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Effect of Zinc-Treated Entamoeba histolytica on the Human Polymorphonuclear Respiratory Burst
Archives of Medical Research 36 (2005) 75–79
BRIEF REPORT
Effect of Zinc-Treated Entamoeba histolytica on the Human Polymorphonuclear Respiratory Burst Gloria B. Vega-Robledo,a Erika Leandro,b Rau´l Silva,b Alfonso Olivosa and Guadalupe Ricob a
Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico (UNAM), Me´xico D.F., Me´xico b Departamento de Inmunologı´a, Hospital de Pediatrı´a, Centro Me´dico Nacional Siglo XXI (CMN-SXXI), Instituto Mexicano del Seguro Social (IMSS), Me´xico D.F., Me´xico Received for publication May 3, 2004; accepted October 1, 2004 (04/101).
One of the mechanisms that Entamoeba histolytica uses to evade host immune response is inhibition of the polymorphonuclear (PMN) leukocyte respiratory burst. In studies previously conducted in a model used in our laboratory, we observed that when treating trophozoites with different zinc concentrations certain amebic functions are inhibited while significantly limiting development of hepatic abscess in golden hamsters (Mesocricetus aureatus). We carried out an in vitro study using a chemoluminescent method to assess the effect zinc-treated amebic trophozoites exercise on respiratory burst in human PMNs. We measured response of PMNs incubated with E. histolytica trophozoites from cultures with TYI-S33 medium alone and with zinc. Zinc concentrations between 0.1 and 1.0 mM did not affect amebic trophozoite viability, and PMNs in contact with these in a zinc-free medium had an oxidative response similar to that obtained with zymosan and significantly greater (p ⬍0.05) than that generated by cells co-incubated with amebas cultured in TYI-S33 medium alone. These results suggest that zinc alters the amebic mechanism that inhibits the oxidative function of human polymorphonuclear leukocyte. 쑖 2005 IMSS. Published by Elsevier Inc. Key Words: Entamoeba histolytica, Ameba, Protozoa, Parasitic, Chemoluminescence, Respiratory burst, Amebic function, Polymorphonuclear, Zinc.
Introduction Entamoeba histolytica is a parasite with great invasive and evasive potential due to its lithic activity, which allows it to penetrate and displace itself through tissues, as well as due to the mechanisms it has developed to evade host immune responses. In this respect, it is known that the ameba, by means of a surface lectin, inhibits the complement membrane attachment complex. The parasite is capable of redistributing the antibodies on its surface and the attached complement in a phenomenon known as capping. Caps can be freed into the medium or internalized, protecting the
Address reprint requests to: G.B. Vega-Robledo, Coordinacio´n de Educacio´n Me´dica Continua, 3er piso, Edificio B, Facultad de Medicina, UNAM, 04510 Me´xico, D.F., Me´xico. Phone: (⫹52) (55) 5623-2504; Fax: (⫹52) (55) 5623-2440; E-mail: [email protected]
0188-4409/05 $–see front matter. Copyright d o i : 1 0 .1 0 16 / j . a rc m e d .2 00 4 .1 0 .0 05
ameba from damage caused by these components (1). Additionally, by means of different mechanisms it induces an increase in calcium in the white cell (2), altering its function immediately and thus modifying important cellular processes including certain host immunity effectors. During acute phase of invasive amebiasis, the following changes have been found in patients: decrease in helper T lymphocyte count (CD4) and increase in cytotoxic T lymphocytes (CD8), as well as decrease in proliferative response of T lymphocytes to amebic antigens (3). Other in vitro assays have shown that amebic trophozoites are able to inhibit polymorphonuclear respiratory bursts (4–7) and to destroy them (8). In a previous study, we observed that zinc (0.1 and 1 mM) inhibited replication and adherence of E. histolytica and significantly diminished development of amebic hepatic abscesses in hamsters (9). Under normal conditions, the ameba alters phagocytic functions and induces death of human leukocytes, suppressing
쑖 2005 IMSS. Published by Elsevier Inc.
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Vega-Robledo et al. / Archives of Medical Research 36 (2005) 75–79
one of our main defense mechanisms. It is then necessary to evaluate other mechanisms that could be disturbed by zinc; therefore, our objective was to study the effect that E. histolytica treated with zinc may have on the respiratory bursts in PMNs.
