema consistent with the local effect of the IUD as it has been previously reported, Horcajadas et al. used a microarray containing more than 16,000 cDNAs to investigate the gene expression profile of receptive versus refractory endometrium in the same women induced by the presence of an IUD and did not find that the gene expression profiles of IL-1b and RANTES significantly dysregulated [6]. So the endometria obtained from five healthy women undergoing removal of intrauterine device were used as control in our study. 2.2. Cell isolation, purification and culture Endometrial and endometrioma biopsies were collected under sterile conditions and transported to the laboratory in DMEM (GIBCO) with 10% FCS. The fresh tissue was cut with scissors into small pieces of approximately 1 mm3, and rinsed carefully to remove blood. Digestion of the tissue was then performed for 1 h at 37 8C, with shaking every 15 min in 0.125% type IA collagenase (Sigma). Debris was removed by 100-mm aperture sieves, and epithelial glands were retained on 40-mm aperture sieves. Stromal cells remaining in the filtrate were plated onto 6-cm dish and allowed to adhere for 45 min, after which blood cells were removed with phosphate-buffered saline rinses. The cells were cultured in DMEM with 10% FCS, penicillin G (100 U/ml) and streptomycin (100 mg/ml). Exogenous steroids were not added to the cultures. Stromal cultures were dissociated with 0.05% trypsin and 0.02% versene in saline, harvested by centrifugation, replated, and allowed to grow to confluence. Purification of the stromal cell population was confirmed by positive staining for mouse anti-Vimentin antibody (Zymed Laboratories Inc.) and negative staining for mouse anti-cytokeratin 17 antibody (Zymed Laboratories Inc.).
compared to reference standards of recombinant human RANTES (R&D Systems). 2.5. Monocyte chemotaxis assay RANTES bioactivity was assessed by a chemotaxis assay using a human histiocytic cell line (U937, purchased from the Cell Bank of the Chinese Academy of Science). U937 cells were treated with 1 mM 8-bromo-cAMP for 48 h to induce a monocyte phenotype and up-regulate oligopeptide chemoattractant receptors. For the measurements, 24-well plates assembled with Boyden chambers (0.4-mm pore size polycarbonate membranes) were used. Sample (600 ml) containing the chemotactic peptide N-formyl-methionylleucyl-phenylalanine (0.1 mM fMLP) (Sigma) in PBS was placed in the lower chamber as positive control for cell migration. Supernatants from IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells were added to the lower chamber to test its activity. After assembly of the Boyden chambers, 200 ml suspensions of U937 cells (1 105 cells) in PBS were placed in the upper chamber. The chambers were then incubated in a humidified CO2 incubator at 37 8C for 90 min. Nonmigrating cells were removed by washes with PBS, and cells migrating across the membrane were fixed, stained, and counted directly. The chemotactic index (CI) was expressed as the multiple of chemotaxis obtained in response to a minimal stimulation with OPTI-MEM alone. To evaluate the monocyte chemotactic activity of RANTES in supernatants from IL-1b treated ectopic and autologous eutopic endometrial stromal cells, the 60-h conditioned media were incubated with serial dose (0.5, 1, 2, 4 and 8 mg/ml) goat antihuman RANTES IgG (R&D Systems) for 30 min at 37 8C before chemotaxis assay, and the chemotactic activity was measured as described above. The goat anti-human RANTES IgG was highly specific for RANTES and not cross-react with monocyte chemotactic protein (MCP)1-3, macrophage inflammatory protein(MIP)1a or b, or many other human chemokines. 2.6. Statistical analysis Software SPSS 11.0 was used in the statistical analysis. Each parameter was presented as mean S.D., and compared using oneway ANOVA analysis of variance. The level of significance was set at P < 0.05.
2.3. Cytokine treatment of cell cultures
3. Results
When the primary stromal cells cultured in six-well plates approached confluence, the complete medium was removed and replaced with fresh low serum Opti-MEM (GIBCO) containing 2.5% FCS for an additional 48 h. Ectopic endometrial stromal cells were treated with IL-1b (Sigma) at 0, 5, 10 and 20 ng/ml dose. Conditioned media were removed and analyzed after 12, 24, 36, 48, 60, 72, 84 and 96 h. Dose–response experiments indicated that 10 ng/ml of IL-1b induced a maximal RANTES response in ectopic endometrial stromal cells. So in the following experiments, the 10 ng/ml of IL-1b was used to induce the expression of RANTES in cultured ectopic, autologous eutopic and normal endometrial stromal cells.
3.1. Immunocytochemistry of purified endometrial stromal cell populations
2.4. Rantes ELISA A specific sandwich ELISA (Quantikine, R&D Systems) was used to quantify RANTES in conditioned media. The assay is specific for human RANTES, with no known cross-reactivity with other cytokines or chemokines. Aliquots of culture supernatants were each tested in duplicate at several dilutions and
The morphologies of ectopic (Fig. 1A), autologous eutopic (Fig. 1B) and normal (Fig. 1C) endometrial stromal cells were polygonal or spindle-shaped. Generally, the size of ectopic endometrial stromal cells was larger than that of autologous eutopic and normal endometrium. The primary ectopic endometrial stromal cells proliferate more slowly compared to autologous eutopic and normal endometrium. Stromal cells were confirmed by positive staining with vimentin antibody (Fig. 1D and E) and negative staining with cytokeratin antibody (Fig. 1F). Extensive characterization of cell cultures prepared using this protocol confirmed that the stromal cells were >95% pure. 3.2. RANTES expression in conditioned media from IL-1b-treated ectopic, autologous eutopic and normal endometrial stromal cells Ectopic endometrial stromal cells were stimulated with IL-1b at 0, 5, 10 and 20 ng/ml dose.
