Title: Plasmodium falciparum-Infected Erythrocytes Adhere Both in the Intervillous Space and on the Villous Surface of Human Placenta by Binding to the Low-Sulfated Chondroitin Sulfate Proteoglycan Receptor
Abstract: In pregnant women infected with Plasmodium falciparum, the parasite-infected red blood cells (IRBCs) sequester in the placenta through chondroitin 4-sulfate (C4S)-mediated adherence. The pattern of IRBC adherence in P. falciparum-infected placenta has been controversial. Moreover, the identity of the chondroitin sulfate proteoglycan (CSPG) receptor, that mediates IRBC adherence, and its location in the placenta have not been established. This study, using immunohistochemical techniques, clearly shows, for the first time, that the low-sulfated CSPGs of the placenta are localized predominantly in the intervillous space. Ex vivo IRBC adherence analyses demonstrate that the IRBCs are adhered to the CSPG receptors in the placenta in a C4S-dependent manner. This IRBC binding pattern was similar to that observed in P. falciparum-infected placentas. These data and the results of dual-fluorescence staining of the endogenous RBCs and syncytiotrophoblasts, and co-localization of CSPG and IRBC adherence unequivocally establish that the low-sulfated CSPGs are the major natural receptors for IRBC adherence in the placenta. Further, it was found that IRBCs adhere mainly in the intervillous space and also at significant levels to the syncytiotrophoblasts. Finally, the ex vivo IRBC adherence method described herein provides a reliable procedure for future studies for the assessment of the efficacy of C4S inhibitors and adhesion inhibitory antibodies. In pregnant women infected with Plasmodium falciparum, the parasite-infected red blood cells (IRBCs) sequester in the placenta through chondroitin 4-sulfate (C4S)-mediated adherence. The pattern of IRBC adherence in P. falciparum-infected placenta has been controversial. Moreover, the identity of the chondroitin sulfate proteoglycan (CSPG) receptor, that mediates IRBC adherence, and its location in the placenta have not been established. This study, using immunohistochemical techniques, clearly shows, for the first time, that the low-sulfated CSPGs of the placenta are localized predominantly in the intervillous space. Ex vivo IRBC adherence analyses demonstrate that the IRBCs are adhered to the CSPG receptors in the placenta in a C4S-dependent manner. This IRBC binding pattern was similar to that observed in P. falciparum-infected placentas. These data and the results of dual-fluorescence staining of the endogenous RBCs and syncytiotrophoblasts, and co-localization of CSPG and IRBC adherence unequivocally establish that the low-sulfated CSPGs are the major natural receptors for IRBC adherence in the placenta. Further, it was found that IRBCs adhere mainly in the intervillous space and also at significant levels to the syncytiotrophoblasts. Finally, the ex vivo IRBC adherence method described herein provides a reliable procedure for future studies for the assessment of the efficacy of C4S inhibitors and adhesion inhibitory antibodies. Of the four species of Plasmodium that infect humans, P. falciparum causes the most fatalities.1Trigg T Kondrachine AV The current global malaria situation.in: Sherman IW Malaria—Parasite Biology, Pathogenesis, and Protection. 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It is well known that, in P. falciparum-infected pregnant women, IRBCs sequester in the placenta,26Duffy PE Fried M Duffy PE Fried M Malaria In Pregnancy: Deadly Parasite, Susceptible Host. Taylor and Francis Press, New York2001: 1-245Crossref Google Scholar and in highly infected placentas, most of the intervillous space is filled with IRBCs. However, the pattern of IRBC adherence is still controversial. Although some investigators have observed IRBC binding to the villous surface, others have found that most IRBCs are localized in the intervillous space and not attached to the syncytiotrophoblasts.22Bray RS Sinden RE The sequestration of Plasmodium falciparum infected erythrocytes in the placenta.Trans R Soc Trop Med Hyg. 1979; 73: 716-719Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 26Duffy PE Fried M Duffy PE Fried M Malaria In Pregnancy: Deadly Parasite, Susceptible Host. Taylor and Francis Press, New York2001: 1-245Crossref Google Scholar, 40Walter PR Garin Y Blot P Placental pathologic changes in malaria: a histologic and ultrastructual study.Am J Pathol. 