Clin. exp. Immunol. (1978) 34, 10-18.
Activation of the alternative pathway of complement by Entamoeba histolytica L. ORTIZ-ORTIZ, RUTH CAPIN, NORMA R. CAPIN, B. SEPULVEDA & G. ZAMACONA Instituto de Investigaciones Biomidicas, Uuiversidad Nacional Antonoma de Mexico, Apartado Postal 70228, Mexico 20 and Servicios de Alergia e Immunologia Clinica y Gastroenterologia, Hospital General, CMN del IMSS, Mexico 7, Mexico
(Received 20 February 1978)
SUMMARY
A cytopathogenic effect was observed when Entamoeba histolytica was exposed to human sera from individuals with no clinical history or laboratory evidence of amoebiasis. Absorption studies showed that the effect was not due to natural antibodies. Studies performed using ethylenediamine tetracetic acid (EDTA), cobra venom factor (CoF) and heat-inactivation at 560C, indicated that the cytopathogenic effect was complement dependent. Furthermore, by using ethylene glycol tetracetic acid (EGTA) and Mg' +, zymosan, heat-inactivation at 50'C to destroy the activity of factor B of the alternative pathway, as well as electrophoretic studies with anti-human factor B, it was possible to determine that E. histolytica activated the properdin pathway. Finally, complement determinations indicated that incubation of E. histolytica with normal human serum consumed complement. The diminution in CH50 correlated with a consumption of C3 but not of Cl, C4 and C2. It was concluded from these results that trophozoites of E. histolytica activate the alternative pathway ofthe human complement system.
INTRODUCTION Entamoeba histolytica is a protozoan found in most parts of the world. The parasite usually confines its activity to the bowel lumen; however, for reasons not well understood, the amoebae may invade the tissues. The amoebae may be carried by the portal blood stream to the liver, where they cause a focal necrosis commonly known as an amoebic liver abscess (ALA) (Elsdon-Dew, 1968). Several papers have been published dealing with the immune response of the host to E. histolytica (Balamuth & Siddiwui, 1970; Kagan, 1973; Krupp & Jung, 1976). In regard to humoral immunity, specific antibodies have been reported to have a cytopathogenic effect (CPE) on amoebae (De la Torre et al., 1973; Sepulveda et al., 1973; Ortiz-Ortiz, Sepulveda & Chevez, 1974), manifested by cell membrane and cytoplasmic alterations which lead to the death of the amoebae after incubation for 60 min at 370C (Ortiz-Ortiz et al., 1974). Unexpectedly, the serum from healthy individuals with no clinical or laboratory evidence of amoebiasis also showed CPE on E. histolytica trophozoites (Ortiz-Ortiz et al., 1974). In this report we demonstrate that the CPE on E. histolytica induced by normal human serum (NHS) is dependent upon the alternative pathway of complement activation (Gotze & Muller-Eberhard, 1971). MATERIALS AND METHODS E. histolytica. A 48 hr axenic culture of E. histolytica, strain HK-9 :NIH (Diamond, 1968), was used in all experiments. This strain was maintained in Diamond's culture media (De la Torre et al., 1971). Before each experiment, the amoebae were washed x 5 with veronal-buffered saline (VBS), pH 7-4. For CPE studies the number of trophozoites used was 5 x 104. Sera. Ten normal human sera (NHS) from healthy individuals with no clinical history or laboratory evidence of amoebiasis Correspondence: Dr L. Ortiz-Ortiz, Instituto de Investigaciones Biomedicas, UNAM, Apartado Postal 70228, Mexico 20, D.F., Mexico. 0099-9104/78/0100-0010$02.00 ©0 1978 Blackwell Scientific Publications
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Complement activation by E. histolytica
1I1
were studied. The sera were used immediately after separation. All sera were checked by counterimmunoelectrophoresis (Sepulveda et al., 1972) and passive haemagglutination tests (Krupp & Powell, 1970), and did not contain antibodies to E. histolytica antigen. Even so, test absorption of NHS was performed twice by incubation of 1 ml of each serum with 108 trophozoites of E. histolytica at 4PC for 60 min and by removal of the parasite by centrifugation at 1000 g for 15 min at 4PC as previously described (Sepulveda et al., 1974). Sera were also obtained from patients with clinical and laboratory diagnosis of amoebic liver abscess (ALA). Diagnosis was established by demonstration of pus in the liver aspirate, X-ray, hepatic gammagraphy, specific antibodies against E. histolytica, and a favourable response to anti-amoebic therapy. To eliminate the specific antibody of these sera, they were absorbed as described above for NHS. The absence of antibodies to E. histolytica antigen in the absorbed sera was determined by counterimmunoelectrophoresis and passive haemagglutination tests. Determination ofcytopathogenic effect (CPE). To test for CPE an equal volume of the suspension of E. histolytica trophozoites in VBS was added to NHS in a test tube, and incubated for 60 min at 370C. The alterations in the cell membrane and cytoplasm induced by either NHS or sera from patients with ALA were followed by phase microscopy of aliquots, taken at 5 min intervals, for wet-mount determination of CPE. CPE at 60 min was determined quantitatively by counting at least 500 amoebae per slide. The number of killed trophozoites was recorded as percentage of CPE, and was calculated with reference to the control, incubated without serum. Trypan blue dye-exclusion was also used for assessing viability in the 60 min reading. This method has the advantage of rapidly scanning suspensions of up to 10,000 or more amoebae per slide. Since we found that the assessment of CPE by phase microscopy was quantitatively similar to the evaluation achieved by the dye-exclusion technique, CPE after 60 min of incubation is reported as percentage ofCPE irrespective ofthe method used. Activators of the alternative complement pathway. Inulin was purchased from Sigma Chemical Company, St. Louis, Missouri; zymosan from Nutritional