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Epidermal Growth Factor (EGF) in the Human Placental Infection with Trypanosoma cruzi
Placenta (2004), 25, 283–286 doi:10.1016/j.placenta.2003.09.008
Epidermal Growth Factor (EGF) in the Human Placental Infection with Trypanosoma cruzi S. Lin*, M. J. Sartori, L. Mezzano and S. P. de Fabro IIa. Ca´tedra de Biologı´ a Celular, Histologı´ a y Embriologı´ a, Facultad de Ciencias Me´dicas, Universidad Nacional de Co´rdoba, Laboratorio B9, Av. Enrique Barros esquina Enfermera Gordillo, Ciudad Universitaria (5016), Co´rdoba, Argentina Paper accepted 22 September 2003
Maternal infection of Trypanosoma cruzi is associated with premature births, abortions and placentitis. A decrease in EGF levels has been suggested to occur in animals infected by T. cruzi, but there is no research about the levels of EGF in human patients with Chagas’ disease. We evaluated serum EGF levels in pregnant women with and without the disease, and with immunological methods detected EGF receptors and EGF in both groups of placentae and in cultures of normal placental villi with and without parasites. PLAP in placentae from those women was also immunologically detected, since EGF can induce the release of PLAP from the trophoblast surface and PLAP is suggested to be a receptor allowing parasite invasion of the placenta. Plasma from women with Chagas’ disease contained lower level of EGF when compared to plasma of healthy women. Placentae from women with Chagas’ disease showed lower PLAP expression but same level of detectable EGF receptors and EGF when compared with placentae from women without the disease. Culture with parasites did not reduce EGFr level. Results suggest a lower availability of EGF in women with Chagas’ disease, which could explain several malfunctions of the placenta associated with maternal Chagas’ disease. Placenta (2004), 25, 283–286 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Epidermal growth factor (EGF) is a small mitogenic polypeptide present in many mammalian species and is distributed throughout a wide number of tissues and body fluids [1]. EGF is a member of a growth factor family which is characterized by the presence of six conserved cysteine motifs that form three disulfide bonds [2]. EGF is structurally homologous to human transforming growth factor alpha, and both exert their actions through EGF receptors. EGF is mitogenic for a variety of epidermal and epithelial cells [3] and can inhibit apoptotic action of TNF- or INF- in trophoblast [4]. Epidermal growth factor stimulates the phosphorylation of a 150 kDa protein. This reaction is supported by Mg2+, Co2+ as well as Mn2+ [5]. EGF receptors (EGFr) are present on trophoblast cells from the blastocyst stage [6]. The number of EGFr increases during gestation but the affinity of the receptors declines later. EGF may stimulate cell proliferation at the initial stage of development and then stimulates differentiation as the tissues mature [7]. Bramley et al. [8] reported no gestationdependent decrease in the specific activity of EGFr or alkaline phosphatase activity in villous membrane fractions between 10–20 weeks of gestation. The connatal Chagas’ disease, caused by the intracellular protozoo Trypanosoma cruzi, is associated with premature *
To whom correspondence should be addressed. +54-351-4334021; E-mail: [email protected] 0143-4004/$–see front matter
Fax:
births, abortions and placentitis. Placental alkaline phosphatase (PLAP; E.C. 3.1.3.1) is suggested to be one of the receptors used by the parasite to trigger a pathway of signals which allow T. cruzi parasite invasion in the human trophoblast [9]. EGF can induce the ATP-dependent release of a fraction of PLAP from syncytiotrophoblast membranes [10]. Exposure of tumour cell lines expressing EGFr to EGF results in suppression of alkaline phosphatase expression [11]; [12]. Host growth factors are also known to induce proliferation of T. cruzi amastigotes. [13]. A decreased level of EGF in animals infected by T. cruzi has been reported [14], however there is no research about the level of EGF in patients with Chagas’ disease. We measured immunohistochemically EGFr and EGF in placental samples from women with and without Chagas’ disease, and in placental villi co-cultured with and without trypomastigotes of T. cruzi. We also measured the level of EGF in plasma of pregnant women with and without Chagas’ disease.