Rochester, NY, USA) 1 × 10⫺6 M and 200 µL opsonized zymosan (12.5 mg/mL). Vials were continuously counted for 30 min using a BioOrbit 1250 luminometer (LKB-Pharmacia) and results were registered in mV and expressed as the area under the curve or as an average number ⫾ standard error (SE) of 1 × 106 mV/106 cells in 1 min at maximum chemoluminescence peak.
Materials and Methods Parasite Culture
Statistical Analysis
E. histolytica HM1-IMSS trophozoites were axenically grown in TYI-S33 medium (10). Amebas (1 × 105) (live trophozoites) were placed in tubes with 10 mL TYI-S33 culture medium supplemented with zinc sulfate (0.1 and 1.0 mM) and were incubated at 37⬚C for 72 h. Amebas were harvested by chilling on ice for 10 min and were subsequently washed three times in cold phosphate-buffered saline solution (PBS) 0.15 M pH 7.4 to eliminate any residual zinc.
Results were analyzed using Fisher exact test (11) for variance and nonparametric Mann–Whitney U test (12).
Results Cell Viability At 180 min PMN incubated alone had a 95% viability, amebic trophozoites alone were 90% alive. The different co-incubation conditions are summarized in Table 1.
Polymorphonuclear Leukocytes Twenty-milliliter samples of heparinized venous blood were obtained from healthy adult donors. The blood, diluted 1:2 with PBS, pH 7.2, was placed over 5 mL of Polymorphoprep d ⫽ 1.113 (Nycomed Pharma AS, Oslo, Norway). The suspension was centrifuged at 400 × g for 40 min at 20⬚C and interphase cells were collected and washed three times with PBS. Remaining erythrocytes were lysed by hypotonic shock with NH4Cl 155 mM and the button was washed twice with PBS. A total of 97% of these cells were PMNs and the remainder, lymphocytes. For respiratory burst assays (measured using chemoluminiscence), PMNs were adjusted to a concentration of 1 × 107 cells per mL and used immediately. Viability Amebic trophozoite and PMN leukocyte viability were 95 and 90%, respectively, as determined using trypan blue exclusion technique. Nonstimulated PMN leukocytes were coincubated for 3 h at 37⬚C with control (wild-type) amebic trophozoites (cell ratio 10:1) or amebic trophozoites previously cultured with zinc (0.1, 1.0 mM). Aliquots of these combinations were dyed and placed on slides for microscopic observation and viability count.
Entamoeba histolytica vs. PMN The observed mortality of PMNs co-incubated with control E. histolytica trophozoites (10:1) during 3 h was 47%, significantly greater (p ⬍0.05) than in trophozoites pre-treated with zinc (0.1 and 1.0 mM), which was 28 and 21%, respectively. PMNs vs. Entamoeba histolytica Amebas pre-treated with zinc (0.1 and 1.0 mM) on interacting with PMNs had a mortality rate of 19 and 25%, respectively, significantly greater (p ⬍0.001) than that observed with control ameba (10%), which did not vary during contact with PMNs. Chemoluminiscence Zymosan-stimulated PMNs emitted luminescence equivalent to 40.3 ⫾ 6.5 mV/106 (Figure 1), which diminished significantly (p ⬍0.05) in cells co-incubated with control
Table 1. Mortality
Chemoluminescence
Co-incubation
Cells were placed in 3-mL polystyrene vials (LKB-Pharmacia, Uppsala, Sweden) with 1 × 106 PMN in 100 µL PBS, or 100 µL PBS with 5 × 103 amebas (grown for 72 h in culture medium TYI-S33 alone or added with zinc and washed). Vials were incubated for 15 min at 37⬚C, and the following were added: 700 µL luminol (Eastman Kodak,
PMN ⫹ E. histolytica control PMN ⫹ E. histolytica pre-treated with 0.1 M Zn PMN ⫹ E. histolytica pre-treated with 1.0 M Zn
15 min
180 min
PMN Amebas PMN
5% 10% 10%
PMN Amebas PMN
47% 10% 28%
Amebas PMN Amebas
12% 10% 12%
Amebas PMN Amebas
19% 21% 25%
Entamoeba histolytica, Zinc, Polymorphonuclear Respiratory Burst
amebic trophozoites (28.4 ⫾ 4.3 mV/106). In PMNs coincubated with amebic trophozoites pre-treated with 0.1 and 1.0 mM zinc, luminescence of 44.5 ⫾ 11.4 and 52.3 ⫾ 9.1 mV/106, respectively, were registered (Figure 1). These values were greater (p ⬍0.05) than those recorded for PMNs co-incubated with control ameba. These did not differ significantly from those obtained from PMNs solely stimulated with zymosan.