Biochemical Corporation, Cleveland, Ohio; and cobra venom factor (CoF) containing 100 U/ml VBS from Cordis Laboratories, Miami, Florida. Activation of complement in NHS. An appropriate amount of zymosan or inulin was washed twice with VBS. After centrifugation at 2000 g for 10 min at 220C, the supernate was removed. Serum deficient in properdin was prepared by incubation of 1 ml of NHS with 1-2 mg of the zymosan pellet at 17°C for 60 min (Blum, Pillemer & Lepow, 1959). Inulin treatment was done by incubation of 1 ml of NHS with 20 mg of the inulin pellet at 370C for 30 min with periodic shaking (Theofilopoulos & Perrin, 1976). In both cases, sera were separated from the zymosan or inulin particles by centrifugation at 2000 g for 15 min at 40C and were used immediately. NHS were also incubated for 30 min at 370C with CoF (100 U/ml) and, after centrifugation at 2000 g for 15 min at 4°C, were used immediately for CPE studies (Theofilopoulos & Perrin, 1976). EDTA or EGTA treatment ofNHS. Treatment of NHS with either EDTA or EGTA was used to distinguish reactions fixing complement via the antibody C142 classical pathway from those proceeding through the C3 shunt (Snyderman & Pike, 1975). Briefly, serum was mixed with either 10 mm of EDTA or with 2 mm of EGTA with or without 0-3 mM MgCl,, and maintained for 20 min at room temperature. Afterwards the serum activity was tested on E. histolytica for CPE activity. Complement assays. Complement activity was determined as described previously (Morrison, 1977). Briefly, 100 microlitres of the suspension of E. histolytica trophozoites in VBS were added to 100 Jl of NHS and incubated for 30 min at 37°C. The treated serum was then serially diluted into VBS, and each 200 pd were mixed with 2 25 ml of VBS containing 5 x 107 sensitized sheep erythrocytes (EA) to measure reduction in haemolytic complement titre. In these assays, the diluted serum plus EA suspension was incubated for an additional 30 min at 37°C, after which samples were chilled at 4°C and centrifuged at 200 g; the absorbance of supernatant fractions was measured at 412 nm. Consumption of the individual complement components Cl, C4, C2 and C3 was titrated by procedures described by Rapp & Borsos (1970). EAC4 were prepared by the technique of Borsos & Rapp (1967), EACI, EAC1,4 and EAC1,4,2 were prepared by standard techniques (Rapp & Borsos, 1970). Immunoelectrophoresis (IEP) was performed according to Scheidegger (1955) in 1-5% agarose using VBS (pH 8-6, ionic strength 0.05) containing 0-01 M EDTA. IEP proceeded for 90-120 min at a potential gradient of 5 mA per slide and was developed with monospecific antisera to factor B (Arroyave, Bhat & Crown, 1976). Monospecific antisera to factor B was a generous gift from Dr C. Arroyave, University of Colorado, Denver, Colorado.
RESULTS CPE ofeither NHS or sera from patients with ALA on E. histolytica Observed initially after 5 min incubation with NHS, the trophozoites exhibited a rapid loss of motility. After 10 min the amoebae became spheroidal and cytoplasm vacuolization began to appear. These changes were reversible and the amoebae could recuperate at this stage; no uptake of trypan blue was yet detected. However, after 15 min cytoplasmic lesions appeared which were irreversible, the most evident of which were swelling and hialinization, which included the nucleus. After 20-30 min most of the organelles disappeared by total lysis, and others were expelled to the medium in a fine granular form (Fig. 1), or were dispersed when the trophozoite was destroyed. All irreversible stages were also characterized by trypan blue uptake. Therefore, only irreversible stages were used in evaluating CPE on E. histolytica. As can be seen in Fig. 2a, incubation of E. histolytica with fresh NHS at dilutions lower
12
L. Ortiz-Ortiz et al.
FiG. 1. Representative CPE of NHS from a healthy individual on E. histolytica. Preparations observed by phase microscopy after 20-30 min of incubation. (a) Trophozoite of E. histolyfica in the presence of NHS previously inactivated by heating at 560C for 30 min. (b) CPE observed after addition of fresh NHS to E. histolytica.
than 1: 8 caused CPE in a high number of trophozoites. CPE was at its maximum at a 1: 2 dilution and had reached completion by 20-30 min as previously mentioned. Dilutions higher than 1: 16 were without effect on the parasite. Interestingly, repeated absorptions at 4VC of NHS with E. histolytica trophozoites had no effect on the CPE indicating that, in spite of the lack of antibody, these sera showed CPE on amoebae. Sera from patients with ALA, however, showed CPE at dilutions ranging from 1: 2 to 1: 80. Their activity was significantly reduced but not abolished after absorption of specific antibodies (Fig. 2b).
13
Complement activation by E. histolytica1
50
/
1/64 1/16
1 1/8
1
If I
1/4
1/2 1/801/20 1/10
1/4
1/2
Serum d ilution
FIG. 2. Effect of the absorption of either NHS or sera from patients with ALA on the CPE on E. histolytica. (a) Mean ± s.e. of unabsorbed NHS (A), and NHS absorbed twice with 10' trophozoites of E. hivtolytica (C). (b) Mean ± s.e. of sera from patients with ALA, before (A&) and after absorption of specific antibody (*). Trhe presence of antibody to E. histolytica was determined by counterimmunoelectrophoresis and passive haernagglutination. In both cases, aliquots of individual sera were mixed with an equal volume of 5 x 104 trophozoites and incubated for 60 min at 37°C. Afterwards CPE was assessed by phase microscopy and the trypan blue dye-exclusion technique.
The CPE induced by non-absorbed sera on the amoebae was similar to that described for NHS, except that the cytoplasmic lesions developed between 10- 15 min. However, some sera showed total membrane lysis without previous immobilization and spheroidal formation, leading to the death of the amoebae within 5 min. The CPE induced by the antibody-depleted serum showed the same kinetics as that showed by NHS.