1982; 109: 330-342PubMed Google Scholar, 41Yamada M Stekete R Abramowsky C Kida M Wirima J Heymann D Rabbege J Breman J Aikawa M Plasmodium falciparum associated placental pathology: a light and electron microscopic and immunohistologic study.Am J Trop Med Hyg. 1989; 41: 161-168Crossref PubMed Scopus (92) Google Scholar In vitro studies using snap-frozen placental tissues showed IRBC binding only on the syncytiotrophoblasts.26Duffy PE Fried M Duffy PE Fried M Malaria In Pregnancy: Deadly Parasite, Susceptible Host. Taylor and Francis Press, New York2001: 1-245Crossref Google Scholar, 42Duffy PE Fried M Turncoat antibodies.Science. 2001; 293: 2009-2010Crossref PubMed Scopus (6) Google Scholar This could be because of the loss of the intervillous space material during the tissue processing and assay procedures, as suggested previously.26Duffy PE Fried M Duffy PE Fried M Malaria In Pregnancy: Deadly Parasite, Susceptible Host. Taylor and Francis Press, New York2001: 1-245Crossref Google Scholar, 42Duffy PE Fried M Turncoat antibodies.Science. 2001; 293: 2009-2010Crossref PubMed Scopus (6) Google Scholar The presence of fibrous filamentous materials and fibrinoid deposits in the intervillous space of the placental histosections has been reported previously,26Duffy PE Fried M Duffy PE Fried M Malaria In Pregnancy: Deadly Parasite, Susceptible Host. Taylor and Francis Press, New York2001: 1-245Crossref Google Scholar, 40Walter PR Garin Y Blot P Placental pathologic changes in malaria: a histologic and ultrastructual study.Am J Pathol. 1982; 109: 330-342PubMed Google Scholar but the possibility that the CSPG receptor present in association with the matrix-like material has not been investigated. It has been proposed that IRBCs present in the intervillous space of P. falciparum-infected placentas represent those dislodged from the placental villous surfaces during labor contractions and delivery26Duffy PE Fried M Duffy PE Fried M Malaria In Pregnancy: Deadly Parasite, Susceptible Host. Taylor and Francis Press, New York2001: 1-245Crossref Google Scholar or IRBCs bound to the C4S chains extending from the syncytiotrophoblasts to the intervillous space.43Miller LH Smith JD Motherhood and malaria.Nat Med. 1998; 4: 1244-1245Crossref PubMed Scopus (22) Google Scholar However, considering the surface area of the syncytiotrophoblasts compared to the volume of intervillous space, binding to syncytiotrophoblasts alone cannot explain massive accumulation of IRBCs in the infected placentas. Moreover, because it is widely believed that intervillous space is simply filled with maternal blood without any matrix-like compounds, there is doubt regarding the presence of immobilized CSPGs in the intervillous space. Therefore, there was no satisfactory explanation for the intervillous space IRBC adherence in the infected placentas. Because of these uncertainties, the identity and location of the major natural receptor for IRBC adherence in the placenta remain unresolved. Human placenta contains three types of CSPGs, high levels of unusually low-sulfated extracellular CSPGs, minor amounts of cell-associated CSPGs, and major amounts of an extracellular dermatan sulfate/chondroitin sulfate proteoglycan (DS/CSPG).44Achur RN Valiyaveettill M Alkhalil A Ockenhouse CF Gowda DC Characterization of proteoglycans of human placenta and identification of unique chondroitin sulfate proteoglycan of the intervillous spaces that mediates the adherence of Plasmodium falciparum-infected erythrocytes to the placenta.J Biol Chem. 2000; 275: 40344-40356Crossref PubMed Scopus (144) Google Scholar Of these proteoglycans, the low-sulfated CSPGs bind IRBCs most efficiently, and therefore, these CSPGs were proposed as the natural receptors.44Achur RN Valiyaveettill M Alkhalil A Ockenhouse CF Gowda DC