MATERIAL AND METHODS Placenta from chagasic women Placentae from women with and without positive serological diagnosis for Chagas’ disease at 38 to 40 weeks of gestation were obtained. Samples of central villi were isolated, washed 2003 Elsevier Ltd. All rights reserved.
284
with PBS and fixed in formol 10 per cent. Plasma from each patient were obtained for the determination of EGF level.
Placenta (2004), Vol. 25
with peroxidase conjugated anti-mouse immunoglobulins (Sigma Co. PN: A9044) and revealed with citrate–phosphate buffer, H2O2 and OPD (10 ml : 10 µl : 4 mg).
Placental cultures PLAP immunodetection Placentae from clinically and serologically healthy women at 38 to 40 weeks of gestation were obtained by Caesarean delivery in order to assure asepsis and preservation of the sample. Immediately, central villi of placental cotyledons were isolated, washed with PBS in order to remove blood, and cut in 1 cm3 pieces. Placental villi were co-cultured with 1106 trypomastigotes in culture media M-199 at 37(C. Controls were maintained in the same conditions without parasites. After 24 h of infection, the cultured villi were fixed with formaldehyde 10 per cent for 24 h, dehydrated with alcohol/xylol and embedded in paraffin for later immunodetection of EGF and EGFr.
Parasites Bloodstream trypomastigotes from Tulahuen strain of T. cruzi were isolated, according to Andrews and Colli [15], from Albino Swiss mice in the peak of parasitaemia. Blood was centrifuged 10 min at 100 gravities; after 1 h at 37(C the plasma was isolated and centrifuged 10 min at 590 gravities. The parasites containing pellet was washed twice and resuspended in M-199 media.
Histological immunodetection of EGF and EGFr in placenta samples Placental villi slides were embedded in PBS, pH 7.2, preincubated with H2O2, rinsed with buffer, incubated either with anti-EGFr (Sigma Co. PN: E3138) or anti EGF monoclonal antibody (Sigma Co. PN: E2520) at 4(C overnight. After three washes in PBS, sections were incubated with peroxidase conjugated anti-mouse immunoglobulins, rinsed with buffer and then Tris–HCl, pretreated with DAB solution (0.5 mg/ml Tris–HCl 0.05 M pH 7.6) and then revealed with DAB solution plus 1 per cent H2O2 under Zeiss Axioskop 20 microscopy. The peroxidase reaction was stopped by rinsing with water.
Elisa detection of EGF in plasma samples Plasma protein content was measured following Lowry et al. [16]. Plasma were diluted at 10 µg protein/µl with carbonate buffer (pH 9.6) and allowed to attach to the wells at 4(C overnight. Samples were blocked with BFS 1 per cent in PBS, then rinsed and incubated either with anti-EGFr or anti EGF monoclonal antibody (Sigma Co. PN: E2520) at 37(C for 45 min. After three washes in PBS, sections were incubated
PLAP location was seen by indirect immunofluorescence assay. Placental villi slides were embedded in PBS, pH 7.2, incubated with anti-PLAP monoclonal antibody (ready to use, BioGenex Ab 228 M) at room temperature for 30 min. After three washes in PBS, sections were incubated with FITC-conjugated antimouse immunoglobulins, washed, and examined by light microscopy.
Image analysis Images stored in .bmp format, were analysed with ‘Image Tool’ UTHSCSA version 3.00, downloadable from http:// ddsdx.uthscsa.edu/dig/download.html. The semi-quantitative evaluation were performed by analysing the line profiles of the villi surface, which were manually drawn. The mean intensity of grey scale or green scale were determined and compared to a relative scale which maximum or minimum was the value of a section on the same image where reaction was considered negative while the maximum or minimum value corresponded to a section considered intensly immunostained. The intensity scale was applied with inverted number value or not depending on the reaction. Positive reaction of peroxidase turns out a dark precipitate while positive reaction of immunofluorescence turns out a green and brilliant colour. A t-test statistic analysis were performed to compare the the relative intensity values considering different images of the same experiment group.