W. Wang, N. Kong / European Journal of Obstetrics & Gynecology and Reproductive Biology 143 (2009) 55–60
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Fig. 1. The morphologies and immunocytochemistry of purified ectopic, autologous eutopic and normal endometrial stromal cells. The morphologies of ectopic (A), autologous eutopic (B) and normal (C), endometrial stromal cells were polygonal or spindle-shaped. Stromal cells were confirmed by positive staining with vimentin antibody (D and E) and negative staining with cytokeratin antibody (F). Vimentin is a member of the intermediate filament family of proteins (D).
As shown in Fig. 2, dose–response experiments showed a maximum stimulation after treatment with IL-1b of 10 ng/ml. Culturing the cells in the presence of IL-1b (10 ng/ml) for 24 h dramatically (P < 0.01) induced RANTES expression in ectopic endometrial stromal cells (Fig. 3), but not in autologous eutopic and normal endometrial stromal cells. Autologous eutopic endometrial stromal cells showed a significant increase (P < 0.05) in RANTES secretion compared to normal endometrium after IL-1b stimulation for 60 h (Fig. 3). 3.3. The chemotactic activity of conditioned supernatants from IL-1btreated ectopic, autologous eutopic and normal endometrial stromal cells Conditioned media from IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells were used for the chemotactic assay in a Boyden chamber. As shown in Fig. 4, the monocyte chemotactic activity of these conditioned media was highly correlated with the immnoreactive RANTES concentration. We observed significantly increased (P < 0.05) monocyte chemotactic activity in conditioned media of ectopic stromal cells compared to autologous eutopic and normal endometrium (Fig. 5).
Fig. 2. RANTES expression in supernatants of ectopic endometrial stromal cells treated with IL-1b at 0, 5, 10 and 20 ng/ml. Dose–response experiments showed a maximum stimulation after treatment with IL-1b of 10 ng/ml. *P < 0.05 vs. 10 ng/ ml of IL-1b; **P < 0.01 vs. 5 ng/ml of IL-1b.
The difference between the autologous eutopic and normal endometrial stromal cells was also statistically significant (P < 0.05) (Fig. 5). To evaluate the contribution of RANTES to the total monocyte chemotactic activity in the conditioned media, we added specific RANTES-neutralizing monoclonal antibodies at serial dose (0.5, 1, 2, 4 and 8 mg/ml) into 60 h conditioned media of ectopic endometrial stromal cells and found that 4 mg/ml of anti-RANTES antibody almost completely abolished the chemotactic activity of RANTES. The 4 mg/ml of anti-RANTES antibody inhibited 62% of the chemotactic activity in conditioned medium of ectopic endometrial stromal cells (Fig. 6) and 55% of that activity in autologous eutopic endometrial stromal cells (Fig. 6). 4. Discussion Endometriosis is a common gynecological disorder, affecting at least 10% of reproductive-aged women. It is characterized by the
Fig. 3. RANTES expression in conditioned media from ectopic, autologous eutopic and normal endometrial stromal cells stimulated with 10 ng/ml of IL-1b. Culturing the cells in the presence of IL-1b (10 ng/ml) for 24 h dramatically (P < 0.01) induced RANTES expression in ectopic endometrial stromal cells, but not in autologous eutopic and normal endometrial stromal cells. Autologous eutopic endometrial stromal cells showed a significant increase (P < 0.05) in RANTES secretion compared to normal endometrium after IL-1b stimulation for 60 h. The data represent the mean S.D. of different subjects in each group. *P < 0.05 vs. normal endometrium; **P < 0.01 vs. autologous eutopic and normal endometriums.
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Fig. 4. The monocyte chemotactic activity of the conditioned media of 60 h from IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells. Conditioned media of 60 h from IL-1b treated ectopic (A), autologous eutopic (B) and normal endometrial (C) stromal cells were used for the chemotactic assay in a Boyden chamber. The monocyte chemotactic activity of these conditioned media was highly correlated with the immnoreactive RANTES concentration. The negative control with Opti-MEM was shown in D.
growth of endometrial tissue outside the uterine cavity. Although multiple theories exist regarding the etiology of endometriosis, the implantation hypothesis of Sampson is the most widely accepted. Since retrograde menstruation is a very common phenomenon among women of reproductive age, there must be other factors that may contribute to the pathophysiology and/or pathogenesis of endometriosis. Genetic predisposition, environmental factors, and alterations in immune and endocrine functions are believed to play significant roles in the establishment and maintenance of endometriosis [7–9]. IL-1, a principal macrophage-derived and
major proinflammatory cytokine, may play a central role in the integrated inflammatory cascade associated with endometriosis and in propagating endometriotic implants through proinflammatory stimuli and synthesis of chemokines, growth factors and angiogenic factors, such as RANTES. Hornung et al. reported peritoneal implants may contribute to the increased pelvic fluid concentrations of RANTES seen in patients with endometriosis and the secretion of RANTES by ectopic implants provides a mechanism for peritoneal macrophage recruitment [3,10]. It is known that ectopic and eutopic endometrium from women with
Fig. 5. The chemotactic activity of conditioned supernatants from IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells. The data represent the mean S.D. of different subjects in each group. The chemotactic index (CI) was expressed as the multiple of chemotaxis obtained in response to a minimal stimulation with OPTI-MEM alone. The CI in conditioned media of ectopic stromal cells significantly increased compared to autologous eutopic and normal endometrial stromal cells. The difference between the autologous eutopic and normal endometrial stromal cells was also statistically significant. *P < 0.05 vs. normal endometrium; **P < 0.01 vs. autologous eutopic endometrium.