Complement dependance and the role of the alternative pathway on the CPE qj'NHS on E. histolytica The data in Table I shows that heat-treatment of NHS at 56°C for 30 min inhibited CPE activity on trophozoites. Similarly, 10 mm EDTA impaired the sera's capacity to destroy trophozoites. Treatment of NHS with CoF for 30 min at 37°C reduced the CPE observed on amoebae. Treatment of sera with 2 mm EGTA inhibited the CPE on trophozoites. However, the CPE against E. histolytica was restored by addition of Mg' + (0 30 mm). Pre-treatment of NHS with zymosan completely abolished the CPE on amoebae. Furthermore, heating NHS at 50°C for 20 min to destroy the activity of factor B of the alternative pathway abolished its lytic activity. Finally, with monospecific antiserum to the properdin pathway complement component factor B. it has also been possible to demonstrate an activation of factor B by E. histolytica. In the (a) well of Fig 3 trophozoite-treated NHS is shown and in the (b) well, untreated NHS control. Anti-human factor B was used in the trough. Factor B conversion to Bs and Ba, both of which show a line of identity with the uncleaved precursor factor B. is seen in the trophozoite-treated NHS, but not in the untreated control. The split products obtained by incubation of NHS with E. histolytica, Fig. 3, well (c) were similar by IEP, to those obtained by treatment of NHS with inulin, Fig. 3, well (d).
Reduction ofhaemolytic complement levels by E. histolytica Various amounts of E. histolytica were added to NHS and incubated at 37°C for 30 min. The reaction mixtures were centrifuged at I1000g9 for 1 5 min and CH 5 in the supernate was titrated. The dose response curve showed that approximately 105 trophozoites per ml NHS fixed about 50%/ of available CH50 (Table 2), and that maximal and almost complete fixation of complement (83%) was achieved by a dose of 6 x 101 parasites per ml NHS. On the other hand, I mg of zymosan per ml was required for maximal
14
L. Ortiz-Ortiz et al. TABLE 1. Complement dependance and the role of the alternative pathway on the cytopathogenic effect (CPE) of normal human sera (NHS) on E. histolytica* Treatment of NHS
CPEt
None None
~~~95-6 (87-100)
560C for 30 min
(0)
17
(0-3)
EDTA, 10 mM
(0-8
CoF
24
40-5
EGTA,2mM
(0-10)
EGTA, 2mM MgCl2, 03 mM
84-5 (75-100)
Zymosan
50'C for 20 min
1-8
(0-10) (010)
* Aliquots of treated or untreated NHS were incubated with an equal volume of trophozoites of E. histolytica. The volume of the reaction mixture was 0 4 ml. The number of parasites added per tube was 5 x 104. t CPE was determined quantitatively after 60 min by examining at least 500 amoebae using phase microscopy and the trypan blue dye-exclusion technique. The latter permitted a rapid scanning of the whole slide. Results obtained by either technique were similar. Mean of 5 to 10 determinations.
complement fixation (96%). Therefore, concomitant to the CPE on E. histolytica exerted by NHS, a reduction in haemolytic complement levels in the amoebae-treated NHS was observed.
Complement component profiles ofthe NHS treated with trophozoites ofE. histolytica The levels of the individual complement components Cl, C4, C2 and C3 were measured in NHS before and after treatment with a dose of 6 x 106 trophozoites per ml. Following treatment of NHS, the level of C3 was significantly diminished, whereas only a negligible decrease in levels of Cl, C4 and C2 was found (Table 3). This profile was essentially the same to that of serum treated with zymosan (Table 3). This finding indiacted that the complement system was activated by E. histolytica through the alternative pathway.
Complement activation by E. histolytica
15
FIG. 3. Conversion of factor B in NHS by trophozoites of E. histolytica. Twenty-five Al of NHS were incubated with an equal volume of either trophozoites of E. histolytica (a) and (c) wells; inulin (d) well, (20 mg/ml) or with VBS (b) well, for 30 min at 370C. Aliquots were then electrophoresed in agarose for 90-120 min at a potential gradient of 5 mA per slide and developed with goat anti-human factor B. The slide was stained with amido black. The cathode was on the right.
TABLE 2. Activation of complement in normal human sera (NHS) by trophozoites of E. histolytica*
Trophozoitest 6 x 106 6 x 105 6 x 104 6 x 103 6 x 102
Reduction of CH50 (%) 83 60 41 26 15
* Aliquots of NHS were incubated with an equal volume of either VBS or VBS containing different concentrations of E. histolytica trophozoites for 30 min at 370C. Residual haemolytic complement was then titrated as described in the Materials and Methods section. t Number of trophozoites per ml of NHS.
DISCUSSION In this report we describe the role of complement in the CPE of sera from healthy individuals upon E. histolytica trophozoites and the contribution of the alternative and classical pathways of complement activation to this process. For this purpose we took advantage of the different requirements of the alternative complement pathway and the classical C142 pathway for Ca' + and/or Mg' +. Complement
16
L. Ortiz-Ortiz et al. TABLE 3. Consumption of early complement components by trophozoites of E. histolytica* Serum treatment E. histolytica
Zymosan
Cl
C4
C2
(%)
(%)
(%)
C3 (%)
2-2t
3-8
(0-6) 0
(0-8) 1-2 (0-6)
2-0 (0-5) 1.0 (0-5)
(75-87) 95 8 (91-98)
812
As determined by haemolytic titration. As compared to baseline values of the untreated NHS tested. Changes in values which were statistically significant are set in italics. Mean of 5 determinations. *
f
