REFERENCES. 1. Desowitz, R. S. 1989. Plasmodium-specific immunoglobulin E in sera from an area of holoendemic malaria. Trans. R. Soc. Trop. Med. Hyg.
INFECTION AND IMMUNITY, Apr. 1996, p. 1432–1433 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 64, No. 4
NOTES Immunoglobulin E Elevation in Plasmodium chabaudi Malaria HELENA HELMBY,* HEDVIG PERLMANN, MARITA TROYE-BLOMBERG,
AND
PETER PERLMANN
Department of Immunology, Stockholm University, Stockholm, Sweden Received 14 September 1995/Returned for modification 22 November 1995/Accepted 9 January 1996
In order to investigate the mechanism of immunoglobulin E (IgE) elevation in malaria we studied mice infected with asexual blood stages of the rodent malaria parasite Plasmodium chabaudi chabaudi for total IgE and IgE antimalarial antibodies. Multiply infected mice had elevated levels of total as well as malaria-specific IgE in their sera. Sera taken from mice 3 weeks after one infection with P. chabaudi showed no IgE elevation, indicating that prolonged or repeated exposure to the parasite is necessary for the induction of an IgE response, which also is induced independently of previous or simultaneous infection with other pathogens such as helminths. In human Plasmodium falciparum malaria, CD41 T cells from immune donors stimulated with malaria antigen in vitro have been demonstrated to secrete gamma interferon and/or interleukin 4 (IL-4). Frequently, production of these cytokines is not correlated, suggesting that the response is regulated by Th1- and Th2-like cells (16). As IL-4 is involved in switching immunoglobulin (Ig) isotype production by B cells from IgM to IgE (8), we have recently investigated IgE levels in the sera of individuals living in areas of high P. falciparum endemicity (11). Approximately 85% of the donors had significantly elevated levels of total IgE. Moreover, some of the IgE had specificity for malarial antigens, suggesting that plasmodial infection may have directly contributed to IgE elevation. Similar findings have also recently been made by others in Papua New Guinea (1, 2). However, helminthic infections, frequently occurring in these study areas, are associated with the induction of Th2-biased immune responses (19) with ensuing IgE elevation (5, 7). Therefore, we could not exclude that IgE elevation in our patients was an IL-4-dependent response initially induced by helminthic infections. For this reason, we decided to look for possible IgE responses in normal mice experimentally infected with asexual blood stages of Plasmodium chabaudi chabaudi, a rodent malaria parasite known to induce a Th1-type response with gamma interferon-producing CD41 T cells in the early phase of infection. After acute infection, the response is dominated by IL-4-producing Th2 cells, which are effective helper cells for antibody production involved in the clearance of parasitemia during the later phases of infection (10, 14, 15, 18). Elevated levels of IgE have also been reported to occur in the blood of such mice (17). BALB/c mice of both sexes received a first subcutaneous injection of 100 ml of P. chabaudi chabaudi (strain AS, originally obtained from D. Walliker, University of Edinburgh)parasitized blood (50 to 70% parasitemia) diluted 1:1,000 in phosphate-buffered saline, pH 7.4. The parasites were highly virulent and killed approximately 70% of the mice. Surviving mice were challenged five times at 2-week intervals with 50 ml
of infected blood (50 to 60% parasitemia). The mice were bled between 1 and 2 weeks after the last challenge, and sera from two to five mice were pooled. Serum pools prepared from sera taken 3 weeks after primary infection and control BALB/c sera from mice hyperimmune to Schistosoma mansoni were also included (9). Total IgE was measured by means of an enzymelinked immunosorbent assay (ELISA) with monoclonal rat anti-mouse IgE antibody (PharMingen, San Diego, Calif.) as the capture antibody and biotinylated monoclonal rat anti-mouse IgE (Fcε specific) as the secondary antibody (Biosource International, Camarillo, Calif.), followed by alkaline phosphataseconjugated streptavidin (Mabtech, Stockholm, Sweden). As shown in Table 1, a significant elevation of total IgE was detected in sera from multiply infected mice (14.7 mg/ml) in comparison with IgE levels in sera from noninfected normal mice (0.5 mg/ml; P 5 0.0001). In contrast, sera taken early (3 weeks) after primary infection showed no significant IgE elevation, indicating that repeated or prolonged exposure is necessary for the induction of an IgE response. To establish the occurrence of IgE antiplasmodial antibodies, a serum pool from multiply infected mice (total IgE level, 9.8 mg/ml) was tested by immunoblotting of P. chabaudi-infected erythrocytes separated on 7.5 to 17.5% gradient sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membrane (Bio-Rad, Hercules, Calif.). The membrane was incubated with mouse serum and after extensive washing incubated with alkaline phosphatase-conjugated polyclonal sheep anti-mouse IgE (The Binding Site, Birmingham, United Kingdom) or goat anti-mouse IgG (Sigma, St. Louis,
TABLE 1. Total IgE in mouse serum Serum
na
IgE (mg/ml [mean 6 SD])
Normal P. chabaudi infected (one infection)b P. chabaudi infected (five infections)
4 2 13
0.5 6 0.21 0.1 6 0.09 14.7 6 5.54c
1
52.6
S. mansoni infected
* Corresponding author. Mailing address: Dept. of Immunology, Stockholm University, S-106 91 Stockholm, Sweden. Phone: 46 8 164170. Fax: 46 8 157356. Electronic mail address: helena@imm2 .su.se.
a
Serum pools were from two to five BALB/c mice and were diluted 1:500. Sera were taken 3 weeks after primary infection. c By the unpaired t test, P 5 0.0001. b
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NOTES
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IgE-mediated pathogenesis in malaria. Although serum IgE levels in cerebral P. falciparum malaria are significantly higher than in uncomplicated malaria, their significance for the pathogenesis of the cerebral complications is not known (11). We are now using the P. chabaudi model to investigate the possible role of IgE for protection and/or pathogenesis in malaria. We thank Klavs Berzins and Go ¨ran Holmqvist for advice and helpful discussions. This work was supported by grants from the Swedish Medical Research Council and the Swedish International Development Cooperation Agency. REFERENCES
FIG. 1. Immunoblotting of IgE (A) and IgG (B) antibodies from normal noninfected mice (lane 1) and mice multiply infected with P. chabaudi (lane 2). Lane 3 is the secondary antibody control with no serum. Molecular mass markers are in kilodaltons.
Mo.) followed by AP-Purple (TSI, Milford, Mass.). The sera were also tested in an ELISA with lysates of P. chabaudiinfected erythrocytes as the coating antigen. The sera displayed a significant binding of IgE to the parasite lysate in the ELISA: for normal serum, the optical density at 405 nm for IgE was 0.036, and for P. chabaudi-infected serum (five infections) the optical density at 405 nm was 0.258 (serum was pooled from two to five BALB/c mice and was diluted 1:10). The sera bound to a variety of antigens ranging from 25 to 250 kDa in immunoblotting assays (Fig. 1A). Sera from noninfected mice gave no specific IgE pattern in comparison with the antibody control (Fig. 1A). Similarly, sera from mice taken 3 weeks after one infection had no detectable IgE antibodies. As in human P. falciparum malaria (11), IgG antibodies from P. chabaudi-infected mice also reacted strongly with parasite antigens (Fig. 1B). Many of these appeared in the blots in positions similar to those of the IgE antibodies, suggesting that the two isotypes at least partially reacted with the same target molecules. Although the IgG antibodies were present at much higher concentrations than were the corresponding IgE antibodies, blocking of the binding of the latter to the antigens in the in vitro assays (ELISA and immunoblotting) was avoided by prolonged incubation, as previously seen for P. falciparum (11). In vivo, it is likely that some of these IgG antibodies, together with the substantial amounts of polyclonal IgE, contribute to prevent IgE-dependent allergic reactions (6). In conclusion, we have here shown that malaria infection may independently of preceding or concomitant helminth infection cause significant elevation of both polyvalent and specific IgE levels. Several investigations have suggested a protective role of IgE and the involvement of IgE binding effector cells in schistosomiasis (3, 4, 12), although the relative importance of this protection is under discussion (13). There is presently no information on either IgE-mediated protection or Editor: S. H. E. Kaufmann
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