Fig. 6. The monocyte chemotactic activity of RANTES in conditioned medium of ectopic and autologous eutopic endometrial stromal cells. The specific RANTESneutralizing monoclonal antibodies at serial dose(0.5, 1, 2, 4 and 8 mg/ml) were added into conditioned medium of 60 h. The 4 mg/ml of anti-RANTES antibody inhibited 62% of the chemotactic activity in conditioned medium of ectopic endometrial stromal cells and 55% of that activity in autologous eutopic endometrial stromal cells. *P < 0.05 vs. 8 mg/ml of anti-RANTES antibody. **P < 0.05 vs. 8 mg/ml of anti-RANTES antibody.
W. Wang, N. Kong / European Journal of Obstetrics & Gynecology and Reproductive Biology 143 (2009) 55–60
endometriosis presents physiological differences with normal endometrium [11–13]. Do differences in expression and chemotactic activity of RANTES exist among ectopic, autologous eutopic and normal endometrium? In this study, we show that the expression and chemotactic activity of RANTES are increased in the ectopic and autologous eutopic endometrium when compared with normal endometrium under the stimulation of IL-1b. In agreement with previous reports, our data showed that the RANTES secreted by ectopic endometrial stromal cells increased significantly compared to normal endometrium after the stimulation of IL-1b. Moreover, we found that the RANTES secreted by autologous eutopic endometrial stromal cells was significantly higher than the normal endometrium and significantly lower than the ectopic endometrium. Until now, it is not clear why misplaced endometrial cells from healthy women do not implant and do not develop into endometriotic lesions as occurs in women that develop endometriosis, indicating that some factor which facilitates their survival and implantation may be involved. Ours and other previous studies showed augmented sensitivity of ectopic and eutopic endometrial cells to the biological actions of IL-1b. These cells displayed increased secretion of RANTES in response to IL-1b. The mechanisms underlying such an accentuated imbalance in the ectopic and autologous eutopic endometrial tissue remain unknown. IL-1 has two known receptors, now designated as IL-1R1 and IL-1R2. Cell activation by IL-1 results from its binding to IL-1R1 which in concert with IL-1R accessory protein (IL-1RAP) is capable of transducing the activation signal. The IL-1R2 has, in contrast to IL-1R1, no signaling properties, but has been described as a potent, specific and natural inhibitor of IL-1. Akoum et al. found that an imbalance in IL-1R1 and IL-1R2 expression occurring in the eutopic endometrium of women with endometriosis and more markedly in the ectopic endometrial tissue. A decreased IL-1R2 expression is predominant in the eutopic endometrium, a concomitant increase in IL-1R1 expression occurs in ectopic endometrial tissue, particularly in the initial and most active implants [14]. Over expression of the activating IL-1R1 combined with depression of the decoy IL-1R2 may represent a key mechanism underlying the augmented sensitivity of ectopic and eutopic endometrial cells to the biological actions of IL-1b. RANTES is a cytokine of the b or ‘‘C–C’’ chemokine family. It is a chemoattractant for monocytes and memory T-cells and may be an important mediator in both acute and chronic inflammation [15,16]. Macrophages play a central role in the immunobiology of endometriosis. Interactions between the endometriotic cells and macrophages are mediated by growth factors, cytokines, and chemokines, leading to the paradoxical survival of implants rather than their demise [17,18]. In our study, the altered expression of RANTES observed in autologous eutopic endometrium suggests their participation in the pathogenesis of endometriosis. Using the U937 chemotaxis assay in a Boyden chamber, our current investigation corroborated the biological activity of RANTES as a monocyte chemokine secreted by cultured endometrial stromal cells, and particularly ectopic and autologous eutopic endometrium. As shown in Fig. 4, the monocyte chemotactic activity of these conditioned media was highly correlated with the immunoreactive RANTES concentration. We observed significantly increased monocyte chemotactic activity in conditioned media of ectopic stromal cells compared to autologous eutopic and normal endometriums. The difference between the autologous eutopic and normal endometrial stromal cells was also statistically significant. To evaluate the contribution of RANTES to the total monocyte chemotactic activity in the conditioned media, we added specific RANTES-neutralizing monoclonal antibodies at serial dose into conditioned media of 60 h and found that 4 mg/ml of antiRANTES antibody almost completely abolished the chemotactic activity of RANTES. We established that RANTES accounts for the
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majority (62%) of the monocyte chemotactic activity in ectopic endometrial stromal cells conditioned media and 55% of that activity in autologous eutopic endometrium. Other monocyte chemokines are likely to contribute the remaining activity of macrophage recruitment. Although the eutopic endometric of women with and without endometriosis are histologically similar, our findings confirm that differential expression and chemotactic activity of RANTES exist between autologous eutopic and normal endometrium. The feedforward inflammatory loop whereby IL-1b from activated macrophages may lead to RANTES production by ectopic and autologous eutopic endometrium and further monocyte chemotaxis facilitates the establishment, progression and inflammatory sequelae of endometriotic tissues. The altered expression of RANTES and monocyte chemotactic activity observed in autologous eutopic endometrium suggests they may contribute to the pathogenesis of endometriosis. Future clinical strategies aimed at neutralizing potentially pathological factors in the endometriosis syndrome must not lose sight of the possible importance of these molecules in normal endometrial physiology. Acknowledgements This work is supported by National Natural Science Foundation of China No. 30600671 (to Wang Xiu-Li), Natural Science Foundation of Jiangsu province No. BK2006577 (to Wang Xiu-Li). References [1] Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75(1):1–10. [2] Kalu E, Sumar N, Giannopoulos T, et al. Cytokine profiles in serum and peritoneal fluid from infertile women with and without endometriosis. J Obstet Gynaecol Res 2007;3(4):490–5. [3] Hornung D, Bentzien F, Wallwiener D, Kiesel L, Taylor RN. Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid. Mol Hum Reprod 2001;7(2):163–8. [4] Hornung D, Klingel K, Dohrn K, Kandolf R, Wallwiener D, Taylor RN. Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal endometrium and endometriotic implants: assessment of autocrine/paracrine regulation by in situ hybridization. Am J Pathol 2001;158(6):1949–54. [5] Wren JD, Wu Y, Guo SW. A system-wide analysis of differentially expressed genes in ectopic and eutopic endometrium. Hum Reprod 2007;22(8): 2093–102. [6] Horcajadas JA, Sharkey AM, Catalano RD, et al. Effect of an intrauterine device on the gene expression profile of the endometrium. J Clin Endocrinol Metab 2006;91(8):3199–207. [7] Siristatidis C, Nissotakis C, Chrelias C, Iacovidou H, Salamalekis E. Immunological factors and their role in the genesis and development of endometriosis. J Obstet Gynaecol Res 2006;32(2):162–70. [8] Bohler HC, Gercel-Taylor C, Lessey BA, Taylor DD. Endometriosis markers: immunologic alterations as diagnostic indicators for endometriosis. Reprod Sci 2007;14(6):595–604. [9] Christodoulakos G, Augoulea A, Lambrinoudaki I, Sioulas V, Creatsas G. Pathogenesis of endometriosis: the role of defective ‘immunosurveillance’. Eur J Contracept Reprod Health Care 2007;12(3):194–202. [10] Hornung D, Ryan IP, Chao VA, Vigne JL, Schriock ED, Taylor RN. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol Metab 1997;82(5):1621–8. [11] Sha G, Wu D, Zhang L, et al. Chen X. Differentially expressed genes in human endometrial endothelial cells derived from eutopic endometrium of patients with endometriosis compared with those from patients without endometriosis. Hum Reprod 2007;22(12):3159–69. [12] Luo Q, Ning W, Wu Y, et al. Altered expression of interleukin-18 in the ectopic and eutopic endometrium of women with endometriosis. J Reprod Immunol 2006;72(1–2):108–17. [13] Collette T, Maheux R, Mailloux J, Akoum A. Increased expression of matrix metalloproteinase-9 in the eutopic endometrial tissue of women with endometriosis. Hum Reprod 2006;21(12):3059–67. [14] Akoum A, Lawson C, Herrmann-Lavoie C, Maheux R. Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis. Hum Reprod 2007;22(5):1464–73. [15] Krensky AM, Ahn YT. Mechanisms of disease: regulation of RANTES (CCL5) in renal disease. Nat Clin Pract Nephrol 2007;3(3):164–70. [16] Mause SF, von Hundelshausen P, Zernecke A, Koenen RR, Weber C. Platelet microparticles: a transcellular delivery system for RANTES promoting mono-
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cyte recruitment on endothelium. Arterioscler Thromb Vasc Biol 2005;25(7):1512–8. [17] Lin YJ, Lai MD, Lei HY, Wing LY. Neutrophils and macrophages promote angiogenesis in the early stage of endometriosis in a mouse model. Endocrinology 2006;147(3):1278–86.
[18] Lebovic DI, Chao VA, Taylor RN. Peritoneal macrophages induce RANTES (regulated on activation, normal T cell expressed and secreted) chemokine gene transcription in endometrial stromal cells. J Clin Endocrinol Metab 2004;89(3):1397–401.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Ectopic, autologous eutopic and normal endometrial stromal cells have altered expression and chemotactic activity of RANTES Fang Chun-Li, Han Su-Ping, Fu Shi-Long, Wang Wei, Kong Na, Wang Xiu-Li* Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, 99 Ding Huai Men Street, 210036 Nanjing, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 March 2008 Received in revised form 28 September 2008 Accepted 1 December 2008
Objective: To evaluate if the expression and chemotactic activity of RANTES are different in IL-1b treated autologous eutopic endometrial stromal cells compared to ectopic and normal endometrium. Study design: Conditioned media from IL-1b-treated ectopic, autologous eutopic and normal endometrial stromal cells were analyzed with a specific sandwich ELISA to quantify RANTES. The monocyte chemotactic activity of RANTES was assayed in a Boyden Chamber. Results: RANTES expression in IL-1b-treated autologous eutopic and normal endometrial stromal cells was significantly lower than ectopic endometrium. Autologous eutopic endometrial stromal cells showed a significant increase in RANTES expression compared to normal endometrium after IL-1b stimulation for 60 h. The monocyte chemotactic activities of these conditioned media were highly correlated with the immunoreactive RANTES concentration. We observed significantly increased monocyte chemotactic activity in conditioned media of ectopic stromal cells compared to autologous eutopic and normal endometrium. The different chemotactic activity of RANTES between the autologous eutopic and normal endometrial stromal cells was also statistically significant. RANTES accounts for the majority (62%) of the monocyte chemotactic activity in ectopic endometrial stromal cells conditioned media and 55% of that activity in autologous eutopic endometrium. Conclusions: Although the eutopic endometric of women with and without endometriosis are histologically similar, our findings confirm that different expression and chemotactic activity of RANTES exist between autologous eutopic and normal endometrium. The altered expression of RANTES and monocyte chemotactic activity observed in ectopic, autologous eutopic and normal endometrium suggest the autologous eutopic endometrium may contribute to the pathogenesis of endometriosis. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: RANTES Ectopic endometrium Eutopic endometrium Macrophage
