Annals of Tropical Medicine & Parasitology

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Complement (C3) levels and activation in rabbits experimentally infected with Trypanosoma evansi U. E. Uche & T. W. Jones To cite this article: U. E. Uche & T. W. Jones (1992) Complement (C3) levels and activation in rabbits experimentally infected with Trypanosoma evansi, Annals of Tropical Medicine & Parasitology, 86:5, 475-480, DOI: 10.1080/00034983.1992.11812696 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812696

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Annals of Tropical Medicine and Parasitology, Vol. 86, No.5, 475-480 (1992)

Complement (C3) levels and activation in rabbits experimentally infected with Trypanosoma evansi

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BYU.E. UCHE Royal Veterinary College, Royal College Street, London NW1 OTU, U.K. ANDT.

W.JONES

Centre for Tropical Veterinary Medicine, Easter Bush, Roslin EH2S 9RG, U.K. Received 14 Apri/1992, Revised 22June 1992, Accepted 23June 1992

Rocket immunoelectrophoresis was used to monitor the levels of the third complement component (C3) in the blood of rabbits experimentally infected with Trypanosoma evansi. Although a reduction in the circulating levels of C3 was associated with C3 activation in rabbits with high levels of parasitaemia, there was no evidence for C3 activation in uninfected rabbits, rabbits with early-stage, light infections or rabbits cleared of infection by drug treatment. Host-tolerance to current infection and tore-exposure to the parasite are probably affected by such changes in C3.

The complement system, which comprises 30 different proteins (Lambris, 1988), has long been known to have important effector functions in the immune response (Staines et a!., 1985). The nine major components of the system (Cl-C9) are directly involved in the complement cascade which, when activated, helps to eliminate pathogens. The third component (C3) plays a central role in the induction of the system and is also important in the generation of the B memory cells (Klaus and Humphrey, 1977) which enable the immune system to recognize a particular antigen during secondary exposure (Irvine, 1979). Studies on the role of the complement system in trypanosomiasis, particularly that ofC3, have been limited. Although decreasing complement levels have sometimes been associated with significant increases in parasitaemia, worsening clinical state and then death of infected animals (Budzko et a!., 197 5; Kobayashi and Tizard, 1976; Cunningham et a!., 1978), Shirazi et a!. (1980) found no demonstrable differences between the parasitaemias of normal and C30003-4983/92/050475 +06 $08.00/0

depleted animals. In view of these equivocal results and the central role C3 plays in the activation of the complement cascade, the aims of the present study were to evaluate the levels and activation of C3 in rabbits experimentally infected with Trypanosoma evansi and to determine the relationship between C3 level and infection before and after drug treatment. MATERIALS AND METHODS Parasite and Infection of Rabbits Mice with fulminating infections of T. evansi TREU 2147 were exsanguinated under ether anaesthesia. The parasites were then separated from the heparinized blood by anion-exchange chromatography (Lanham and Godfrey, 1970), resuspended in ice-cold phosphate saline glucose and injected, 4 x 10 5 parasites/rabbit, into the ear veins of four adult female New Zealand White rabbits. Twenty-four days postinfection, each rabbit was treated with 7 mg (active principle)/kg bodyweight of diaminezene aceturate (Berenil). © 1992 Liverpool School of Tropical Medicine

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Serum and Plasma Preparation Two blood samples were collected from each of the four rabbits before infection, seven, II, 18, 21 and 24 days post-infection and eight and 14 days after drug treatment. One of these samples was collected in a bottle with ethylene diamine tetraacetic acid (EDT A) as anticoagulant and used to estimate parasite density (Paris et a!., 1982) and for the preparation of plasma. The other sample was collected without anticoagulant and held on ice from 20 minutes so that the serum could be aspirated. Each serum sample was then centrifuged at 2500 g for 35 minutes at 4oC. Both serum and plasma samples were divided into 300 J.ll aliquots before storage at - 79°C. Estimation of Relative C3 Levels The C3 levels in the plasma samples were measured by rocket immunoelectrophoresis (Laurell, 1966) in a 1% agarose gel, pH 8·6, in barbitone acetate buffer containing 2· 5% sheep anti-rabbit-C3 serum (ICN Immunobiologicals) and 3% polyethylene glycol 6000 (PEG 6000). Briefly, 10 J.ll of plasma was placed in each 5-mm diameter well cut in the gel with a metal punch. A constant voltage of 150 V (volts) was applied and the proteins allowed to run across the gel for about 18 hours. The gels were then washed in phosphate buffered saline, pH 7·3, (PBS) for 24 hours, fixed in 25% methanol in 10% glacial acetic acid for one hour and then dried in a stream of hot air from a hair-dryer. Components that bound to the gel were visualized by Coomassie Blue staining. C3 Activation Assay One- and two-dimensional immunoelectrophoresis were used to assay for the cleavage products of C3 in the serum samples from the rabbits (Johnstone and Thorpe, 1987). Immunoelectrophoresis in the first dimension was conducted as for the estimation ofC3levels in plasma (see above), except that, for each run, 250 V were applied for seven hours and no anti-C3 antiserum was used in the gel. For the second dimension, each track of proteins separated in the one-dimensional electrophoresis was cut from the gel and laid on one edge of a Gel bond film before 15 ml of warm 1%

agarose in barbitone acetate buffer, pH 8·6, containing 2·5% sheep anti-rabbit C3 and 3% PEG 6000 were poured on the film, ensuring that the agarose merged smoothly with the solidified track of proteins in the agarose. These proteins were then drawn across the agarose bed containing anti-C3 antiserum by electrophoresis for 16 hours at 150 V. The gels were then washed, fixed, dried and stained as before.