Characterization of proteoglycans of human placenta and identification of unique chondroitin sulfate proteoglycan of the intervillous spaces that mediates the adherence of Plasmodium falciparum-infected erythrocytes to the placenta.J Biol Chem. 2000; 275: 40344-40356Crossref PubMed Scopus (144) Google Scholar However, there was no direct evidence for the type and location of CSPGs that mediate the IRBC adherence in the placenta. Therefore, this study was undertaken to resolve the above-mentioned controversies and to provide experimental proof that low-sulfated CSPGs are present in the intervillous space and serve as major receptors for IRBC adherence. Immunohistochemical analysis and ex vivo IRBC adherence studies using a modified procedure showed, for the first time, that the low-sulfated CSPGs are localized in the intervillous space, and that these are the major natural receptors for IRBC adherence in the placenta. Further, the results of dual-fluorescence staining of the endogenous RBCs and syncytiotrophoblasts, and co-localization of CSPG and IRBC adherence firmly establish that the IRBCs, by binding to the low-sulfated CSPGs, sequester predominantly in the intervillous space and at low but significant levels on the syncytiotrophoblast surface. Additionally, the ex vivo adherence assay developed in this study overcomes the problems associated with the preservation of the intervillous space materials and loss of bound IRBCs from the tissue section before examination under the microscope. Thus, the assay procedure is useful for studies testing the efficacy of potential inhibitors of IRBC adhesion and IRBC-adhesion inhibitory antibodies. The blood and placenta tissue samples were collected from the term placentas of P. falciparum-infected Cameroonian women, who were admitted for delivery at the Central Hospital, Yaounde, Cameroon. The nature of the project was explained and informed consent was obtained before the collection of the samples. The collection of samples was approved by the Ethical Committee, Ministry of Health, Cameroon, and by the institutional review board at Georgetown University, Washington, DC. Blood samples were cryopreserved in glycerolyte solution, transported, and stored until used. Placental tissues (1 × 1 cm) were fixed with 10% neutral buffered formalin in phosphate-buffered saline (PBS), pH 7.2. Tissues were cut into 5-μm-thick sections, mounted on glass slides, stained with hematoxylin and eosin (H&E), and examined under light microscope. Normal term placenta tissues were collected, under the Pennsylvania State University College of Medicine IRB approved protocol, from the healthy women who delivered babies at the Hershey Medical Center, Hershey, PA. Protease-free Proteus vulgaris chondroitinase ABC (120 U/mg), anti-Δdi-4S mouse monoclonal IgG, and anti-Δdi-6S mouse monoclonal IgM were purchased from Seikagaku America, Falmouth, MA. Anti-Δdi-4S and anti-Δdi-6S antibodies specifically recognize the unsaturated chondroitin sulfate disaccharide stubs, Δdi-4S and Δdi-6S, that formed at C4S and C6S chain attachment regions on core proteins when the proteoglycans were treated with chondroitinase ABC. Bovine tracheal chondroitin sulfate A (52% 4-sulfate, 39% 6-sulfate, and 9% nonsulfate), mouse anti-human glycophorin A and B monoclonal IgG, and 1-propyl gallate were from Sigma Chemical Co., St. Louis, MO. SYBR Green I, 4,6-diamidino-2-phenylindole (DAPI), Alexa Fluor 568-conjugated goat anti-mouse IgG (H + L), and goat anti-rabbit IgG (H + L) were from Molecular Probes, Eugene, OR. Titermax adjuvant was from CytRx Corp, Norcross, GA. Alkaline phosphatase-conjugated goat anti-rabbit IgG (H + L), horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L), and ABTS substrate were from Kirkegaard Perry Laboratories, Gaithersburg, MD. The Vectastain Elite ABC kit (containing biotinylated goat anti-mouse IgG, goat anti-mouse IgM and goat anti-rabbit IgG, horseradish peroxidase-conjugated avidin, and diaminobenzidine tetrahydrochloride substrate), hematoxylin, eosin, and Methyl Green were from Vector Laboratories, Burlingame, CA. Human blood and serum for parasite culturing were