Discussion Zinc prevents inhibition of PMN respiratory burst by E. histolytica, as we observed when these cells came into contact with trophozoites pre-treated with this metal. Under normal conditions, E. histolytica alters neutrophil function, and it has been reported that amebic proteins induce LTB4 production in this cell (13). The LTB4 molecule, as well as gamma interferon, tumor necrosis factor, and bacterial lipopolysaccharides, stimulate neutrophil respiratory burst through protein kinase C-dependent pathways. This initial stimulus (13), which could be responsible in part for the granulomatous reaction observed in amebic injuries, is rapidly followed by inhibition through diverse mechanisms that the ameba exercises on white-cell respiratory burst (13). One mechanism could be increase of prostaglandins and AMPc that E. histolytica induces on the host cell, capable of inhibiting protein kinase C. Another mechanism could
77
be through its oxygen-free radical inactivating enzymes including iron-dependent superoxide dismutase (FeSOD) that catalyzes superoxide (14), acid phosphatase or tyrosinphosphatase, similar to Francisella tularensis (15), or the Leishmania lipophosphoglucans, in inhibiting neutrophil respiratory burst (16), or as in the case of amebic catalase, peroxidase (17) and alkyhydroxyperoxide reductase (18). The phagocyte appears to respond differently depending on the site selected by the molecule that unites to it, as inferred from observations recorded from the principal phagocyte receptors involved in the infection. Participating elements include immunoglobin G (FcgammaR) Fc receptors that involve the Rac GTPase regulator of the NADPH oxidase enzyme, capable of stimulating respiratory burst, and complement (CR3) receptors that involve Rho GTPase and result in phagocyte respiratory burst inhibition (19). In addition, these Rac and Rho genes appear to possess activating or inhibiting effects, respectively, on integrin-dependent kB nuclear factor (NF-kB) signaling (20). Genes of Rac (21–23) and Rho (24) protein kinase families have been characterized in E. histolytica. From this, we infer that the infected organisms are capable of sending signals of a different type from the cell surface (25). These could participate in modulation of certain phagocyte functions, as occurs with respiratory burst that is modified by a considerable number of microorganisms, among them the ameba (4,16,26,27). If so, zinc, with its ability to unite with cysteine, could induce alterations in principal surface amebic proteins that modify signal-sending during contact with white cells. Another possibility
Figure 1. Data are expressed as means ⫾ standard error (SE) of 10 separate experiments. Bars represent data from four different groups: a) polymorphonuclear phagocytes (PMNs) with opsonized zymosan (OZ); b) PMNs ⫹ normal E. histolytica with OZ; c) PMNs ⫹ E. histolytica pre-treated with 0.01 mM of Zn in presence of OZ, and d) PMNs ⫹ E. histolytica pre-treated with 1.0 mM Zn in presence of OZ. Amebas alone in presence of zymosan did not emit luminescence. Statistical analysis: a vs. b, p ⬍0.05; b vs. c, d, p ⬍0.05; a vs. c, d, not significant (N.S.); c vs. d, N.S.