fixation via the C142 pathway requires both ionized magnesium and ionized calcium and is inhibited by EGTA, while the alternative pathway requires only ionized Mg and proceeds in the presence of EGTA and a minimal addition of Mg' + (0 3 mM). Both pathways of complement fixation are inhibited by EDTA (Snyderman & Pike, 1975). Our data indicate that the CPE on E. histolytica requires complement as shown by its inhibition by either EDTA or heat-inactivation at 560C for 30 min. The results obtained with CoF are also indicative of a complement requirement. Similar results with CoF have been reported with the culture form of another protozoan, Trypanosoma cruzi (Budzko, Pizzimenti & Kierszenbaum, 1975; Nogueira, Bianco & Cohn, 1975). The dependence of the CPE on Mg' 'and not Ca' + (Pillemer et al., 1954), and the inhibition of CEP by the selective removal of most if not all of the properdin with zymosan (Pillemer et al., 1956), both indicate that the alternative pathway of complement activation is sufficient to generate CPE activity. Furthermore, heating NHS at 50'C for 20 min to destroy the activity of factor B of the alternative pathway abolished its CPE activity. In addition, as determined by electrophoretic criteria, factor B had been activated in NHS following incubation at 370C with trophozoites of E. histolytica. This conversion of factor B by amoebae resembled that observed with inulin (Morrison, 1977). Results reported elsewhere (Capin et al., 1978) indicate that the activation of the alternative complement pathway is not an exclusive property of the HK-9: NIH strain of E. histolytica. Other E. histolytica isolated from human cases and adapted to axenic growth in our laboratory i.e., HM-1 :IMSS, and HM-3 :IMSS, have also shown the same properties. The virulence of HK-9:NIH has been found to be of an intermediate level in either the newborn (Mattern & Keister, 1977) or the young adult hamster (Diamond, Phillips & Bartgis, 1974). The quantification of CH50 indicated a decrease in complement titre in all trophozoite-treated NHS samples tested (37°C). The diminution in CH50 correlated with a consumption of C3. In contrast, no statistically significant decreases in Cl, C4 and C2 were found in the same samples, indicating that E. histolytica activated the properdin pathway. All the previous data and the fact that repeated absorption with the trophozoites at 4°C left the CPE activity of NHS intact, indicated that specific or cross-reacting natural antibody is not essential for CPE (absorption experiments, Fig. 2a). Although the alternative pathway was capable of generating CPE by itself under these conditions, CPE will occur in the presence of specific antibodies with the consequent activation of the classical pathway (Ortiz-Ortiz, et al., 1974). Absorption studies of sera from patients with ALA showed that after removal of the specific antibodies, CPE on amoebae was still in evidence. The reduction in the CPE to levels lower than those observed in NHS may be due to complement fixation by antigen-antibody complexes formed during absorption at 4°C (Cunniff & Stollar, 1968). This would further support the participation of the alternative pathway of complement on the CPE on amoebae. It is possible that surface components of the trophozoite forms trigger than C3 activator system. This
Complement activation by E. histolytica
17
mechanism of complement activation may come into play in vivo before an immune response to amoebae makes sufficient amounts of antibody available for CPE by the classical complement reactions. The possibility exists that conditions such as repeated or recurrent infections or other human disorders associated with complement deficiencies (Cooper, 1976) are very likely responsible for the appearance of the invasive form of the infection. Studies are in progress to determine complement levels as well as participation ofthe alternative pathway in patients with the invasive form ofthe disease. In conclusion, the mechanism by which trophozoites of E. histolytica are destroyed by the sera of healthy individuals is a complement-dependent process, activated by the alternative pathway. The CPE exerted by the sera of healthy individuals upon E. histolytica points to the importance of the alternative pathway as a defence mechanism in humans against amoebae and perhaps against other protozoa, such as Trypanosoma cruzi (Kierszenbaum, Ivanyi & Budzko, 1976) and Toxoplasma (Raffel, 1961). We thank Dr Kaethe Willms and Miss Veronica Yakoleff for reviewing this manuscript.
REFERENCES ARROYAVE, C., BATH, K.N. & CROWN R. (1976) Activation GOTZE, 0. & MULLER-EBERHARD, H. (1971) The C3 activaof the alternative pathway of the complement system by tor system: an alternate pathway of complement activation. . exp. Med. 134, suppl. 90. radiographic contrast media. J. Immunol. 117, 1866. BALAmuTH, W. & SIDDIWUI, W.A. (1970) Amoebas and KAGAN, I.G. (1973) The immunology of amebiasis. Arch. other intestinal protozoa. Immunity to Parasitic Animals Invest. Mid. (Mix.) 4, suppl. 177. Vol. 2 (ed. by G. J. Jackson, R. Herman and I. Singer), KIERSZENCAUM, F., IvANYI, J. & BuDzKo, D.B. (1976) p. 249. Appleton-Century-Crofts, Chicago. Mechanisms of natural resistance to trypanosomal infecBLUM, L., PILLEMER, L. & LEPOW, I.H. (1959) The protion. Role of complement in avian resistance to Trypanoperdin system and immunity. XI. Studies on the intersoma cruzi infection. Immunology, 30, 1. action of zymosan with the properdin system. Z. Immuni- KRUPP, I.M., & POWELL, S.J. (1970) Modification of the taetsforsch. Exp. Ther. 118,313. indirect haemagglutination test for amebiasis. 