RESULTS Immunodetection of EGFr performed on placentae from women with and without Chagas’ disease showed positive expression of the receptor in both cases. Although the mean value for the placentae from women with Chagas’ disease (48.75 per cent of the surface) is lower than those from women without the disease (67.68 per cent), the t-test indicated no statistical differences between groups. Placental villi cultured with trypomastigotes of T. cruzi showed lower expression of EGFr (37.25 per cent) than villi cultured without parasites (46.43 per cent), but the difference was not statistically significant. Image analysis showed no significant difference of expression of EGFr in placental villi cultured in vitro when compared to placentae without culture. The immunodetection of placental EGF from women with and without Chagas’ disease revealed a higher immunostaining of EGF on placenta from women without (45.11 per cent versus 40.42 per cent) but this difference is also not significant using the t-test. The immunodetection of placental EGF from
Lin et al.: EGF in the Human Placental Infection with T. cruzi
285
Figure 1. Elisa optical density (OD) values of EGF level in plasma of normal and chagasic pregnant women. Plasma of normal pregnant women showed higher level of detectable EGF (n=8, s.d.).
women with Chagas’ disease was positive not only on the surface of syncytiotrophoblast but also in the mesenchyme. EGF levels in the plasma of pregnant women with and without Chagas’ disease was detemined by ELISA. Plasma from women with the disease presented a statistically significant decrease (25 per cent) of detectable EGF through this method (Figure 1). Immunodetection of PLAP was performed on placentae from women with and without Chagas’ disease. PLAP was present on the surface of syncytiotrophoblasts and on the endothelia in both groups. Placentae from healthy women showed a more uniform expression and statistically significant higher expression of PLAP (Figure 2A) while placentae from women with Chagas’ disease presented lower mean expression of PLAP and large areas where PLAP was absent (Figure 2B).
Figure 2. Immunodetection for PLAP on placenta from women with (B) and without (A) Chagas’ disease. Normal placentae showed regular expression of PLAP on the trophoblast surface, while the expression of PLAP on chagasic ones is interrupted. Original magnification: 400. Scale bar represents 5 µm.
DISCUSSION Silva et al. [14] suggested that a decreased level of EGF could happen in animals infected by T. cruzi. Our results showed a statistically significant decrease of plasma EGF levels of women with Chagas’ disease, which would provide positive evidence for this. The immunodetection of EGF in placentae from women with and without Chagas’ disease did not show statistical differences according to the results of image analysis. Studies on EGFr did not present any difference between placentae from women with or without the disease. There was also no difference between placentae before and after culture. EGF can induce the ATP-dependent release of a fraction of PLAP from syncytiotrophoblast membranes [10]. Exposure of tumour cell lines expressing EGFr to EGF results in suppression of alkaline phosphatase expression [11]; [12]. The induction of the release of PLAP by EGF could be accomplished through activation of phospholipase D activity [10], [17].
As the level of EGF in the plasma of women with Chagas’ disease was found to be lower than women without, it could be expected that PLAP levels in chagasic placenta to be high, because the lower level of plasma EGF would activate less EGF receptors and thus not favour the release of PLAP. However we detected a lower level of PLAP expression in placentae from women with Chagas disease. This lower expression of PLAP on the surface of placenta from women with Chagas’ disease could be explained by the presence of the parasite. The trypomastigotes are able to release PLAP from the trophoblast surface itself through the action of its phospholipase C [18]; [19]; [20]. In this way, the level of PLAP expression on the surface of trophoblast from women with Chagas’ disease could be independent from the level of EGF. Host growth factors are known to induce proliferation of T. cruzi amastigotes [13]. The receptors for EGF are present on amastigote stage of the parasite, which means that newly released parasites from infected cells could compete for the EGF and thus diminish the real available level of EGF for the
286
placental tissue. The presence of the parasite could then explain the lower level of EGF observed in women with Chagas’ disease and the deficiency on EGF signalization could explain the complex and variable symptoms associated to the connatal Chagas’ disease, namely those related to the malfunction of the placenta. The lower availability of EGF would thus diminish the release of PLAP, which is suggested to be one of the placental receptors used by T. cruzi for the placenta invasion, but on the other hand the parasite self is able to release the PLAP from
Placenta (2004), Vol. 25
the syncytiotrophoblast surface [9] thus the relationship among parasite, receptor and the molecules implied are somehow balanced. EGF induces actin depolymerization [21]; [22]; [23]; [24] which was also demonstrated in T. cruzi invasion to the placental villi [25]. It would be interesting to find out if EGFr could be another receptor pathway for the trypomastigotes to invade the placental villi since EGFr and PLAP seem to share elements of the same intracellular signalling network, probably the dual Calcium-IP3 signal pathway [25], [26].