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Vega-Robledo et al. / Archives of Medical Research 36 (2005) 75–79
is action of the metal specifically on the cysteine-rich 29 kDa-alkylhydroxyperoxidoreductase protein. In a previous study (28), we observed that zinc modified the dynamics of atypical tubulin in the parasite, a key protein involved in the cytoskeleton function, participating in replication, movement, receptor formation, and exocytosis (29). It must be added that there is antagonism between zinc and calcium that induces polymerization of the tubulin but in presence of zinc, this metal has greater affinity for the tubulin. Thus, Zn is the metal that regulates union and brake polymerization (9), manifested mainly as changes in replication and adherence (8). It is feasible that zinc, due to its ability to induce changes in some enzymes participating in amebic oxidative activity, blocks their inhibiting effect on the white cell, thus allowing for expression of respiratory burst. PMN leukocytes co-incubated with control ameba (1:10) had a 47% mortality rate [in a similar study, Salata observed lysis in 67% (30), a divergence that could be due to the cell ratio the investigators used (1:300)], while in PMNs coincubated with trophozoites pre-treated with zinc (0.1 and 1.0 mM), mortality diminished significantly. Nonetheless, zinc-treated trophozoites could not inhibit PMN respiratory burst and were only capable of eliminating a certain percentage of these (21–28%). It is important to note that PMNs capable of normally expressing their respiratory burst had a greater number of survivors, although we must bear in mind that they faced a trophozoite that had been functionally altered by zinc. On the other hand, mortality of these trophozoites in the face of PMNs was greater than that of the control trophozoite, but not 100%, even when PMNs expressed their respiratory burst and the trophozoite was altered. We know that in elimination of parasite by host, cytokine-activated macrophages kill E. histolytica using oxygen- and nonoxygen-dependent mechanisms (13), which in accordance with our results, the oxygen-dependent mechanism occurred in PMNs. The doses used in vitro (0.1, 1.0 mM) to inhibit amebic function are greater than values of physiologic levels of Zn in plasma (0.015 mM). According to the literature and the viability assays, this dose is nontoxic (31,32). The importance of the mechanisms expressed by parasites evading the immune response should direct our attention toward elements that block this inhibiting effect and allow for a normal host immune response.
Acknowledgments This work was supported by Consejo Nacional de Ciencia y Tecnologı´a (CONACYT, Me´xico) grant no. 400309-5-27671.
References 1. Braga L, Ninomiya H, McCoy J, Eacher S, Wiedner T, Pham C, Wood S, Sonis P, Petri W. Inhibition of the complement membrane attach complex by the galactose-specific adhesin of Entamoeba histolytica. Arch Med Res 1992;23:133–139.
2. Ravdin J, Moreau F, Sullivan J, Petri W, Mandell G. Relationship of free intracellular calcium to the cytolytic activity of Entamoeba histolytica. Infect Immun 1988;56:1505–1512. 3. Salata R, Martı´nez-Palomo A. Patients treated for amebic liver abscess develop cell mediated immune responses effective in vitro against E. histolytica. J Immunol 1986;136:2633–2639. 4. Salata R, Pearson R, Ravdin J. Interaction of human leukocytes and Entamoeba histolytica killing of virulent amebae by the activated macrophages. J Clin Invest 1985;76:491–499. 5. Denis M, Chadee K. Immunopathology of Entamoeba histolytica infections. Parasitol Today 1988;4:828–831. 6. Arbo A, Hoefsloot M, Ramı´rez A, Santos JI. Entamoeba histolytica inhibits the respiratory burst of polymorphonuclear leukocytes. Arch Invest Med 1990;21:57–61. 7. Rico G, Dı´az G, Gime´nez S, Kretschmer R. Effect of the monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica upon the respiratory burst of human leucocytes. Arch Med Res 1992;23(2):157–159. 8. Jarumilinta R, Kradolfer F. The toxic effect of Entamoeba histolytica on leukocytes. Ann Trop Med Parasitol 1964;58:375–380. 9. Vega-Robledo G, Carrero J, Ortiz-Ortiz L. Effect of zinc on Entamoeba histolytica pathogenicity. Parasitol Res 1999;85:487–492. 10. Diamond L. Axenic cultivation of Entamoeba histolytica. Science 1962;134:336–339. 11. Fisher RA. Statistical Methods for Research Workers. Edinburg, UK: Oliver & Boyd;1954. 12. Mann H, Whitney D. On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat 1947;18:50–65. 13. Denis M, Chadee K. Cytokine activation of murine macrophages for in vitro killing of Entamoeba histolytica trophozoites. Infect Immun 1989;57:1750–1755. 14. Tannich E, Bruchhaus Y, Walter R, Horstmann R. Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase. Mol Biochem Parasitol 1991;49:61–67. 