3. Clin. BORSOS, T. & RAPP, H.J. (1967) Immune hemolysis: A Path. 22, 530. simplified method for the preparation of EAC'4 with KRUPP, I.M. & JUNG, R.C. (1976) Immunity of amoebic guinea pig or with human complement. J. Immunol. infection. Immunology of Parasitic Infections (ed. by S. 99, 263. Cohen and E. H. Sadun), p. 163. Blackwell, Oxford. BuDzKo, D.F., PIZZIMENTI, M.C. & KIERSZENBAUM, F. MATTERN, C.F.T. & KEISTER, D.B. (1977) Experimental (1975) Effects of complement depletion in experimental amebiasis. II. Hepatic amebiasis in the newborn hamster. Chagas' disease: immune lysis of virulent blood forms Amer.7. trop. Med. Hyg. 26,402. of Trypanosoma cruzi. Infect. Immun. 11, 86. MORRISON, D. (1977) Activation of the classical and proCAPiN, N.R., CAPfN, R., ZAMACONA, G. & ORTIZ-ORTIZ, L. perdin pathways of complement by bacterial lipopoly(1978) Activacion de la via alterna del complemento por saccharides (LPS).J. Immunol. 118,362. varias cepas axenicas de Entamoeba histolytica. Arch. NOGUEIRA, N., BIANCO, C. & COHN, Z. (1975) Studies on Invest. Mid. (Mix.) 9, suppl. 297. the selective lysis and purification of Trypanosoma cruzi. COOPER, N.R. (1976) The complement system. Basic and 3. exp. Med. 142,224. Clinical Immunology (ed. by H. H. Fudenberg, D. P. ORTIZ-ORTIZ, L., SEPULVEDA, B. & CHiEvz, A. (1974) Stites, J. L. Caldwell and J. V. Wells), p. 58. Lange, Nuevos estudios acerca de la accion de sueros humanos Los Altos, California. normales e inmunes sobre el trofozoito de E. histolytica. CUNNIFF, R.V. & STOLLAR, B.D. (1968) Properties of 19S Arch. Invest. Me'd. (Mex.) 5, suppl. 337. antibodies in complement fixation. I. Temperature depen- PILLEMER, L., BLUM, L., LEPOW, I.H., Ross, O.A., TODD, dence and role of antigen structure. J. Immunol. 100, 7. E.W. & WARDLAw, A.C. (1954) The properdin system DE LA TORRE, M., DE LA Hoz, R., LANDA, L. & SEPULVEDA, and immunity: I. Demonstration and isolation of a new B. (1971) Cultivos axenicos de Entamoeba histolytica. serum protein, properdin, and its role in immune pheArch. Invest. Mid. (Mix.) 2, 165. nomena. Science, 120,279. DE LA TORRE, M., ORTIZ-ORTIZ, L., DE LA Hoz, R. & PILLEMER, L., BLUM, L., LEPOW, I.H., WURZ, L. & TODD, SEPULVEDA, B. (1973) Accion del suero humano inmune y E.W. (1956) The properdin system and immunity. III. de la gammaglobulina antiamibiana sobre cultivos de The zymosan assay ofproperdin. J. exp. Med. 103, 1. E. histolytica. Arch. Invest. Mid. (Mex.) 4, suppl. 67. RAFFEL, S. (1961) Immunity, p. 6. Appleton-CenturyDIAMOND, L.S. (1968) Techniques of axenic cultivation of Crofts, New York. E. histolytica Schaundinn, 1903 and E. histolytica-like RAPP, H.J. & BORSOS, T. (1970) Molecular Basis of Compleamebae. J. Parasit. 54, 1047. ment Action. Appleton-Century-Crofts, New York. DIAMOND, L.S., PHILLIPS, B.P. & BARTGIS, I.L. (1974) SCHEIDEGGER, J.J. (1955) Une micromethod de l'immunoA comparison of the virulence of nine strains of axenically electrophorese. Int. Arch. Allergy Appl. Immunol. 7, 103. cultivated E. histolytica in hamster liver. Arch. Invest. SEP6LVEDA, B., AUBANEL, M., LANDA, L., & VELAZQUEZ, Mid. (Mex.) 5, suppl. 423. G. (1972) Avances en la tecnica de contrainmunoELSDON-DEW, R. (1968) The epidemiology of amoebiasis. electroforesis para el estudio serologico de la amibiasis. Adv. Parasit. 6, 1. Arch. Invest. Mid. (Me'x.) 3, 363. B
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SEPJLVEDA, B., CH£vEz, A., ITURBE-ALESSIO, I. & ORTIZORTIZ, L. (1973) Efecto de la gammaglobulina inmune sobre el trofozoito de E. histolytica. Arch. Invest. Mid. (Mix.) 4, suppl. 79. SEPfiLVEDA, B., ORTIz-ORTIz, L., CQEvEz, A. & SEGURA, M. (1974) Comprobaci6n de la naturaleza inmunologica del efecto del suero y de la gammaglobulina inmunes sobre el trofozoito de E. histolytica. Arch. Invest. Mid. (Mix.) 5, 343.
SNYDERMAN, R. & PiKE, M.C. (1975) Interaction of complex polysaccharides with the complement system: effect of calcium depletion on terminal component consumption. Infect. Immunity, 11, 273. THEOFILOPOULOS, A.N. & PERRIN, L.H. (1976) Binding of components of the properdin system to cultured human lymphoblastoid cells and B lymphocytes. J. exp. Med.
143,271.
Activation of the alternative pathway of complement by Entamoeba histolytica L. ORTIZ-ORTIZ, RUTH CAPIN, NORMA R. CAPIN, B. SEPULVEDA & G. ZAMACONA Instituto de Investigaciones Biomidicas, Uuiversidad Nacional Antonoma de Mexico, Apartado Postal 70228, Mexico 20 and Servicios de Alergia e Immunologia Clinica y Gastroenterologia, Hospital General, CMN del IMSS, Mexico 7, Mexico
(Received 20 February 1978)
SUMMARY
A cytopathogenic effect was observed when Entamoeba histolytica was exposed to human sera from individuals with no clinical history or laboratory evidence of amoebiasis. Absorption studies showed that the effect was not due to natural antibodies. Studies performed using ethylenediamine tetracetic acid (EDTA), cobra venom factor (CoF) and heat-inactivation at 560C, indicated that the cytopathogenic effect was complement dependent. Furthermore, by using ethylene glycol tetracetic acid (EGTA) and Mg' +, zymosan, heat-inactivation at 50'C to destroy the activity of factor B of the alternative pathway, as well as electrophoretic studies with anti-human factor B, it was possible to determine that E. histolytica activated the properdin pathway. Finally, complement determinations indicated that incubation of E. histolytica with normal human serum consumed complement. The diminution in CH50 correlated with a consumption of C3 but not of Cl, C4 and C2. It was concluded from these results that trophozoites of E. histolytica activate the alternative pathway ofthe human complement system.