ACKNOWLEDGEMENTS The authors are thankful to Dr Patricia Paglini and Biol. Walter Rivarola for providing trypomastigotes of T. cruzi, to Miriam Rabino for technical support and to Dion Whitehead for proof reading the manuscript. This work was supported by grants from Consejo de Investigaciones Cientı´ficas y Tecnolo´gicas de la Provincia de Co´rdoba (CONICOR), Repu´blica Argentina and Secretarı´a de Ciencia y Tecnologı´a de la Universidad Nacional de Co´rdoba (SeCyT).
REFERENCES [1] Carpenter G, Cohen S. Epidermal growth factor. Annual Reviews Biochemistry 1979;48:193–216. [2] George Nascimento C, Gyenes A, Halloran SM, Merryweather J, Valenzuela P, Steimer KS et al. Characterization of recombinant human epidermal growth factor produced in yeast. Biochemistry 1988;27(2): 797–802. [3] Todaro GJ, Fryling C, De Larco JE. Transforming growth factors produced by certain human tumour cells: polypeptides that interact with epidermal growth factor receptors. Proceedings of National Academy of Science of the United States of America 1980;77(9):5258–62. [4] Smith S, Francis R, Guilbert L, Baker PN. Growth factor rescue of cytokine mediated trophoblast apoptosis. Placenta 2002;23(4):322–30 doi:10.1053/plac.2001.0783. [5] Avruch J, Nemenoff RA, Blackshear PJ, Pierce MW, Osathanondh R. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in detergent extracts of human placental membranes. Comparison to epidermal growth factor-stimulated phosphorilation. Journal of Biological Chemistry 1982;257(24):15162–6. [6] Hock RA, Hollenberg MD. Characterization of the receptor for epidermal growth factor-urogastrone in human placenta membranes. Journal of Biological Chemistry 1980;255(22):10731–6. [7] Adamson ED, Meek J. The ontogeny of epidermal growth factor receptors during mouse development. Developmental Biology 1984;103(1):62–70. [8] Bramley TA, McPhie CA, Menzies GS. Human placental gonadotrophin-releasing hormone (GnRH) binding sites: III. Changes in GnRH binding levels with stage of gestation. Placenta 1994;15(7):733–45. [9] Sartori MJ, Lin S, Frank FM, Malchiodi EL, de Fabro SP. Role of placental alkaline phosphatase in the interaction between human placental trophoblast and Trypanosoma cruzi. Experimental Molecular Pathology 2002;72(1):84–90 doi:10.1006/exmp.2001.2412. [10] Kenton P, Johnson PM. Growth factor-induced release of placental alkaline phosphatase from human syncytiotrophoblast membranes. Journal of Reproduction and Fertility 1994;100(1):71–6. [11] Dabare AA, Nouri AM, Cannell H, Moss T, Nigam AK, Oliver RT. Profile of placental alkaline phosphatase expression in human malignancies: effect of tumour cell activation on alkaline phosphatase expression. Urologia Internationalis 1999;63(3):168–74 doi:10.1159/ 000030441. [12] Nouri AM, Thompson C, Cannell H, Symes M, Purkiss S, Amirghofran Z. Profile of epidermal growth factor receptor (EGFr) expression in human malignancies: effects of exposure to EGF and its biological influence on established human tumour cell lines. International Journal of Molecular Medicine 2000;6(4):495–500. [13] Ghansah TJ, Ager EC, Freeman-Junior P, Villalta F, Lima MF. Epidermal growth factor binds to a receptor on Trypanosoma cruzi