15. Reilly T, Baron G, Nano F, Kublenschmidt M. Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis. J Biol Chem 1996;271:10973–10983. 16. McNeely T, Rosen G, Londner M, Turco S. Inhibitory effects on protein kinase C activity by lipophosphoglycan fragments and glycosylphosphatidylinositol antigens of the protozoan parasite Leishmania. Biochem J 1989;259:601–608. 17. Jinfu C, Jinglong C, Xiang H, Youyao L, Gengsun D, Wenlie C. The detoxicating enzymes of Entamoeba histolytica and their detoxifying roles. Arch Med Res 1997;28:93–95. 18. Bruchhaus Y, Richter Y, Tannich E. Characterization of two Entamoeba histolytica proteins that inactivate reactive oxygen species. Arch Med Res 1997;28:591–592. 19. Caron E, Hall A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPasas. Science 1998;282:1717– 1721. 20. Reyes-Reyes M, Mora N, Zentella A, Rosales C. Phosphatidylinositol 3-kinase mediates integrin-dependent NF-kB and MAPK activation through separate signaling pathways. J Cell Sci 2001;114:1579–1589. 21. Que X, Reed S. Expression and characterization of a rac family protein kinase of Entamoeba histolytica. Mol Biochem Parasitol 1994;58: 177–181. 22. Castellanos S, Montcourrier P, Chaurier P. Membrane recruitment of Rac triggers phagocytosis. J Cell Sci 2000;112:2955–2961. 23. Greenberg S, Grinstein S. Phagocytosis and innate immunity. Curr Op Immunol 2002;14:136–145. 24. Lohia A, Samuelson J. Molecular cloning of a rho family gene of Entamoeba histolytica. Mol Biochem Parasitol 1993;58:177–181. 25. Texeira J, Mann B. Entamoeba histolytica induced dephosphorylation in host cells. Infect Immun 2002;70:1816–1823.
Entamoeba histolytica, Zinc, Polymorphonuclear Respiratory Burst 26. Tirada L, Johansen K, Nowar S, Vasil A, Vasil M. Pseudomona aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity. Infect Immun 1999;67:2371–2376. 27. Leiro J, Iglesias R, Parama´ M, Sanmartı´n L, Ubeira F. Respiratory burst responses of rat macrophages to microsporidian spores. Exp Parasitol 2001;98:1–9. 28. Vega-Robledo G, Carrero J. Zinc: interference with the calcium function in Entamoeba histolytica. Arch Med Res 1997;28:110–112. 29. Kativar S, Endlind T. Entamoeba histolytica encodes a highly divergent β tubulina. J Eukaryot Microbiol 1996;43:31–34.
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30. Salata R, Ravdin J. The interaction of human neutrophils and Entamoeba histolytica increases cytopathogenicity for liver cell monolayers. J Infect Dis 1986;154:19–26. 31. Boukaiba N, Flament C, Acher S, Chappuis P, Piau A, Fusselier M, Dardenne M, Lemmonier D. A physiological amount of zinc supplementation: effects on nutritional liquid and thymic status in an elderly population. Am J Clin Nutr 1993;57:566–572. 32. Wasney M, Aamodt R, Rumble W, Henkin R. Kinetic analysis of zinc metabolism and its regulation in normal humans. Am J Physiol 1986;251:398–408.
BRIEF REPORT
Effect of Zinc-Treated Entamoeba histolytica on the Human Polymorphonuclear Respiratory Burst Gloria B. Vega-Robledo,a Erika Leandro,b Rau´l Silva,b Alfonso Olivosa and Guadalupe Ricob a
Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico (UNAM), Me´xico D.F., Me´xico b Departamento de Inmunologı´a, Hospital de Pediatrı´a, Centro Me´dico Nacional Siglo XXI (CMN-SXXI), Instituto Mexicano del Seguro Social (IMSS), Me´xico D.F., Me´xico Received for publication May 3, 2004; accepted October 1, 2004 (04/101).
One of the mechanisms that Entamoeba histolytica uses to evade host immune response is inhibition of the polymorphonuclear (PMN) leukocyte respiratory burst. In studies previously conducted in a model used in our laboratory, we observed that when treating trophozoites with different zinc concentrations certain amebic functions are inhibited while significantly limiting development of hepatic abscess in golden hamsters (Mesocricetus aureatus). We carried out an in vitro study using a chemoluminescent method to assess the effect zinc-treated amebic trophozoites exercise on respiratory burst in human PMNs. We measured response of PMNs incubated with E. histolytica trophozoites from cultures with TYI-S33 medium alone and with zinc. Zinc concentrations between 0.1 and 1.0 mM did not affect amebic trophozoite viability, and PMNs in contact with these in a zinc-free medium had an oxidative response similar to that obtained with zymosan and significantly greater (p ⬍0.05) than that generated by cells co-incubated with amebas cultured in TYI-S33 medium alone. These results suggest that zinc alters the amebic mechanism that inhibits the oxidative function of human polymorphonuclear leukocyte. 쑖 2005 IMSS. Published by Elsevier Inc. Key Words: Entamoeba histolytica, Ameba, Protozoa, Parasitic, Chemoluminescence, Respiratory burst, Amebic function, Polymorphonuclear, Zinc.