RESULTS Relative C3 Levels There was a small initial increase (5-7%) in the levels of circulating C3 in all animals by day 7 post-infection, followed by a gradual decrease to day 11 and then a rapid fall until about day 21 (Fig. 1). The maximum overall decrease in C3 level up to day 21 was 70%, observed in the one rabbit which showed a rise in C3 level on day 24 which coincided with a fall in the level of parasitaemia [Fig. l(A)]. The levels of circulating C3 rose in all the rabbits following the drug treatment on day 24 (Fig. 1).

Changes in Parasitaemia Parasites were first detected in the blood of each rabbit on day 7 post-infection and the levels of parasitaemia then rose until day 11. After day 11, the levels of parasitaemia fluctuated and varied between rabbits until they all fell after drug treatment on day 24; no parasites could be detected in any rabbit when examined on days 32 and 38 (eight and 14 days post-treatment, respectively (Fig. 1).

C3 Activation Although only a single precipitation peak was observed after immunoelectrophoresis of serum samples collected pre-infection (day 0), seven days post-infection (day 7) and post-treatment (day 32), two peaks were observed when sera collected on days 21 or 24 were used (shown in Fig. 2 for the rabbit in which parasite levels fell and blood C3 levels increased pre-treatment).

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Fig. 2. Activation ofC3 in the plasma and serum of a rabbit experimentally infected with Trypanosoma evansi. The one-dimensional rocket immunoelectrophoresis (top) indicates (left to right) the changing levels ofC3 in plasma taken pre-infection (day 0) and 7, II, 18, 21, 24,32 and 38 days post-infection. The rabbit was treated with Berenil on day 24. The two-dimensional electrophoresis gels, numbers 9-13, were of serum samples taken on days 0, 7, 21, 24 and 32 (i.e. eight days post-treatment), respectively. Number 14 is a schematic representation of gel 11.

DISCUSSION A consistent feature observed in all four rabbits infected with T. evansi was a fall in the blood C3 levels as the infections progressed; similar observations have been made on infections with other trypanosome species (Jarvinen and Dalmasso, 1976; Kobayashi and Tizard, 1976; Nielsen et al., 1978). One possible cause of this fall in C3 level could be C3 activation by

the antigen-antibody complexes (Musoke and Barbet, 1977) which are produced as parasites are eliminated by the host immune system through complement-dependent effector mechanisms (Murray and Urquhart, 1977). In the present study, the lowest levels of C3 were observed at the same times as the highest levels of parasitaemia, C3 levels increased following chemotherapy and, in one rabbit, an increase in the blood level ofC3 coincided with a

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TRYPANOSOME-INDUCED CHANCES IN C3

fall in the level of parasitaemia pre-treatment. Parasite burden and C3 level therefore seem inversely linked, possibly as the result of the trypanosomes releasing large quantities of complement-activating factor (Cunningham et a!., 1978). During the initial stages of the infection C3 levels increased slightly {5-7%) and a similar, though much larger (two- to three-fold) increase was observed by Shirazi et a!. (1980). This initial rise in C3 level may be caused by the large amounts of C3 produced during the acute phase of infections (Horning and Arquembourg, 1965) and/or the high numbers and level of activity, particularly in the liver, of mononuclear phagocytes, during the initial stages of trypanosome infection (Longstaffe, 1974; Murray et a!., 1974 ). Mononuclear phagocytes are known to secrete C3, 90% of which is produced in the liver (Lambris, 1988). The presence of more than one precipitation peak after immunoelectrophoresis of serum samples taken on days 21 and 24 showed that C3 was activated at those times (Pryjma et a!., 1974). The C3a and C3b activation products of C3 move faster than native C3 over the electrophoretic field (Clarke and Freeman, 1968;

479

Sandberg and Osler, 1971) but appear as a single peak as they have shared antigenic determinants (Hudson and Hay, 1976). The observation ofC3 activation during infection in the present study indicates that C3 hypocomplementaemia in T. evansi-infected rabbits may be caused by activation of C3, as has been suggested for infections with other trypanosome species (Musoke and Barbet, 1977). Demand for C3 activation may be linked to level of parasitaemia, as C3 activation appeared to be greatest, in the present study, when levels ofparasitaemia were high. The depletion of circulating C3 by T. evansi could not only have far-reaching effects on how the host reacts to an ongoing infection (Staines et al., 1985) but could also compromise the host's ability to respond to subsequent infections, as C3 has been shown to play in important role in the development of immunological memory (Klaus and Humphrey, 1977).