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amastigotes inducing signal transduction events and cell proliferation. Journal of Eukaryotic Microbiology 2002;49(5):383–90. Silva RC, Cardoso JE, Silva GA, Moreira A, Alves JB. Histological and histoquantitative study of the rat paroid gland after Trypanosoma cruzi infection. Parasite 2000;7(2):109–13. Andrews NW, Colli W. Adhesion and interiorization of Trypanosoma cruzi in mammalian cells. Journal of Protozoology 1982;29:264–9. Lowry OH, Rosebrough MJ, Farr AJ, Fandall RJ. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 1951; 93:265–75. Hughes WE, Larijani B, Parker PJ. Detecting protein-phospholipid interactions. Epidermal growth factor-induced activation of phospholipase D 1b in situ. Journal of Biological Chemistry 2002;277(2):2974–9 doi:10.1074/jbc.M201391200. Low MG, Zilversmit DB. Role of phosphatidylinositol in attachment of alkaline phosphatase to membranes. Biochemistry 1980;19:3913–8. Malik AS, Low M.G. Conversion of human placental alkaline phosphatase from a high Mr form to a low Mr form during butanol extraction. An investigation of the role of endogenous phosphoinositide-specific phospholipases. Biochemical Journal 1986;240:519–27. Hawrylak K, Stinson RA. The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis. Journal of Biological Chemistry 1988;263(28):14368–73. Diakonova M, Payrastre B, van Velzen AG, Hage WJ, van Bergen en Henegouwen PM, Boonstra J et al. Epidermal growth factor induces rapid and transient association of phospholipase C-gamma 1 with EGF-receptor and filamentous actin at membrane ruffles of A431 cells. Journal of Cell Science 1995;108(Pt 6):2499–509. Rijken PJ, Hage WJ, van Bergen en Henegouwen PM, Verkleij AJ, Boonstra J. Epidermal growth factor induces rapid reorganization of the actin microfilament system in human A431 cells. Journal of Cell Science 1991;100(Pt3):491–9. Rijken PJ, Post SM, Hage WJ, van Bergen en Henegouven PM, Verkleij AJ, Boonstra J. Actin polymerization localizes to the activated epidermal growth factor receptor in the plasma membrane, independent of the cytosolic free calcium transient. Experimental Cell Research 1995; 218:223–32 doi:10.1006/excr.1995.1150. Hazan RB, Norton L. The epidermal growth factor receptor modulates the interaction of E-cadherin with the actin cytoskeleton. Journal of Biological Chemistry 1998;273:9078–84 doi:10.1074/jbc.273.15.9078. Sartori MJ, Pons P, Mezzano L, Lin S, de Fabro SP. Trypanosoma cruzi infection induces microfilament depletion in human placenta cytotrophoblast. Placenta 2003;24(7):767–71. Munaron L.. Calcium signalling and control of cell proliferation by tyrosine kinase receptors. International Journal of Molecular Medicine 2002;10:671–6 Review.