INTRODUCTION Entamoeba histolytica is a protozoan found in most parts of the world. The parasite usually confines its activity to the bowel lumen; however, for reasons not well understood, the amoebae may invade the tissues. The amoebae may be carried by the portal blood stream to the liver, where they cause a focal necrosis commonly known as an amoebic liver abscess (ALA) (Elsdon-Dew, 1968). Several papers have been published dealing with the immune response of the host to E. histolytica (Balamuth & Siddiwui, 1970; Kagan, 1973; Krupp & Jung, 1976). In regard to humoral immunity, specific antibodies have been reported to have a cytopathogenic effect (CPE) on amoebae (De la Torre et al., 1973; Sepulveda et al., 1973; Ortiz-Ortiz, Sepulveda & Chevez, 1974), manifested by cell membrane and cytoplasmic alterations which lead to the death of the amoebae after incubation for 60 min at 370C (Ortiz-Ortiz et al., 1974). Unexpectedly, the serum from healthy individuals with no clinical or laboratory evidence of amoebiasis also showed CPE on E. histolytica trophozoites (Ortiz-Ortiz et al., 1974). In this report we demonstrate that the CPE on E. histolytica induced by normal human serum (NHS) is dependent upon the alternative pathway of complement activation (Gotze & Muller-Eberhard, 1971). MATERIALS AND METHODS E. histolytica. A 48 hr axenic culture of E. histolytica, strain HK-9 :NIH (Diamond, 1968), was used in all experiments. This strain was maintained in Diamond's culture media (De la Torre et al., 1971). Before each experiment, the amoebae were washed x 5 with veronal-buffered saline (VBS), pH 7-4. For CPE studies the number of trophozoites used was 5 x 104. Sera. Ten normal human sera (NHS) from healthy individuals with no clinical history or laboratory evidence of amoebiasis Correspondence: Dr L. Ortiz-Ortiz, Instituto de Investigaciones Biomedicas, UNAM, Apartado Postal 70228, Mexico 20, D.F., Mexico. 0099-9104/78/0100-0010$02.00 ©0 1978 Blackwell Scientific Publications
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Complement activation by E. histolytica
1I1
were studied. The sera were used immediately after separation. All sera were checked by counterimmunoelectrophoresis (Sepulveda et al., 1972) and passive haemagglutination tests (Krupp & Powell, 1970), and did not contain antibodies to E. histolytica antigen. Even so, test absorption of NHS was performed twice by incubation of 1 ml of each serum with 108 trophozoites of E. histolytica at 4PC for 60 min and by removal of the parasite by centrifugation at 1000 g for 15 min at 4PC as previously described (Sepulveda et al., 1974). Sera were also obtained from patients with clinical and laboratory diagnosis of amoebic liver abscess (ALA). Diagnosis was established by demonstration of pus in the liver aspirate, X-ray, hepatic gammagraphy, specific antibodies against E. histolytica, and a favourable response to anti-amoebic therapy. To eliminate the specific antibody of these sera, they were absorbed as described above for NHS. The absence of antibodies to E. histolytica antigen in the absorbed sera was determined by counterimmunoelectrophoresis and passive haemagglutination tests. Determination ofcytopathogenic effect (CPE). To test for CPE an equal volume of the suspension of E. histolytica trophozoites in VBS was added to NHS in a test tube, and incubated for 60 min at 370C. The alterations in the cell membrane and cytoplasm induced by either NHS or sera from patients with ALA were followed by phase microscopy of aliquots, taken at 5 min intervals, for wet-mount determination of CPE. CPE at 60 min was determined quantitatively by counting at least 500 amoebae per slide. The number of killed trophozoites was recorded as percentage of CPE, and was calculated with reference to the control, incubated without serum. Trypan blue dye-exclusion was also used for assessing viability in the 60 min reading. This method has the advantage of rapidly scanning suspensions of up to 10,000 or more amoebae per slide. Since we found that the assessment of CPE by phase microscopy was quantitatively similar to the evaluation achieved by the dye-exclusion technique, CPE after 60 min of incubation is reported as percentage ofCPE irrespective ofthe method used. Activators of the alternative complement pathway. Inulin was purchased from Sigma Chemical Company, St. Louis, Missouri; zymosan from Nutritional Biochemical Corporation, Cleveland, Ohio; and cobra venom factor (CoF) containing 100 U/ml VBS from Cordis Laboratories, Miami, Florida. Activation of complement in NHS. An appropriate amount of zymosan or inulin was washed twice with VBS. After centrifugation at 2000 g for 10 min at 220C, the supernate was removed. Serum deficient in properdin was prepared by incubation of 1 ml of NHS with 1-2 mg of the zymosan pellet at 17°C for 60 min (Blum, Pillemer & Lepow, 1959). Inulin treatment was done by incubation of 1 ml of NHS with 20 mg of the inulin pellet at 370C for 30 min with periodic shaking (Theofilopoulos & Perrin, 1976). In both cases, sera were separated from the zymosan or inulin particles by centrifugation at 2000 g for 15 min at 40C and were used immediately. NHS were also incubated for 30 min at 370C with CoF (100 U/ml) and, after centrifugation at 2000 g for 15 min at 4°C, were used immediately for CPE studies (Theofilopoulos & Perrin, 1976). EDTA or EGTA treatment ofNHS. Treatment of NHS with either EDTA or EGTA was used to distinguish reactions fixing complement via the antibody C142 classical pathway from those proceeding through the C3 shunt (Snyderman & Pike, 1975). Briefly, serum was mixed with either 10 mm of EDTA or with 2 mm of EGTA with or without 0-3 mM MgCl,, and maintained for 20 min at room temperature. Afterwards the serum activity was tested on E. histolytica for CPE activity. Complement assays. Complement activity was determined as described previously (Morrison, 1977). Briefly, 100 microlitres of the suspension of E. histolytica trophozoites in VBS were added to 100 Jl of NHS and incubated for 30 min at 37°C. The treated serum was then serially diluted into VBS, and each 200 pd were mixed with 2 25 ml of VBS containing 5 x 107 sensitized sheep erythrocytes (EA) to measure reduction in haemolytic complement titre. In these assays, the diluted serum plus EA suspension was incubated for an additional 30 min at 37°C, after which samples were chilled at 4°C and centrifuged at 200 g; the absorbance of supernatant fractions was measured at 412 nm. Consumption of the individual complement components Cl, C4, C2 and C3 was titrated by procedures described by Rapp & Borsos (1970). EAC4 were prepared by the technique of Borsos & Rapp (1967), EACI, EAC1,4 and EAC1,4,2 were prepared by standard techniques (Rapp & Borsos, 1970). Immunoelectrophoresis (IEP) was performed according to Scheidegger (1955) in 1-5% agarose using VBS (pH 8-6, ionic strength 0.05) containing 0-01 M EDTA. IEP proceeded for 90-120 min at a potential gradient of 5 mA per slide and was developed with monospecific antisera to factor B (Arroyave, Bhat & Crown, 1976). Monospecific antisera to factor B was a generous gift from Dr C. Arroyave, University of Colorado, Denver, Colorado.