Introduction Entamoeba histolytica is a parasite with great invasive and evasive potential due to its lithic activity, which allows it to penetrate and displace itself through tissues, as well as due to the mechanisms it has developed to evade host immune responses. In this respect, it is known that the ameba, by means of a surface lectin, inhibits the complement membrane attachment complex. The parasite is capable of redistributing the antibodies on its surface and the attached complement in a phenomenon known as capping. Caps can be freed into the medium or internalized, protecting the
Address reprint requests to: G.B. Vega-Robledo, Coordinacio´n de Educacio´n Me´dica Continua, 3er piso, Edificio B, Facultad de Medicina, UNAM, 04510 Me´xico, D.F., Me´xico. Phone: (⫹52) (55) 5623-2504; Fax: (⫹52) (55) 5623-2440; E-mail: [email protected]
0188-4409/05 $–see front matter. Copyright d o i : 1 0 .1 0 16 / j . a rc m e d .2 00 4 .1 0 .0 05
ameba from damage caused by these components (1). Additionally, by means of different mechanisms it induces an increase in calcium in the white cell (2), altering its function immediately and thus modifying important cellular processes including certain host immunity effectors. During acute phase of invasive amebiasis, the following changes have been found in patients: decrease in helper T lymphocyte count (CD4) and increase in cytotoxic T lymphocytes (CD8), as well as decrease in proliferative response of T lymphocytes to amebic antigens (3). Other in vitro assays have shown that amebic trophozoites are able to inhibit polymorphonuclear respiratory bursts (4–7) and to destroy them (8). In a previous study, we observed that zinc (0.1 and 1 mM) inhibited replication and adherence of E. histolytica and significantly diminished development of amebic hepatic abscesses in hamsters (9). Under normal conditions, the ameba alters phagocytic functions and induces death of human leukocytes, suppressing
쑖 2005 IMSS. Published by Elsevier Inc.
76
Vega-Robledo et al. / Archives of Medical Research 36 (2005) 75–79
one of our main defense mechanisms. It is then necessary to evaluate other mechanisms that could be disturbed by zinc; therefore, our objective was to study the effect that E. histolytica treated with zinc may have on the respiratory bursts in PMNs.
Rochester, NY, USA) 1 × 10⫺6 M and 200 µL opsonized zymosan (12.5 mg/mL). Vials were continuously counted for 30 min using a BioOrbit 1250 luminometer (LKB-Pharmacia) and results were registered in mV and expressed as the area under the curve or as an average number ⫾ standard error (SE) of 1 × 106 mV/106 cells in 1 min at maximum chemoluminescence peak.
Materials and Methods Parasite Culture
Statistical Analysis
E. histolytica HM1-IMSS trophozoites were axenically grown in TYI-S33 medium (10). Amebas (1 × 105) (live trophozoites) were placed in tubes with 10 mL TYI-S33 culture medium supplemented with zinc sulfate (0.1 and 1.0 mM) and were incubated at 37⬚C for 72 h. Amebas were harvested by chilling on ice for 10 min and were subsequently washed three times in cold phosphate-buffered saline solution (PBS) 0.15 M pH 7.4 to eliminate any residual zinc.
Results were analyzed using Fisher exact test (11) for variance and nonparametric Mann–Whitney U test (12).
Results Cell Viability At 180 min PMN incubated alone had a 95% viability, amebic trophozoites alone were 90% alive. The different co-incubation conditions are summarized in Table 1.