Epidermal Growth Factor (EGF) in the Human Placental Infection with Trypanosoma cruzi S. Lin*, M. J. Sartori, L. Mezzano and S. P. de Fabro IIa. Ca´tedra de Biologı´ a Celular, Histologı´ a y Embriologı´ a, Facultad de Ciencias Me´dicas, Universidad Nacional de Co´rdoba, Laboratorio B9, Av. Enrique Barros esquina Enfermera Gordillo, Ciudad Universitaria (5016), Co´rdoba, Argentina Paper accepted 22 September 2003
Maternal infection of Trypanosoma cruzi is associated with premature births, abortions and placentitis. A decrease in EGF levels has been suggested to occur in animals infected by T. cruzi, but there is no research about the levels of EGF in human patients with Chagas’ disease. We evaluated serum EGF levels in pregnant women with and without the disease, and with immunological methods detected EGF receptors and EGF in both groups of placentae and in cultures of normal placental villi with and without parasites. PLAP in placentae from those women was also immunologically detected, since EGF can induce the release of PLAP from the trophoblast surface and PLAP is suggested to be a receptor allowing parasite invasion of the placenta. Plasma from women with Chagas’ disease contained lower level of EGF when compared to plasma of healthy women. Placentae from women with Chagas’ disease showed lower PLAP expression but same level of detectable EGF receptors and EGF when compared with placentae from women without the disease. Culture with parasites did not reduce EGFr level. Results suggest a lower availability of EGF in women with Chagas’ disease, which could explain several malfunctions of the placenta associated with maternal Chagas’ disease. Placenta (2004), 25, 283–286 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Epidermal growth factor (EGF) is a small mitogenic polypeptide present in many mammalian species and is distributed throughout a wide number of tissues and body fluids [1]. EGF is a member of a growth factor family which is characterized by the presence of six conserved cysteine motifs that form three disulfide bonds [2]. EGF is structurally homologous to human transforming growth factor alpha, and both exert their actions through EGF receptors. EGF is mitogenic for a variety of epidermal and epithelial cells [3] and can inhibit apoptotic action of TNF- or INF- in trophoblast [4]. Epidermal growth factor stimulates the phosphorylation of a 150 kDa protein. This reaction is supported by Mg2+, Co2+ as well as Mn2+ [5]. EGF receptors (EGFr) are present on trophoblast cells from the blastocyst stage [6]. The number of EGFr increases during gestation but the affinity of the receptors declines later. EGF may stimulate cell proliferation at the initial stage of development and then stimulates differentiation as the tissues mature [7]. Bramley et al. [8] reported no gestationdependent decrease in the specific activity of EGFr or alkaline phosphatase activity in villous membrane fractions between 10–20 weeks of gestation. The connatal Chagas’ disease, caused by the intracellular protozoo Trypanosoma cruzi, is associated with premature *
To whom correspondence should be addressed. +54-351-4334021; E-mail: [email protected] 0143-4004/$–see front matter
Fax:
births, abortions and placentitis. Placental alkaline phosphatase (PLAP; E.C. 3.1.3.1) is suggested to be one of the receptors used by the parasite to trigger a pathway of signals which allow T. cruzi parasite invasion in the human trophoblast [9]. EGF can induce the ATP-dependent release of a fraction of PLAP from syncytiotrophoblast membranes [10]. Exposure of tumour cell lines expressing EGFr to EGF results in suppression of alkaline phosphatase expression [11]; [12]. Host growth factors are also known to induce proliferation of T. cruzi amastigotes. [13]. A decreased level of EGF in animals infected by T. cruzi has been reported [14], however there is no research about the level of EGF in patients with Chagas’ disease. We measured immunohistochemically EGFr and EGF in placental samples from women with and without Chagas’ disease, and in placental villi co-cultured with and without trypomastigotes of T. cruzi. We also measured the level of EGF in plasma of pregnant women with and without Chagas’ disease.
MATERIAL AND METHODS Placenta from chagasic women Placentae from women with and without positive serological diagnosis for Chagas’ disease at 38 to 40 weeks of gestation were obtained. Samples of central villi were isolated, washed 2003 Elsevier Ltd. All rights reserved.
284
with PBS and fixed in formol 10 per cent. Plasma from each patient were obtained for the determination of EGF level.
Placenta (2004), Vol. 25
with peroxidase conjugated anti-mouse immunoglobulins (Sigma Co. PN: A9044) and revealed with citrate–phosphate buffer, H2O2 and OPD (10 ml : 10 µl : 4 mg).
Placental cultures PLAP immunodetection Placentae from clinically and serologically healthy women at 38 to 40 weeks of gestation were obtained by Caesarean delivery in order to assure asepsis and preservation of the sample. Immediately, central villi of placental cotyledons were isolated, washed with PBS in order to remove blood, and cut in 1 cm3 pieces. Placental villi were co-cultured with 1106 trypomastigotes in culture media M-199 at 37(C. Controls were maintained in the same conditions without parasites. After 24 h of infection, the cultured villi were fixed with formaldehyde 10 per cent for 24 h, dehydrated with alcohol/xylol and embedded in paraffin for later immunodetection of EGF and EGFr.