1. Introduction The inflammatory milieu in the peritoneal cavity of women with endometriosis has been extensively characterized. It was noted that the macrophage was the most abundant leukocyte in the pelvic fluid of women with endometriosis and plays a central role in the immunobiology of endometriosis [1]. Peritoneal fluid specimens from women with endometriosis have elevated levels of a variety of cytokines, including IL-1b, tumor necrosis factor a (TNF-a), IL-6, IL-8, and RANTES (regulated on activation, normal T cell expressed and secreted), compared to normal controls [2]. RANTES is an 8-kDa secreted protein with chemoattractant actions on monocytes, natural killer cells, T cells, and eosinophils. Hornung et al. reported that the concentration of RANTES is elevated in the peritoneal cavity of women with endometriosis and correlates
with the severity of the disease [3]. Hornung et al. have shown that a predominant activated macrophage product, IL-1b, induces RANTES gene expression in ectopic endometrial stromal cells [4]. Although the endometria of women with and without endometriosis are histologically similar, studies revealed that there are many differences between these two tissues [5]. In the current study, we examined the expression and chemotactic activity of RANTES in IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells. Our results indicated that there were significant differences of RANTES expression and chemotactic activity among ectopic, autologous eutopic and normal endometrium. 2. Materials and methods 2.1. Sources of tissues
* Corresponding author. Tel.: +86 13813382652; fax: +86 25 86227420. E-mail address: [email protected] (X.-L. Wang). 0301-2115/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2008.12.001
Ovulatory women, who had not received hormones or GnRH agonist therapy for at least 3 months before surgery, were
56
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recruited after they had provided written informed consent under a study protocol approved by the First Affiliated Hospital of Nanjing Medical University. Ectopic and autologous eutopic endometrium were obtained from women with ovarian endometriomata (n = 11, stage III or IV) who have been identified pathologically and staged according to a modification of the revised American Fertility Society system. Control endometrial tissue was obtained from five healthy women undergoing removal of intrauterine device. All of the tissues were acquired during the midproliferative phase of the menstrual cycle. Although most of the endometrial biopsies immediately after IUD removal showed inflammatory changes and edema consistent with the local effect of the IUD as it has been previously reported, Horcajadas et al. used a microarray containing more than 16,000 cDNAs to investigate the gene expression profile of receptive versus refractory endometrium in the same women induced by the presence of an IUD and did not find that the gene expression profiles of IL-1b and RANTES significantly dysregulated [6]. So the endometria obtained from five healthy women undergoing removal of intrauterine device were used as control in our study. 2.2. Cell isolation, purification and culture Endometrial and endometrioma biopsies were collected under sterile conditions and transported to the laboratory in DMEM (GIBCO) with 10% FCS. The fresh tissue was cut with scissors into small pieces of approximately 1 mm3, and rinsed carefully to remove blood. Digestion of the tissue was then performed for 1 h at 37 8C, with shaking every 15 min in 0.125% type IA collagenase (Sigma). Debris was removed by 100-mm aperture sieves, and epithelial glands were retained on 40-mm aperture sieves. Stromal cells remaining in the filtrate were plated onto 6-cm dish and allowed to adhere for 45 min, after which blood cells were removed with phosphate-buffered saline rinses. The cells were cultured in DMEM with 10% FCS, penicillin G (100 U/ml) and streptomycin (100 mg/ml). Exogenous steroids were not added to the cultures. Stromal cultures were dissociated with 0.05% trypsin and 0.02% versene in saline, harvested by centrifugation, replated, and allowed to grow to confluence. Purification of the stromal cell population was confirmed by positive staining for mouse anti-Vimentin antibody (Zymed Laboratories Inc.) and negative staining for mouse anti-cytokeratin 17 antibody (Zymed Laboratories Inc.).
compared to reference standards of recombinant human RANTES (R&D Systems). 2.5. Monocyte chemotaxis assay RANTES bioactivity was assessed by a chemotaxis assay using a human histiocytic cell line (U937, purchased from the Cell Bank of the Chinese Academy of Science). U937 cells were treated with 1 mM 8-bromo-cAMP for 48 h to induce a monocyte phenotype and up-regulate oligopeptide chemoattractant receptors. For the measurements, 24-well plates assembled with Boyden chambers (0.4-mm pore size polycarbonate membranes) were used. Sample (600 ml) containing the chemotactic peptide N-formyl-methionylleucyl-phenylalanine (0.1 mM fMLP) (Sigma) in PBS was placed in the lower chamber as positive control for cell migration. Supernatants from IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells were added to the lower chamber to test its activity. After assembly of the Boyden chambers, 200 ml suspensions of U937 cells (1 105 cells) in PBS were placed in the upper chamber. The chambers were then incubated in a humidified CO2 incubator at 37 8C for 90 min. Nonmigrating cells were removed by washes with PBS, and cells migrating across the membrane were fixed, stained, and counted directly. The chemotactic index (CI) was expressed as the multiple of chemotaxis obtained in response to a minimal stimulation with OPTI-MEM alone. To evaluate the monocyte chemotactic activity of RANTES in supernatants from IL-1b treated ectopic and autologous eutopic endometrial stromal cells, the 60-h conditioned media were incubated with serial dose (0.5, 1, 2, 4 and 8 mg/ml) goat antihuman RANTES IgG (R&D Systems) for 30 min at 37 8C before chemotaxis assay, and the chemotactic activity was measured as described above. The goat anti-human RANTES IgG was highly specific for RANTES and not cross-react with monocyte chemotactic protein (MCP)1-3, macrophage inflammatory protein(MIP)1a or b, or many other human chemokines. 2.6. Statistical analysis Software SPSS 11.0 was used in the statistical analysis. Each parameter was presented as mean S.D., and compared using oneway ANOVA analysis of variance. The level of significance was set at P < 0.05.
