Ann. Immunol. (Inst. Pasteur) 1984, 135 O, 231-240

OF

BLOOD-STAGE MA LA RIA I N B I O Z Z I H I G H

AND LOW A N T I B O D Y R E S P O N D E R MICE:

T H E I MP ORTANCE

A N T I B O D Y AND MACROPHAGES IN I M M U N I T Y

by I. N. Brown 0), H. M. Dockrell (2), j. B. de Souza (2) and J. H. L. Playfair (2)

(1) Department o/Medical Microbiology, Wright-Fleming Institute, St. Mary's Hospital Medical School, London W2, and (2) Departmenl o[ Immunology, Arthur Stanley House,

The Middlesex Hospital Medical School, London W I P 9PG

SUMMARY

Mice from Biozzi high (H) and low (L) antibody responder lines were infected either with non-lethal Plasmodium yoelii 17X, with a lethal variant of the same parasite or with P. chabaudi (non-lethal). H mice recovered from infection with both P. yoelii and its normally lethal variant, whereas L mice died from both. However, L mice showed better initial control of parasitaemia with all three infections. Both H and L mice infected with P. chabaudi showed a similar rapid reduction of parasitaemia once it had reached 40-50%, but L mice were unable to clear the infection completely. When infected with either non-lethal P. yoelii or P. chabaudi, H mice made substantially more antibody than L mice, especially around the time of recovery. The courses of parasitaemia suggest a role for non-antibody factors in early control of infection and for antibody in recovery from P. yoelii. Although antibody was apparently necessary for complete recovery from P. chabaudi, the first rapid fall in parasitaemia appeared to be unrelated to antibody levels.

KEY-WORDS: Immune response, Plasmodium, Parasitaemia; Macro- phage, Antibody, Biozzi mice.

INTRODUCTION

The Biozzi high (H) and low (L) antibody responder lines of mice were produced by bidirectional selective breeding for antibody titres against

Manuscrit re~u le 20 dficembre 1983, accept~ le 9 ffivrier 1984.

232 I. N. BROWN AND COLL.

sheep and pigeon erythrocytes [1, 2]. Subsequently, it was found tha t H mice also produced increased antibody responses to other unrelated antigens such as bovine serum albumin (BSA), pneumococcal polysaccharide (S III) and the dinitrophenyl hapten (DNP) [2]. However, L mice had more active macrophages than H mice, leading to faster antigen catabolism, while antigen persisted longer in H mice [19]. T-lymphocyte functions such as delayed-type hypersensit ivity (DTH) were similar in both H and L mice [2].

These lines of mice have been used as a tool to investigate the relative importance of antibody and macrophage activity in various infections. For example, H mice are more resistant to infection with Trgpanosoma cruzi [ 11] and Nemalospiroides dubius [ 10], implicating antibody in recovery, while L mice showed greater resistance to Salmonella tgphimurium [12] and to intracellular parasites such as Leishmania tropica [7], Mgcobaclerium boris BCG, and M. lepraemurium (I. N. Brown, unpublished results). Earlier studies with P. berghei malaria confirmed tha t the antibody response to P. berghei antigens was 8 to 10-fold higher in H than in L lines of mice; vaccination induced effective protection in H mice, while only weak protection was achieved in L mice, indicating the importance of anti- body in this model [8, 9].

In this study, we have infected H and L mice with three other malaria parasites: P. goelii, which produces a non-lethal infection in most strains of mice, a generally lethal variant of P. goelii, and P. chabaudi, which gives an initial peak of parasitaemia which resolves, followed by recrudescences. The results of our experiments indicate that , although macrophages may be involved in early control of infection, antibody is required both for recovery from P. goelii and for complete clearance of parasitaemia with P. chabaudi, as judged by blood film counts; however, both H and L mice reacted simi- larly at the first crisis.

MATERIALS AND METHODS

Mice.

All the mice used in this study were females derived by brother-sister mating at St.-Mary's Hospital Medical School from stock originally obtained from Dr Biozzi by Pr A. A. Glynn. The lines have been tested periodica]ly for antibody responsi- veness to sheep erythrocytes.

Parasites.

P. goelii 1 7 X a n d a l e tha l v a r i a n t d e r i v e d in th i s l a b o r a t o r y [14] were m a i n t a i n e d b y l i m i t e d b lood passage or w e r e s to r ed in l iqu id n i t rogen . P. chabaudi 2 7 2 2 A S was k i n d l y p r o v i d e d by D r W. J a r r a , T h e N a t i o n a l I n s t i t u t e for Med ica l R e s e a r c h , Mil l Hi l l , L o n d o n . I n f ec t i ons w e r e i n i t i a t e d w i t h 1 • 104 p a r a s i t i z e d red cells i n j ec -

BSA = bovine serum albumin. DNP = dinitrophenyl hapten. DTH = delayed-type hypersensitivity. H = high (responder).

i .v . = intravenous(ly). L = low (responder). PBS ~ phosphate-buffered saline.