Polymorphonuclear Leukocytes Twenty-milliliter samples of heparinized venous blood were obtained from healthy adult donors. The blood, diluted 1:2 with PBS, pH 7.2, was placed over 5 mL of Polymorphoprep d ⫽ 1.113 (Nycomed Pharma AS, Oslo, Norway). The suspension was centrifuged at 400 × g for 40 min at 20⬚C and interphase cells were collected and washed three times with PBS. Remaining erythrocytes were lysed by hypotonic shock with NH4Cl 155 mM and the button was washed twice with PBS. A total of 97% of these cells were PMNs and the remainder, lymphocytes. For respiratory burst assays (measured using chemoluminiscence), PMNs were adjusted to a concentration of 1 × 107 cells per mL and used immediately. Viability Amebic trophozoite and PMN leukocyte viability were 95 and 90%, respectively, as determined using trypan blue exclusion technique. Nonstimulated PMN leukocytes were coincubated for 3 h at 37⬚C with control (wild-type) amebic trophozoites (cell ratio 10:1) or amebic trophozoites previously cultured with zinc (0.1, 1.0 mM). Aliquots of these combinations were dyed and placed on slides for microscopic observation and viability count.
Entamoeba histolytica vs. PMN The observed mortality of PMNs co-incubated with control E. histolytica trophozoites (10:1) during 3 h was 47%, significantly greater (p ⬍0.05) than in trophozoites pre-treated with zinc (0.1 and 1.0 mM), which was 28 and 21%, respectively. PMNs vs. Entamoeba histolytica Amebas pre-treated with zinc (0.1 and 1.0 mM) on interacting with PMNs had a mortality rate of 19 and 25%, respectively, significantly greater (p ⬍0.001) than that observed with control ameba (10%), which did not vary during contact with PMNs. Chemoluminiscence Zymosan-stimulated PMNs emitted luminescence equivalent to 40.3 ⫾ 6.5 mV/106 (Figure 1), which diminished significantly (p ⬍0.05) in cells co-incubated with control
Table 1. Mortality
Chemoluminescence
Co-incubation
Cells were placed in 3-mL polystyrene vials (LKB-Pharmacia, Uppsala, Sweden) with 1 × 106 PMN in 100 µL PBS, or 100 µL PBS with 5 × 103 amebas (grown for 72 h in culture medium TYI-S33 alone or added with zinc and washed). Vials were incubated for 15 min at 37⬚C, and the following were added: 700 µL luminol (Eastman Kodak,
PMN ⫹ E. histolytica control PMN ⫹ E. histolytica pre-treated with 0.1 M Zn PMN ⫹ E. histolytica pre-treated with 1.0 M Zn
15 min
180 min
PMN Amebas PMN
5% 10% 10%
PMN Amebas PMN
47% 10% 28%
Amebas PMN Amebas
12% 10% 12%
Amebas PMN Amebas
19% 21% 25%
Entamoeba histolytica, Zinc, Polymorphonuclear Respiratory Burst
amebic trophozoites (28.4 ⫾ 4.3 mV/106). In PMNs coincubated with amebic trophozoites pre-treated with 0.1 and 1.0 mM zinc, luminescence of 44.5 ⫾ 11.4 and 52.3 ⫾ 9.1 mV/106, respectively, were registered (Figure 1). These values were greater (p ⬍0.05) than those recorded for PMNs co-incubated with control ameba. These did not differ significantly from those obtained from PMNs solely stimulated with zymosan.