2.3. Cytokine treatment of cell cultures
3. Results
When the primary stromal cells cultured in six-well plates approached confluence, the complete medium was removed and replaced with fresh low serum Opti-MEM (GIBCO) containing 2.5% FCS for an additional 48 h. Ectopic endometrial stromal cells were treated with IL-1b (Sigma) at 0, 5, 10 and 20 ng/ml dose. Conditioned media were removed and analyzed after 12, 24, 36, 48, 60, 72, 84 and 96 h. Dose–response experiments indicated that 10 ng/ml of IL-1b induced a maximal RANTES response in ectopic endometrial stromal cells. So in the following experiments, the 10 ng/ml of IL-1b was used to induce the expression of RANTES in cultured ectopic, autologous eutopic and normal endometrial stromal cells.
3.1. Immunocytochemistry of purified endometrial stromal cell populations
2.4. Rantes ELISA A specific sandwich ELISA (Quantikine, R&D Systems) was used to quantify RANTES in conditioned media. The assay is specific for human RANTES, with no known cross-reactivity with other cytokines or chemokines. Aliquots of culture supernatants were each tested in duplicate at several dilutions and
The morphologies of ectopic (Fig. 1A), autologous eutopic (Fig. 1B) and normal (Fig. 1C) endometrial stromal cells were polygonal or spindle-shaped. Generally, the size of ectopic endometrial stromal cells was larger than that of autologous eutopic and normal endometrium. The primary ectopic endometrial stromal cells proliferate more slowly compared to autologous eutopic and normal endometrium. Stromal cells were confirmed by positive staining with vimentin antibody (Fig. 1D and E) and negative staining with cytokeratin antibody (Fig. 1F). Extensive characterization of cell cultures prepared using this protocol confirmed that the stromal cells were >95% pure. 3.2. RANTES expression in conditioned media from IL-1b-treated ectopic, autologous eutopic and normal endometrial stromal cells Ectopic endometrial stromal cells were stimulated with IL-1b at 0, 5, 10 and 20 ng/ml dose.
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Fig. 1. The morphologies and immunocytochemistry of purified ectopic, autologous eutopic and normal endometrial stromal cells. The morphologies of ectopic (A), autologous eutopic (B) and normal (C), endometrial stromal cells were polygonal or spindle-shaped. Stromal cells were confirmed by positive staining with vimentin antibody (D and E) and negative staining with cytokeratin antibody (F). Vimentin is a member of the intermediate filament family of proteins (D).
As shown in Fig. 2, dose–response experiments showed a maximum stimulation after treatment with IL-1b of 10 ng/ml. Culturing the cells in the presence of IL-1b (10 ng/ml) for 24 h dramatically (P < 0.01) induced RANTES expression in ectopic endometrial stromal cells (Fig. 3), but not in autologous eutopic and normal endometrial stromal cells. Autologous eutopic endometrial stromal cells showed a significant increase (P < 0.05) in RANTES secretion compared to normal endometrium after IL-1b stimulation for 60 h (Fig. 3). 3.3. The chemotactic activity of conditioned supernatants from IL-1btreated ectopic, autologous eutopic and normal endometrial stromal cells Conditioned media from IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells were used for the chemotactic assay in a Boyden chamber. As shown in Fig. 4, the monocyte chemotactic activity of these conditioned media was highly correlated with the immnoreactive RANTES concentration. We observed significantly increased (P < 0.05) monocyte chemotactic activity in conditioned media of ectopic stromal cells compared to autologous eutopic and normal endometrium (Fig. 5).
Fig. 2. RANTES expression in supernatants of ectopic endometrial stromal cells treated with IL-1b at 0, 5, 10 and 20 ng/ml. Dose–response experiments showed a maximum stimulation after treatment with IL-1b of 10 ng/ml. *P < 0.05 vs. 10 ng/ ml of IL-1b; **P < 0.01 vs. 5 ng/ml of IL-1b.
The difference between the autologous eutopic and normal endometrial stromal cells was also statistically significant (P < 0.05) (Fig. 5). To evaluate the contribution of RANTES to the total monocyte chemotactic activity in the conditioned media, we added specific RANTES-neutralizing monoclonal antibodies at serial dose (0.5, 1, 2, 4 and 8 mg/ml) into 60 h conditioned media of ectopic endometrial stromal cells and found that 4 mg/ml of anti-RANTES antibody almost completely abolished the chemotactic activity of RANTES. The 4 mg/ml of anti-RANTES antibody inhibited 62% of the chemotactic activity in conditioned medium of ectopic endometrial stromal cells (Fig. 6) and 55% of that activity in autologous eutopic endometrial stromal cells (Fig. 6). 4. Discussion Endometriosis is a common gynecological disorder, affecting at least 10% of reproductive-aged women. It is characterized by the
Fig. 3. RANTES expression in conditioned media from ectopic, autologous eutopic and normal endometrial stromal cells stimulated with 10 ng/ml of IL-1b. Culturing the cells in the presence of IL-1b (10 ng/ml) for 24 h dramatically (P < 0.01) induced RANTES expression in ectopic endometrial stromal cells, but not in autologous eutopic and normal endometrial stromal cells. Autologous eutopic endometrial stromal cells showed a significant increase (P < 0.05) in RANTES secretion compared to normal endometrium after IL-1b stimulation for 60 h. The data represent the mean S.D. of different subjects in each group. *P < 0.05 vs. normal endometrium; **P < 0.01 vs. autologous eutopic and normal endometriums.