ACKNOWLEDGEMENTS. This investigation was

supported by a Commonwealth Scholarship granted to U.E.U., for which he is very thankful. We are also grateful toR. Munroe for the preparation of Figure 2.

REFERENCES BUDZKO, D. B., PIZZIMENTI, M. C. & KIERSZENBAUN, F. (1975). Effects of complement depletion in experimental chagas disease. Immune lysis of virulent blood forms of Trypanosoma cruzi. Infection and Immunity, 11, 86--91. CLARKE, H. G. M. & FREEMAN, T. (1968). Quantitative immunoelectrophoresis of human serum proteins. Clinical Science, 35,403-413. CuNNINGHAM, D. S., CRAIG, W. H. & KUHN, R. E. (1978). Reduction of complement levels in mice infected with Trypanosoma cruzi. Journal of Parasitology, 64, 1044-1049. HoRNING, M. & ARQUEMBOURG, R. C. (1965). PiC-globulin: an acute phase serum reactant of human serum. Journal of Immunology, 94, 307-316. HuDSON, L. & HAY, F. C. (1976). Practical Immunology. Oxford: Blackwell Scientific. IRVINE, J. (1979). Medical Immunology. Edinburgh: Teviot Scientific. JARVINEN, A. ]. & DALMASSO, A. P. (1976). Complement in experimental Trypanosoma lewisi infections in rats. Infection and Immunity, 14, 894-902. JoHNSTONE, A. & THORPE, R. ( 1987). Immunochemistry in Practice. Oxford: Blackwell Scientific. KLAUS, G. G. B. & HUMPHREY, J. H. (1977). The generation of memory cells. 1: The role of C3 in the generation ofB memory cells. Immunology, 33, 31-40. KoBOYASHI, A. & TIZARD, I. R. (1976). The response to Trypanosoma congolense infections in calves: determination oflmmunoglobulins IgG 1, IgG2, lgM and C3 levels and the complement fixing antibody titres during the course of infection. Tropical Medicine and Parasitology, 27, 411-417. LAMBRIS, ]. D. (1988). The multifunctional role of C3, the third component of complement. Immunology Today, 9, 387-393.

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LANHAM, S. M. & GoDFREY, D. G. (1970). Isolation of salivarian trypanosomes from man and other mammalians using DEAE-cellulose. Experimental Parasitology, 28, 521-534. LAURELL, C. B. (1966). Quantitative estimation of proteins by electrophoresis in agarose-gel containing antibodies. Analytical Biochemistry, 15, 45-52. LONGSTAFFE, J. A. ( 1974). Effects of infection with Trypanosoma brucei brucei on the macrophage of the mouse spleen. Parasitology, 69, xxiv. MuRRAY, P. K., jENNINGS, F. W., MURRAY, M. & URQUHART, G. M. (1974). The nature of immunosuppression in Trypanosoma brucei infections in mice. 1. The role of the macrophage. Immunology, 27, 815-824. MURRAY, M. & URQUHART, G. M. (1977). lmmunoprophylaxis against African trypanosomiasis. In Immunity to Blood Parasites of Animal and Man, eds. Miller, L. H., Pino, ]. A. & Mckevery, J. J. New York, Plenum Press. MusOKE, A. J. & BARBET, A. F. (1977). Activation of complement by variant-specific surface antigen of Trypanosoma brucei. Nature, 270, 438-440. NIELSEN, K., SHEPPARD, J., HoLMES, W. & TIZARD, I. (1978). Experimental bovine trypanosomiasis: changes in serum immunoglobulins, complement and complement components in infected animals. Immunology, 35, 817-826. PARIS,]., MuRRAY, M. & Mc0DIMBA, F. (1982). A comparative evaluation of the parasitological techniques currently available for the diagnosis of African trypanosomiasis in cattle. Acta Tropica, 39,307-316. PRYJMA, ]., HuMPHREY, J. H. & KLAUS, G. G. B. (1974). C3 activation and T -independent B cell stimulation. Nature, 252, 505-506. SANDBERG, A. L. & OSLER, A. G. (1971). Dual pathways of complement interaction with guinea-pig immunoglobulins. Journal of Immunology, 107, 1268-1273. SHIRAZI, M. F., HOLMAN, M., HuDSON, K. M., KLAUS, G. G. B. & TERRY, R. J. (1980). Complement (C3) levels and effect of C3 depletion in infections of Trypanosoma brucei in mice. Parasite Immunology, 2, 155-161. STAINES, N., BROSTOFF, J. & ]AMES, K. (1985). Introducing Immunology. London: Gower Medical.

Complement (C3) levels and activation in rabbits experimentally infected with Trypanosoma evansi.

Rocket immunoelectrophoresis was used to monitor the levels of the third complement component (C3) in the blood of rabbits experimentally infected wit...
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