Parasites Bloodstream trypomastigotes from Tulahuen strain of T. cruzi were isolated, according to Andrews and Colli [15], from Albino Swiss mice in the peak of parasitaemia. Blood was centrifuged 10 min at 100 gravities; after 1 h at 37(C the plasma was isolated and centrifuged 10 min at 590 gravities. The parasites containing pellet was washed twice and resuspended in M-199 media.
Histological immunodetection of EGF and EGFr in placenta samples Placental villi slides were embedded in PBS, pH 7.2, preincubated with H2O2, rinsed with buffer, incubated either with anti-EGFr (Sigma Co. PN: E3138) or anti EGF monoclonal antibody (Sigma Co. PN: E2520) at 4(C overnight. After three washes in PBS, sections were incubated with peroxidase conjugated anti-mouse immunoglobulins, rinsed with buffer and then Tris–HCl, pretreated with DAB solution (0.5 mg/ml Tris–HCl 0.05 M pH 7.6) and then revealed with DAB solution plus 1 per cent H2O2 under Zeiss Axioskop 20 microscopy. The peroxidase reaction was stopped by rinsing with water.
Elisa detection of EGF in plasma samples Plasma protein content was measured following Lowry et al. [16]. Plasma were diluted at 10 µg protein/µl with carbonate buffer (pH 9.6) and allowed to attach to the wells at 4(C overnight. Samples were blocked with BFS 1 per cent in PBS, then rinsed and incubated either with anti-EGFr or anti EGF monoclonal antibody (Sigma Co. PN: E2520) at 37(C for 45 min. After three washes in PBS, sections were incubated
PLAP location was seen by indirect immunofluorescence assay. Placental villi slides were embedded in PBS, pH 7.2, incubated with anti-PLAP monoclonal antibody (ready to use, BioGenex Ab 228 M) at room temperature for 30 min. After three washes in PBS, sections were incubated with FITC-conjugated antimouse immunoglobulins, washed, and examined by light microscopy.
Image analysis Images stored in .bmp format, were analysed with ‘Image Tool’ UTHSCSA version 3.00, downloadable from http:// ddsdx.uthscsa.edu/dig/download.html. The semi-quantitative evaluation were performed by analysing the line profiles of the villi surface, which were manually drawn. The mean intensity of grey scale or green scale were determined and compared to a relative scale which maximum or minimum was the value of a section on the same image where reaction was considered negative while the maximum or minimum value corresponded to a section considered intensly immunostained. The intensity scale was applied with inverted number value or not depending on the reaction. Positive reaction of peroxidase turns out a dark precipitate while positive reaction of immunofluorescence turns out a green and brilliant colour. A t-test statistic analysis were performed to compare the the relative intensity values considering different images of the same experiment group.
RESULTS Immunodetection of EGFr performed on placentae from women with and without Chagas’ disease showed positive expression of the receptor in both cases. Although the mean value for the placentae from women with Chagas’ disease (48.75 per cent of the surface) is lower than those from women without the disease (67.68 per cent), the t-test indicated no statistical differences between groups. Placental villi cultured with trypomastigotes of T. cruzi showed lower expression of EGFr (37.25 per cent) than villi cultured without parasites (46.43 per cent), but the difference was not statistically significant. Image analysis showed no significant difference of expression of EGFr in placental villi cultured in vitro when compared to placentae without culture. The immunodetection of placental EGF from women with and without Chagas’ disease revealed a higher immunostaining of EGF on placenta from women without (45.11 per cent versus 40.42 per cent) but this difference is also not significant using the t-test. The immunodetection of placental EGF from
Lin et al.: EGF in the Human Placental Infection with T. cruzi
285
Figure 1. Elisa optical density (OD) values of EGF level in plasma of normal and chagasic pregnant women. Plasma of normal pregnant women showed higher level of detectable EGF (n=8, s.d.).
women with Chagas’ disease was positive not only on the surface of syncytiotrophoblast but also in the mesenchyme. EGF levels in the plasma of pregnant women with and without Chagas’ disease was detemined by ELISA. Plasma from women with the disease presented a statistically significant decrease (25 per cent) of detectable EGF through this method (Figure 1). Immunodetection of PLAP was performed on placentae from women with and without Chagas’ disease. PLAP was present on the surface of syncytiotrophoblasts and on the endothelia in both groups. Placentae from healthy women showed a more uniform expression and statistically significant higher expression of PLAP (Figure 2A) while placentae from women with Chagas’ disease presented lower mean expression of PLAP and large areas where PLAP was absent (Figure 2B).