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Fig. 4. The monocyte chemotactic activity of the conditioned media of 60 h from IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells. Conditioned media of 60 h from IL-1b treated ectopic (A), autologous eutopic (B) and normal endometrial (C) stromal cells were used for the chemotactic assay in a Boyden chamber. The monocyte chemotactic activity of these conditioned media was highly correlated with the immnoreactive RANTES concentration. The negative control with Opti-MEM was shown in D.
growth of endometrial tissue outside the uterine cavity. Although multiple theories exist regarding the etiology of endometriosis, the implantation hypothesis of Sampson is the most widely accepted. Since retrograde menstruation is a very common phenomenon among women of reproductive age, there must be other factors that may contribute to the pathophysiology and/or pathogenesis of endometriosis. Genetic predisposition, environmental factors, and alterations in immune and endocrine functions are believed to play significant roles in the establishment and maintenance of endometriosis [7–9]. IL-1, a principal macrophage-derived and
major proinflammatory cytokine, may play a central role in the integrated inflammatory cascade associated with endometriosis and in propagating endometriotic implants through proinflammatory stimuli and synthesis of chemokines, growth factors and angiogenic factors, such as RANTES. Hornung et al. reported peritoneal implants may contribute to the increased pelvic fluid concentrations of RANTES seen in patients with endometriosis and the secretion of RANTES by ectopic implants provides a mechanism for peritoneal macrophage recruitment [3,10]. It is known that ectopic and eutopic endometrium from women with
Fig. 5. The chemotactic activity of conditioned supernatants from IL-1b treated ectopic, autologous eutopic and normal endometrial stromal cells. The data represent the mean S.D. of different subjects in each group. The chemotactic index (CI) was expressed as the multiple of chemotaxis obtained in response to a minimal stimulation with OPTI-MEM alone. The CI in conditioned media of ectopic stromal cells significantly increased compared to autologous eutopic and normal endometrial stromal cells. The difference between the autologous eutopic and normal endometrial stromal cells was also statistically significant. *P < 0.05 vs. normal endometrium; **P < 0.01 vs. autologous eutopic endometrium.
Fig. 6. The monocyte chemotactic activity of RANTES in conditioned medium of ectopic and autologous eutopic endometrial stromal cells. The specific RANTESneutralizing monoclonal antibodies at serial dose(0.5, 1, 2, 4 and 8 mg/ml) were added into conditioned medium of 60 h. The 4 mg/ml of anti-RANTES antibody inhibited 62% of the chemotactic activity in conditioned medium of ectopic endometrial stromal cells and 55% of that activity in autologous eutopic endometrial stromal cells. *P < 0.05 vs. 8 mg/ml of anti-RANTES antibody. **P < 0.05 vs. 8 mg/ml of anti-RANTES antibody.
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endometriosis presents physiological differences with normal endometrium [11–13]. Do differences in expression and chemotactic activity of RANTES exist among ectopic, autologous eutopic and normal endometrium? In this study, we show that the expression and chemotactic activity of RANTES are increased in the ectopic and autologous eutopic endometrium when compared with normal endometrium under the stimulation of IL-1b. In agreement with previous reports, our data showed that the RANTES secreted by ectopic endometrial stromal cells increased significantly compared to normal endometrium after the stimulation of IL-1b. Moreover, we found that the RANTES secreted by autologous eutopic endometrial stromal cells was significantly higher than the normal endometrium and significantly lower than the ectopic endometrium. Until now, it is not clear why misplaced endometrial cells from healthy women do not implant and do not develop into endometriotic lesions as occurs in women that develop endometriosis, indicating that some factor which facilitates their survival and implantation may be involved. Ours and other previous studies showed augmented sensitivity of ectopic and eutopic endometrial cells to the biological actions of IL-1b. These cells displayed increased secretion of RANTES in response to IL-1b. The mechanisms underlying such an accentuated imbalance in the ectopic and autologous eutopic endometrial tissue remain unknown. IL-1 has two known receptors, now designated as IL-1R1 and IL-1R2. Cell activation by IL-1 results from its binding to IL-1R1 which in concert with IL-1R accessory protein (IL-1RAP) is capable of transducing the activation signal. The IL-1R2 has, in contrast to IL-1R1, no signaling properties, but has been described as a potent, specific and natural inhibitor of IL-1. Akoum et al. found that an imbalance in IL-1R1 and IL-1R2 expression occurring in the eutopic endometrium of women with endometriosis and more markedly in the ectopic endometrial tissue. A decreased IL-1R2 expression is predominant in the eutopic endometrium, a concomitant increase in IL-1R1 expression occurs in ectopic endometrial tissue, particularly in the initial and most active implants [14]. Over expression of the activating IL-1R1 combined with depression of the decoy IL-1R2 may represent a key mechanism underlying the augmented sensitivity of ectopic and eutopic endometrial cells to the biological actions of IL-1b. RANTES is a cytokine of the b or ‘‘C–C’’ chemokine family. It is a chemoattractant for monocytes and memory T-cells and may be an important mediator in both acute and chronic inflammation [15,16]. Macrophages