from Hershey Medical Center, Pennsylvania State University, Hershey, PA. The proteoglycans, BCSPG-2 (low-sulfated CSPGs) and DS/CSPG-2 (DS/CSPG), were isolated from the normal human term placentas and purified by CsBr density gradient centrifugation followed by gel filtration on Sepharose CL-4B as described previously.44Achur RN Valiyaveettill M Alkhalil A Ockenhouse CF Gowda DC Characterization of proteoglycans of human placenta and identification of unique chondroitin sulfate proteoglycan of the intervillous spaces that mediates the adherence of Plasmodium falciparum-infected erythrocytes to the placenta.J Biol Chem. 2000; 275: 40344-40356Crossref PubMed Scopus (144) Google Scholar To prepare preimmune serum, blood was collected from each rabbit before immunization. For the production of anti-CSPG antibody, the animals were immunized with 600 μg of the low-sulfated CSPGs in 300 μl of PBS, pH 7.2, emulsified with 300 μl of Titermax adjuvant. The animals were boosted after 2, 4, 7, 10, and 13 weeks with similar amounts of antigen. For anti-DS/CSPG antibody production, the rabbits were immunized with DS/CSPG (500 μg) in 250 μl of PBS, pH 7.2, emulsified with 250 μl of Titermax. The booster injections were given after 3, 5, and 6 weeks with the similar amounts of DS/CSPG. In both cases, the antibody responses were monitored by enzyme-linked immunosorbent assay. The antibody titers for CSPGs and DS/CSPG were maximal after 15 and 8 weeks of initial immunization, respectively. The IgG from the antisera were isolated by Protein A-Sepharose CL-4B affinity chromatography. Briefly, antisera were diluted with an equal volume of 100 mmol/L Tris-HCl buffer, pH 7.0, and chromatographed on Protein A-Sepharose CL-4B columns. After washing the columns, the bound antibodies were eluted with 0.1 mol/L of glycine, pH 3.0, and the pH adjusted to 7.0. The IgG concentrations were determined by absorption at 280 nm. Ninety-six-well microtiter plates were coated with 50 μl/well of the low-sulfated CSPGs (20 μg/ml) and DS/CSPG (10 μg/ml) in 100 mmol/L bicarbonate buffer, pH 9.0, at 4°C overnight. The wells were then blocked with 150 μl of 0.5% casein in 50 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5, containing 0.05% Tween 20 (TBST) at room temperature for 2 hours. In another set of experiment, the wells coated with proteoglycans were blocked with casein and incubated with chondroitinase ABC (5 mU) in 50 μl of 100 mmol/L Tris-HCl, 30 mmol/L NaOAc, pH 8.0, containing 0.01% bovine serum albumin (BSA) at 37°C for 5 hours.45Oike Y Kimata K Shinomura T Nakazawa K Suzuki S Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-beta-D-galactosidase (keratinases).Biochem J. 1980; 191: 193-207Crossref PubMed Scopus (201) Google Scholar The plates were washed with TBST, and incubated with 50 μl of sera serially diluted with TBST. The bound antibodies were measured using horseradish peroxidase-conjugated goat anti-rabbit IgG and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) substrate. The purified low-sulfated CSPGs or DS/CSPG (50 μg each) were treated with chondroitinase ABC (20 mU) in 50 μl of 100 mmol/L Tris-HCl, 30 mmol/L NaOAc, pH 8.0, containing 0.01% BSA at 37°C for 5 hours,45Oike Y Kimata K Shinomura T Nakazawa K Suzuki S Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-beta-D-galactosidase (keratinases).Biochem J. 1980; 191: 193-207Crossref PubMed Scopus (201) Google Scholar and used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. SDS-PAGE was performed according to the procedure of Laemmli46Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar using 4 to 15% gradient polyacrylamide mini gels under reducing conditions. The gels were stained with Coomassie Blue followed by Alcian Blue and ammoniacal silver.47Krueger Jr, RC Schwartz NB An improved method of sequential Alcian Blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gel.Anal Biochem. 1987; 167: 295-300Crossref PubMed Scopus (44) Google Scholar The untreated and chondroitinase ABC-treated placental proteoglycans (10 to 15 μg) were electrophoresed as above and transferred onto polyvinylidene difluoride membranes. The membranes