Discussion Zinc prevents inhibition of PMN respiratory burst by E. histolytica, as we observed when these cells came into contact with trophozoites pre-treated with this metal. Under normal conditions, E. histolytica alters neutrophil function, and it has been reported that amebic proteins induce LTB4 production in this cell (13). The LTB4 molecule, as well as gamma interferon, tumor necrosis factor, and bacterial lipopolysaccharides, stimulate neutrophil respiratory burst through protein kinase C-dependent pathways. This initial stimulus (13), which could be responsible in part for the granulomatous reaction observed in amebic injuries, is rapidly followed by inhibition through diverse mechanisms that the ameba exercises on white-cell respiratory burst (13). One mechanism could be increase of prostaglandins and AMPc that E. histolytica induces on the host cell, capable of inhibiting protein kinase C. Another mechanism could
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be through its oxygen-free radical inactivating enzymes including iron-dependent superoxide dismutase (FeSOD) that catalyzes superoxide (14), acid phosphatase or tyrosinphosphatase, similar to Francisella tularensis (15), or the Leishmania lipophosphoglucans, in inhibiting neutrophil respiratory burst (16), or as in the case of amebic catalase, peroxidase (17) and alkyhydroxyperoxide reductase (18). The phagocyte appears to respond differently depending on the site selected by the molecule that unites to it, as inferred from observations recorded from the principal phagocyte receptors involved in the infection. Participating elements include immunoglobin G (FcgammaR) Fc receptors that involve the Rac GTPase regulator of the NADPH oxidase enzyme, capable of stimulating respiratory burst, and complement (CR3) receptors that involve Rho GTPase and result in phagocyte respiratory burst inhibition (19). In addition, these Rac and Rho genes appear to possess activating or inhibiting effects, respectively, on integrin-dependent kB nuclear factor (NF-kB) signaling (20). Genes of Rac (21–23) and Rho (24) protein kinase families have been characterized in E. histolytica. From this, we infer that the infected organisms are capable of sending signals of a different type from the cell surface (25). These could participate in modulation of certain phagocyte functions, as occurs with respiratory burst that is modified by a considerable number of microorganisms, among them the ameba (4,16,26,27). If so, zinc, with its ability to unite with cysteine, could induce alterations in principal surface amebic proteins that modify signal-sending during contact with white cells. Another possibility
Figure 1. Data are expressed as means ⫾ standard error (SE) of 10 separate experiments. Bars represent data from four different groups: a) polymorphonuclear phagocytes (PMNs) with opsonized zymosan (OZ); b) PMNs ⫹ normal E. histolytica with OZ; c) PMNs ⫹ E. histolytica pre-treated with 0.01 mM of Zn in presence of OZ, and d) PMNs ⫹ E. histolytica pre-treated with 1.0 mM Zn in presence of OZ. Amebas alone in presence of zymosan did not emit luminescence. Statistical analysis: a vs. b, p ⬍0.05; b vs. c, d, p ⬍0.05; a vs. c, d, not significant (N.S.); c vs. d, N.S.
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is action of the metal specifically on the cysteine-rich 29 kDa-alkylhydroxyperoxidoreductase protein. In a previous study (28), we observed that zinc modified the dynamics of atypical tubulin in the parasite, a key protein involved in the cytoskeleton function, participating in replication, movement, receptor formation, and exocytosis (29). It must be added that there is antagonism between zinc and calcium that induces polymerization of the tubulin but in presence of zinc, this metal has greater affinity for the tubulin. Thus, Zn is the metal that regulates union and brake polymerization (9), manifested mainly as changes in replication and adherence (8). It is feasible that zinc, due to its ability to induce changes in some enzymes participating in amebic oxidative activity, blocks their inhibiting effect on the white cell, thus allowing for expression of respiratory burst. PMN leukocytes co-incubated with control ameba (1:10) had a 47% mortality rate [in a similar study, Salata observed lysis in 67% (30), a divergence that could be due to the cell ratio the investigators used (1:300)], while in PMNs coincubated with trophozoites pre-treated with zinc (0.1 and 1.0 mM), mortality diminished significantly. Nonetheless, zinc-treated trophozoites could not inhibit PMN respiratory burst and were only capable of eliminating a certain percentage of these (21–28%). It is important to note that PMNs capable of normally expressing their respiratory burst had a greater number of survivors, although we must bear in mind that they faced a trophozoite that had been functionally altered by zinc. On the other hand, mortality of these trophozoites in the face of PMNs was greater than that of the control trophozoite, but not 100%, even when PMNs expressed their respiratory burst and the trophozoite was altered. We know that in elimination of parasite by host, cytokine-activated macrophages kill E. histolytica using oxygen- and nonoxygen-dependent mechanisms (13), which in accordance with our results, the oxygen-dependent mechanism occurred in PMNs. The doses used in vitro (0.1, 1.0 mM) to inhibit amebic function are greater than values of physiologic levels of Zn in plasma (0.015 mM). According to the literature and the viability assays, this dose is nontoxic (31,32). The importance of the mechanisms expressed by parasites evading the immune response should direct our attention toward elements that block this inhibiting effect and allow for a normal host immune response.
Acknowledgments This work was supported by Consejo Nacional de Ciencia y Tecnologı´a (CONACYT, Me´xico) grant no. 400309-5-27671.
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