RESULTS CPE ofeither NHS or sera from patients with ALA on E. histolytica Observed initially after 5 min incubation with NHS, the trophozoites exhibited a rapid loss of motility. After 10 min the amoebae became spheroidal and cytoplasm vacuolization began to appear. These changes were reversible and the amoebae could recuperate at this stage; no uptake of trypan blue was yet detected. However, after 15 min cytoplasmic lesions appeared which were irreversible, the most evident of which were swelling and hialinization, which included the nucleus. After 20-30 min most of the organelles disappeared by total lysis, and others were expelled to the medium in a fine granular form (Fig. 1), or were dispersed when the trophozoite was destroyed. All irreversible stages were also characterized by trypan blue uptake. Therefore, only irreversible stages were used in evaluating CPE on E. histolytica. As can be seen in Fig. 2a, incubation of E. histolytica with fresh NHS at dilutions lower
12
L. Ortiz-Ortiz et al.
FiG. 1. Representative CPE of NHS from a healthy individual on E. histolytica. Preparations observed by phase microscopy after 20-30 min of incubation. (a) Trophozoite of E. histolyfica in the presence of NHS previously inactivated by heating at 560C for 30 min. (b) CPE observed after addition of fresh NHS to E. histolytica.
than 1: 8 caused CPE in a high number of trophozoites. CPE was at its maximum at a 1: 2 dilution and had reached completion by 20-30 min as previously mentioned. Dilutions higher than 1: 16 were without effect on the parasite. Interestingly, repeated absorptions at 4VC of NHS with E. histolytica trophozoites had no effect on the CPE indicating that, in spite of the lack of antibody, these sera showed CPE on amoebae. Sera from patients with ALA, however, showed CPE at dilutions ranging from 1: 2 to 1: 80. Their activity was significantly reduced but not abolished after absorption of specific antibodies (Fig. 2b).
13
Complement activation by E. histolytica1
50
/
1/64 1/16
1 1/8
1
If I
1/4
1/2 1/801/20 1/10
1/4
1/2
Serum d ilution
FIG. 2. Effect of the absorption of either NHS or sera from patients with ALA on the CPE on E. histolytica. (a) Mean ± s.e. of unabsorbed NHS (A), and NHS absorbed twice with 10' trophozoites of E. hivtolytica (C). (b) Mean ± s.e. of sera from patients with ALA, before (A&) and after absorption of specific antibody (*). Trhe presence of antibody to E. histolytica was determined by counterimmunoelectrophoresis and passive haernagglutination. In both cases, aliquots of individual sera were mixed with an equal volume of 5 x 104 trophozoites and incubated for 60 min at 37°C. Afterwards CPE was assessed by phase microscopy and the trypan blue dye-exclusion technique.
The CPE induced by non-absorbed sera on the amoebae was similar to that described for NHS, except that the cytoplasmic lesions developed between 10- 15 min. However, some sera showed total membrane lysis without previous immobilization and spheroidal formation, leading to the death of the amoebae within 5 min. The CPE induced by the antibody-depleted serum showed the same kinetics as that showed by NHS.
Complement dependance and the role of the alternative pathway on the CPE qj'NHS on E. histolytica The data in Table I shows that heat-treatment of NHS at 56°C for 30 min inhibited CPE activity on trophozoites. Similarly, 10 mm EDTA impaired the sera's capacity to destroy trophozoites. Treatment of NHS with CoF for 30 min at 37°C reduced the CPE observed on amoebae. Treatment of sera with 2 mm EGTA inhibited the CPE on trophozoites. However, the CPE against E. histolytica was restored by addition of Mg' + (0 30 mm). Pre-treatment of NHS with zymosan completely abolished the CPE on amoebae. Furthermore, heating NHS at 50°C for 20 min to destroy the activity of factor B of the alternative pathway abolished its lytic activity. Finally, with monospecific antiserum to the properdin pathway complement component factor B. it has also been possible to demonstrate an activation of factor B by E. histolytica. In the (a) well of Fig 3 trophozoite-treated NHS is shown and in the (b) well, untreated NHS control. Anti-human factor B was used in the trough. Factor B conversion to Bs and Ba, both of which show a line of identity with the uncleaved precursor factor B. is seen in the trophozoite-treated NHS, but not in the untreated control. The split products obtained by incubation of NHS with E. histolytica, Fig. 3, well (c) were similar by IEP, to those obtained by treatment of NHS with inulin, Fig. 3, well (d).
Reduction ofhaemolytic complement levels by E. histolytica Various amounts of E. histolytica were added to NHS and incubated at 37°C for 30 min. The reaction mixtures were centrifuged at I1000g9 for 1 5 min and CH 5 in the supernate was titrated. The dose response curve showed that approximately 105 trophozoites per ml NHS fixed about 50%/ of available CH50 (Table 2), and that maximal and almost complete fixation of complement (83%) was achieved by a dose of 6 x 101 parasites per ml NHS. On the other hand, I mg of zymosan per ml was required for maximal
14
L. Ortiz-Ortiz et al. TABLE 1. Complement dependance and the role of the alternative pathway on the cytopathogenic effect (CPE) of normal human sera (NHS) on E. histolytica* Treatment of NHS
CPEt
None None
~~~95-6 (87-100)
560C for 30 min
(0)
17
(0-3)
EDTA, 10 mM
(0-8
CoF
24
40-5
EGTA,2mM
(0-10)
EGTA, 2mM MgCl2, 03 mM
84-5 (75-100)
Zymosan
50'C for 20 min
1-8
(0-10) (010)
* Aliquots of treated or untreated NHS were incubated with an equal volume of trophozoites of E. histolytica. The volume of the reaction mixture was 0 4 ml. The number of parasites added per tube was 5 x 104. t CPE was determined quantitatively after 60 min by examining at least 500 amoebae using phase microscopy and the trypan blue dye-exclusion technique. The latter permitted a rapid scanning of the whole slide. Results obtained by either technique were similar. Mean of 5 to 10 determinations.
complement fixation (96%). Therefore, concomitant to the CPE on E. histolytica exerted by NHS, a reduction in haemolytic complement levels in the amoebae-treated NHS was observed.
Complement component profiles ofthe NHS treated with trophozoites ofE. histolytica The levels of the individual complement components Cl, C4, C2 and C3 were measured in NHS before and after treatment with a dose of 6 x 106 trophozoites per ml. Following treatment of NHS, the level of C3 was significantly diminished, whereas only a negligible decrease in levels of Cl, C4 and C2 was found (Table 3). This profile was essentially the same to that of serum treated with zymosan (Table 3). This finding indiacted that the complement system was activated by E. histolytica through the alternative pathway.