play a central role in the immunobiology of endometriosis. Interactions between the endometriotic cells and macrophages are mediated by growth factors, cytokines, and chemokines, leading to the paradoxical survival of implants rather than their demise [17,18]. In our study, the altered expression of RANTES observed in autologous eutopic endometrium suggests their participation in the pathogenesis of endometriosis. Using the U937 chemotaxis assay in a Boyden chamber, our current investigation corroborated the biological activity of RANTES as a monocyte chemokine secreted by cultured endometrial stromal cells, and particularly ectopic and autologous eutopic endometrium. As shown in Fig. 4, the monocyte chemotactic activity of these conditioned media was highly correlated with the immunoreactive RANTES concentration. We observed significantly increased monocyte chemotactic activity in conditioned media of ectopic stromal cells compared to autologous eutopic and normal endometriums. The difference between the autologous eutopic and normal endometrial stromal cells was also statistically significant. To evaluate the contribution of RANTES to the total monocyte chemotactic activity in the conditioned media, we added specific RANTES-neutralizing monoclonal antibodies at serial dose into conditioned media of 60 h and found that 4 mg/ml of antiRANTES antibody almost completely abolished the chemotactic activity of RANTES. We established that RANTES accounts for the
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majority (62%) of the monocyte chemotactic activity in ectopic endometrial stromal cells conditioned media and 55% of that activity in autologous eutopic endometrium. Other monocyte chemokines are likely to contribute the remaining activity of macrophage recruitment. Although the eutopic endometric of women with and without endometriosis are histologically similar, our findings confirm that differential expression and chemotactic activity of RANTES exist between autologous eutopic and normal endometrium. The feedforward inflammatory loop whereby IL-1b from activated macrophages may lead to RANTES production by ectopic and autologous eutopic endometrium and further monocyte chemotaxis facilitates the establishment, progression and inflammatory sequelae of endometriotic tissues. The altered expression of RANTES and monocyte chemotactic activity observed in autologous eutopic endometrium suggests they may contribute to the pathogenesis of endometriosis. Future clinical strategies aimed at neutralizing potentially pathological factors in the endometriosis syndrome must not lose sight of the possible importance of these molecules in normal endometrial physiology. Acknowledgements This work is supported by National Natural Science Foundation of China No. 30600671 (to Wang Xiu-Li), Natural Science Foundation of Jiangsu province No. BK2006577 (to Wang Xiu-Li). References [1] Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75(1):1–10. [2] Kalu E, Sumar N, Giannopoulos T, et al. Cytokine profiles in serum and peritoneal fluid from infertile women with and without endometriosis. J Obstet Gynaecol Res 2007;3(4):490–5. [3] Hornung D, Bentzien F, Wallwiener D, Kiesel L, Taylor RN. Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid. Mol Hum Reprod 2001;7(2):163–8. [4] Hornung D, Klingel K, Dohrn K, Kandolf R, Wallwiener D, Taylor RN. Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal endometrium and endometriotic implants: assessment of autocrine/paracrine regulation by in situ hybridization. Am J Pathol 2001;158(6):1949–54. [5] Wren JD, Wu Y, Guo SW. A system-wide analysis of differentially expressed genes in ectopic and eutopic endometrium. Hum Reprod 2007;22(8): 2093–102. [6] Horcajadas JA, Sharkey AM, Catalano RD, et al. Effect of an intrauterine device on the gene expression profile of the endometrium. J Clin Endocrinol Metab 2006;91(8):3199–207. [7] Siristatidis C, Nissotakis C, Chrelias C, Iacovidou H, Salamalekis E. Immunological factors and their role in the genesis and development of endometriosis. J Obstet Gynaecol Res 2006;32(2):162–70. [8] Bohler HC, Gercel-Taylor C, Lessey BA, Taylor DD. Endometriosis markers: immunologic alterations as diagnostic indicators for endometriosis. Reprod Sci 2007;14(6):595–604. [9] Christodoulakos G, Augoulea A, Lambrinoudaki I, Sioulas V, Creatsas G. Pathogenesis of endometriosis: the role of defective ‘immunosurveillance’. Eur J Contracept Reprod Health Care 2007;12(3):194–202. [10] Hornung D, Ryan IP, Chao VA, Vigne JL, Schriock ED, Taylor RN. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol Metab 1997;82(5):1621–8. [11] Sha G, Wu D, Zhang L, et al. Chen X. Differentially expressed genes in human endometrial endothelial cells derived from eutopic endometrium of patients with endometriosis compared with those from patients without endometriosis. Hum Reprod 2007;22(12):3159–69. [12] Luo Q, Ning W, Wu Y, et al. Altered expression of interleukin-18 in the ectopic and eutopic endometrium of women with endometriosis. J Reprod Immunol 2006;72(1–2):108–17. [13] Collette T, Maheux R, Mailloux J, Akoum A. Increased expression of matrix metalloproteinase-9 in the eutopic endometrial tissue of women with endometriosis. Hum Reprod 2006;21(12):3059–67. [14] Akoum A, Lawson C, Herrmann-Lavoie C, Maheux R. Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis. Hum Reprod 2007;22(5):1464–73. [15] Krensky AM, Ahn YT. Mechanisms of disease: regulation of RANTES (CCL5) in renal disease. Nat Clin Pract Nephrol 2007;3(3):164–70. [16] Mause SF, von Hundelshausen P, Zernecke A, Koenen RR, Weber C. Platelet microparticles: a transcellular delivery system for RANTES promoting mono-
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