MALARIA IN HIGH AND LOW Ig RESPONDER MICE 233

ted i. v. Parasitized red cells were always prepared from animals in the ascending phase of parasitaemia. Tail-blood films were prepared daily and the parasitaemia counted after staining with Giemsa's solution. Results from the two lines were compared using Student's t test.

Measurement o/antibodg titres.

Total anti-parasite antibody was measured by the slide fluorescence method [17] using homologous parasitized blood from an early infection in CBA or (C57BL xBALB/c)F1 mice as the antigen. Two-fold dilutions of serum in phosphate- buffered saline (PBS) were applied to the antigen-coated slides at 20 o C for 30 rain; the slides were then washed with PBS and treated with fluorescein-conjugated polyvalent rabbit anti-mouse immunoglobulin for another 30 min. The fluorescence was read in a UV microscope with incident light. The end-point was taken as the last serum dilution showing clear fluorescence.

R E S U L T S

Course of infection with P. yoelii.

Mice from the H and L lines were infected with P. goelii in two separate experiments. Figure la and b shows tha t during the first 14 days of infection, H mice showed a slight bu t significant increase in parasitaemia compared to L mice. However, H mice were able to clear the infection, while in L mice, parasi taemia continued to rise and all mice died. In another experiment, the lethal variant of P. goelii was used. Figure lc shows that , al though parasi- taemia again developed more slowly in L mice, it then continued to rise and all mice were dead by day 24. H mice had slightly lower parasi taemia between days 7 and 12; parasitaemia fell rapidly after day 16 and all mice had recovered by day 23.

Course of infection with P. chabaudi.

When H mice were infected with P. chabaudi, parasitaemia increased to about 50 % and then fell sharply to approximate ly 1 ~ in the next three days in all bu t two mice, which died on days 11 and 12; the remaining mice were clear of demonstrable parasites by day 20 and a typical recru- descence was observed around day 38 (fig. 2a). L mice gave a similar bu t slightly delayed response, with a similar sharp fall once the parasi taemia had reached 50% and no deaths; they failed, however, to completely clear the parasites from the blood, maintaining parasi taemia between 0.1 and 1 ~o over the next two weeks. At this stage (and during the early phase of the infection), the parasites were entirely restricted to mature erythrocytes . A second experiment produced similar results (fig. 2b). This suggests tha t the initial rapid drop in parasi taemia was not related to an t ibody levels, al though ant ibody may be needed for subsequent clearance of pa ten t para- sitaemia.

Table I summarizes the outcome of the 3 infections in H and L mice, showing the first day on which no parasites could be detected in the blood

234

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I. N. B R O W N A N D COLL.

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Day of Infection

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Day of Infection

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F I G . 1

MALARIA IN HIGH AND LOW Ig RESPONDER MICE 235

TABLE I. - - The outcome of infection with 3 species of malaria in individual H and L mice.

D a y of D a y of D a y of D a y of High responder line recovery death Low responder l ine recovery death

P. goetii (non- le thal) 16, 17, 18, 10 P. yoelii (non- le thal) 18, 26, 26, 19, 20, 25, 28, 31, 31, 25. 31, 31.

P. goelii (lethal) 20, 21, 23, P. goelii (lethal) 13, 17, 17, 23, 23. 21, 24.

P. chabaudi 18, 19, 20, l l , 12. P. chabaudi 13, 16, 18, 20, 20, 20, ~ 2 8 , ~ 2 8 , 26. 30, 41,

59, ~ 6 2 .

or the day of death. Mice of the H strain all recovered from lethal P. yoelii between days 20 and 23. Seven out of eight mice recovered from infection with the non-lethal P. yoelii between days 16 and 25; the eighth mouse died on day 10 with a high parasitaemia. Also, two H mice died of P. chabaudi infection on days 11 and 12; the remaining mice cleared their primary parasitaemia between days 18 and 26. In contrast, only two L mice with P. chabaudi showed a negative blood film by day 25, and three mice still showed patent parasitaemia on day 48. However, there were no deaths. Low responder mice all died from the normally non-lethal P. yoelii between days 18 and 31 and from the lethal variant of P. yoelii between days 13 and 24.

Measurement of antibody titres.

Total serum anti-parasite antibody was measured in a small group of H and L mice on day 22 of infection with non-lethal P. yoelii and on days 15, 28 and 48 of infection with P. ehabaudi (table II). On day 22 of infection with P. yoelii, H mice had antibody titres 16-fold higher than L mice. Similarly, on day 15 of infection with P. chabaudi, antibody titres were 8-fold higher

FIG. 1. - - P. yoeli i in/ection in high responder (solid circles) and low responder (open circles) mice.

Groups of five mice were infected in travenous ly w i th 10' parasit ized red cells. Pane l s a and b show two separate exper iments w i th P. yoelii 17 X (,, non- l e tha l ~,); pane l c shows a single exper iment wi th the lethal var iant of P. yoelii 17 X. Paras i taemias were fo l lowed on stained ta i l -blood films. Two mice infected wi th non- le tha l P. yoelii 17 X were killed on day 22 to test an t ibody levels; detai ls of the day of recovery or death of the remaining mice are given in table I. Resul t s are expressed as geometric m e a n • standard error. The paras i taemias of the two lines are s ignif icant ly different ( p < 0 . 0 1 ) in panel a on day 8, panel b on days 10-11 and 15, and panel c on days 3-5 and 7-8.