Figure 2. Immunodetection for PLAP on placenta from women with (B) and without (A) Chagas’ disease. Normal placentae showed regular expression of PLAP on the trophoblast surface, while the expression of PLAP on chagasic ones is interrupted. Original magnification: 400. Scale bar represents 5 µm.
DISCUSSION Silva et al. [14] suggested that a decreased level of EGF could happen in animals infected by T. cruzi. Our results showed a statistically significant decrease of plasma EGF levels of women with Chagas’ disease, which would provide positive evidence for this. The immunodetection of EGF in placentae from women with and without Chagas’ disease did not show statistical differences according to the results of image analysis. Studies on EGFr did not present any difference between placentae from women with or without the disease. There was also no difference between placentae before and after culture. EGF can induce the ATP-dependent release of a fraction of PLAP from syncytiotrophoblast membranes [10]. Exposure of tumour cell lines expressing EGFr to EGF results in suppression of alkaline phosphatase expression [11]; [12]. The induction of the release of PLAP by EGF could be accomplished through activation of phospholipase D activity [10], [17].
As the level of EGF in the plasma of women with Chagas’ disease was found to be lower than women without, it could be expected that PLAP levels in chagasic placenta to be high, because the lower level of plasma EGF would activate less EGF receptors and thus not favour the release of PLAP. However we detected a lower level of PLAP expression in placentae from women with Chagas disease. This lower expression of PLAP on the surface of placenta from women with Chagas’ disease could be explained by the presence of the parasite. The trypomastigotes are able to release PLAP from the trophoblast surface itself through the action of its phospholipase C [18]; [19]; [20]. In this way, the level of PLAP expression on the surface of trophoblast from women with Chagas’ disease could be independent from the level of EGF. Host growth factors are known to induce proliferation of T. cruzi amastigotes [13]. The receptors for EGF are present on amastigote stage of the parasite, which means that newly released parasites from infected cells could compete for the EGF and thus diminish the real available level of EGF for the
286
placental tissue. The presence of the parasite could then explain the lower level of EGF observed in women with Chagas’ disease and the deficiency on EGF signalization could explain the complex and variable symptoms associated to the connatal Chagas’ disease, namely those related to the malfunction of the placenta. The lower availability of EGF would thus diminish the release of PLAP, which is suggested to be one of the placental receptors used by T. cruzi for the placenta invasion, but on the other hand the parasite self is able to release the PLAP from
Placenta (2004), Vol. 25
the syncytiotrophoblast surface [9] thus the relationship among parasite, receptor and the molecules implied are somehow balanced. EGF induces actin depolymerization [21]; [22]; [23]; [24] which was also demonstrated in T. cruzi invasion to the placental villi [25]. It would be interesting to find out if EGFr could be another receptor pathway for the trypomastigotes to invade the placental villi since EGFr and PLAP seem to share elements of the same intracellular signalling network, probably the dual Calcium-IP3 signal pathway [25], [26].
ACKNOWLEDGEMENTS The authors are thankful to Dr Patricia Paglini and Biol. Walter Rivarola for providing trypomastigotes of T. cruzi, to Miriam Rabino for technical support and to Dion Whitehead for proof reading the manuscript. This work was supported by grants from Consejo de Investigaciones Cientı´ficas y Tecnolo´gicas de la Provincia de Co´rdoba (CONICOR), Repu´blica Argentina and Secretarı´a de Ciencia y Tecnologı´a de la Universidad Nacional de Co´rdoba (SeCyT).
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