Complement activation by E. histolytica
15
FIG. 3. Conversion of factor B in NHS by trophozoites of E. histolytica. Twenty-five Al of NHS were incubated with an equal volume of either trophozoites of E. histolytica (a) and (c) wells; inulin (d) well, (20 mg/ml) or with VBS (b) well, for 30 min at 370C. Aliquots were then electrophoresed in agarose for 90-120 min at a potential gradient of 5 mA per slide and developed with goat anti-human factor B. The slide was stained with amido black. The cathode was on the right.
TABLE 2. Activation of complement in normal human sera (NHS) by trophozoites of E. histolytica*
Trophozoitest 6 x 106 6 x 105 6 x 104 6 x 103 6 x 102
Reduction of CH50 (%) 83 60 41 26 15
* Aliquots of NHS were incubated with an equal volume of either VBS or VBS containing different concentrations of E. histolytica trophozoites for 30 min at 370C. Residual haemolytic complement was then titrated as described in the Materials and Methods section. t Number of trophozoites per ml of NHS.
DISCUSSION In this report we describe the role of complement in the CPE of sera from healthy individuals upon E. histolytica trophozoites and the contribution of the alternative and classical pathways of complement activation to this process. For this purpose we took advantage of the different requirements of the alternative complement pathway and the classical C142 pathway for Ca' + and/or Mg' +. Complement
16
L. Ortiz-Ortiz et al. TABLE 3. Consumption of early complement components by trophozoites of E. histolytica* Serum treatment E. histolytica
Zymosan
Cl
C4
C2
(%)
(%)
(%)
C3 (%)
2-2t
3-8
(0-6) 0
(0-8) 1-2 (0-6)
2-0 (0-5) 1.0 (0-5)
(75-87) 95 8 (91-98)
812
As determined by haemolytic titration. As compared to baseline values of the untreated NHS tested. Changes in values which were statistically significant are set in italics. Mean of 5 determinations. *
f
fixation via the C142 pathway requires both ionized magnesium and ionized calcium and is inhibited by EGTA, while the alternative pathway requires only ionized Mg and proceeds in the presence of EGTA and a minimal addition of Mg' + (0 3 mM). Both pathways of complement fixation are inhibited by EDTA (Snyderman & Pike, 1975). Our data indicate that the CPE on E. histolytica requires complement as shown by its inhibition by either EDTA or heat-inactivation at 560C for 30 min. The results obtained with CoF are also indicative of a complement requirement. Similar results with CoF have been reported with the culture form of another protozoan, Trypanosoma cruzi (Budzko, Pizzimenti & Kierszenbaum, 1975; Nogueira, Bianco & Cohn, 1975). The dependence of the CPE on Mg' 'and not Ca' + (Pillemer et al., 1954), and the inhibition of CEP by the selective removal of most if not all of the properdin with zymosan (Pillemer et al., 1956), both indicate that the alternative pathway of complement activation is sufficient to generate CPE activity. Furthermore, heating NHS at 50'C for 20 min to destroy the activity of factor B of the alternative pathway abolished its CPE activity. In addition, as determined by electrophoretic criteria, factor B had been activated in NHS following incubation at 370C with trophozoites of E. histolytica. This conversion of factor B by amoebae resembled that observed with inulin (Morrison, 1977). Results reported elsewhere (Capin et al., 1978) indicate that the activation of the alternative complement pathway is not an exclusive property of the HK-9: NIH strain of E. histolytica. Other E. histolytica isolated from human cases and adapted to axenic growth in our laboratory i.e., HM-1 :IMSS, and HM-3 :IMSS, have also shown the same properties. The virulence of HK-9:NIH has been found to be of an intermediate level in either the newborn (Mattern & Keister, 1977) or the young adult hamster (Diamond, Phillips & Bartgis, 1974). The quantification of CH50 indicated a decrease in complement titre in all trophozoite-treated NHS samples tested (37°C). The diminution in CH50 correlated with a consumption of C3. In contrast, no statistically significant decreases in Cl, C4 and C2 were found in the same samples, indicating that E. histolytica activated the properdin pathway. All the previous data and the fact that repeated absorption with the trophozoites at 4°C left the CPE activity of NHS intact, indicated that specific or cross-reacting natural antibody is not essential for CPE (absorption experiments, Fig. 2a). Although the alternative pathway was capable of generating CPE by itself under these conditions, CPE will occur in the presence of specific antibodies with the consequent activation of the classical pathway (Ortiz-Ortiz, et al., 1974). Absorption studies of sera from patients with ALA showed that after removal of the specific antibodies, CPE on amoebae was still in evidence. The reduction in the CPE to levels lower than those observed in NHS may be due to complement fixation by antigen-antibody complexes formed during absorption at 4°C (Cunniff & Stollar, 1968). This would further support the participation of the alternative pathway of complement on the CPE on amoebae. It is possible that surface components of the trophozoite forms trigger than C3 activator system. This
Complement activation by E. histolytica
17
mechanism of complement activation may come into play in vivo before an immune response to amoebae makes sufficient amounts of antibody available for CPE by the classical complement reactions. The possibility exists that conditions such as repeated or recurrent infections or other human disorders associated with complement deficiencies (Cooper, 1976) are very likely responsible for the appearance of the invasive form of the infection. Studies are in progress to determine complement levels as well as participation ofthe alternative pathway in patients with the invasive form ofthe disease. In conclusion, the mechanism by which trophozoites of E. histolytica are destroyed by the sera of healthy individuals is a complement-dependent process, activated by the alternative pathway. The CPE exerted by the sera of healthy individuals upon E. histolytica points to the importance of the alternative pathway as a defence mechanism in humans against amoebae and perhaps against other protozoa, such as Trypanosoma cruzi (Kierszenbaum, Ivanyi & Budzko, 1976) and Toxoplasma (Raffel, 1961). We thank Dr Kaethe Willms and Miss Veronica Yakoleff for reviewing this manuscript.
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