Ann. Immunol. (Inst. Pasteur), 135 C, ,l ~ 2, 1984, 16

2 3 6 I. N . B R O W N A N D C O L L .

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F I 6 . 2 . - - P. c h a b a u d i infection in high responder (solid circles) and low responder (open circles) mice.

Groups of five mice were infected i n t r a v e n o u s l y w i t h 10 4 pa ras i t i zed red cells. Panels a and b show two sepa ra te exper imen t s . P a r a s i t a e m i a s were fo l lowed on s ta ined ta i l -b lood films. Some mice were ki l led to t e s t a n t i b o d y levels on days 15, 28 and 48; de ta i l s of t he d a y of r ecove ry or dea th of the r e m a i n i n g mice are given in t ab l e I. Resu l t s are expressed as geome- t r i c mean : t :1 s t anda rd error. The pa ra s i t a emias of the two l ines are s ign i f ican t ly different (p<:0.01) in pane l a on days 6-9 and 11, and in pane l b on days 6-11 and 13-15.

MALARIA IN HIGH AND LOW Ig RESPONDEB MICE 237

TABLE II . - - A n t i b o d y t i tres in H and L m i c e i n f e c t e d with e P. yoelii >> (non-lethal) or (( P. chabaudi >>.

Parasite

Day of

infection

High responders Low responders

individual mean individual mean

P. yoelii (non-lethal)

P. chabaudi

22 > 16,384 16,384 512 1,024 22 16,384 2,048

15 2,048 2,048 256 256 2,048 512 2,048 256

28 1,024-2,048 1,024-2,048 1,024 2,048 1,024-2,048 4,096

48 4,096 8,192-16,384 8,192 16,384 8,192 8,192-16,384 --16,384

in H than in L mice. However , later in the infection with P. chabaudi, on days 28 and 48, the an t ibody titres were similar in H and L mice, even though the H mice were controlling their parasi taemia significantly better .

DISCUSSION

The three parasite strains chosen for infection of Biozzi H and L respon- der mice in this s tudy have been widely used in studies of immuni ty to malaria. Our results with P. goelii, showing tha t H mice recovered from infection while making substant ial ly more specific an t ibody than L mice, agree with previous studies in which an t ibody was shown to be impor tan t in recovery [13]. It is interesting tha t H mice also recovered from the normally lethal P. yoelii infection. In this respect, the Biozzi H responder line resembles other resistant strains of mice such as the CBA, which can occasionally recover, ra ther than more susceptible strains such as B A L B / e or C57BL, which invar iably die from this infection. One factor which could be involved is the rate of erythropoiesis, as the H line may be able to very efficiently replace ery throcytes [16].

Ant ibody seems to be far less impor tan t in recovery from some other murine malaria infections. The innate resistance of H and L mice to P. berghei was not found to differ significantly [8] and we have now shown that , with the exception of two H mice which died with high parasi taemias, bo th H and L mice infected with P. chabaudi showed a similar rapid drop in parasi taemia once it had reached 50%. This occurred despite an 8-fold difference in total an t ibody levels at this t ime. Although we have measured total anti-P, chabaudi ant ibody rather than protect ive ant ibody, this result agrees with earlier studies using p-suppressed mice which recovered from P. chabaudi without significant an t ibody production [6]. In addition,

238 I. N. BROWN AND COLL.

L mice were unable to totally clear parasites from their blood, although making similar levels of antibody to H mice. Whether the waves of parasi- taemia seen in L mice relate to antigenically distinct populations, as has been postulated for the recrudeseent populations in P. knowlesi infections in monkeys [3], remains to be investigated. Splenectomy or total body irra- diation of infected CBA mice following the first sharp fall in parasitaemia also produces similar chronic parasitaemias (R. S. Phillips, personal com- munication).

We conclude that a non-antibody mechanism is responsible for the rapid fall and clearance of parasitaemia with P. chabaudi, although protec- tive antibody and the distribution of antibody subclasses still need to be measured. Macrophages may be responsible for the initial delay in parasi- taemia seen in all three infections in L mice, but though some of their macrophage functions have been shown to increase relative to H mice [19], L mice are still unable to recover faster than H mice. It is possible that non-specific factors such as tumour necrosis factor [15, 17] or products of the oxidative burst [4, 5] might be secreted by macrophages or other cells in both lines of mice, and tha t their production does not correlate with the other macrophage functions measured to date. We have shown that P. cha- baudi can be killed by both hydrogen peroxide [5] and tumour necrosis serum given in oivo (J. Taverne, unpublished results) and we are currently investigating (1) the potential of both lines of mice to secrete such non- specific factors, and (2) the possibility that vaccination can accelerate reco- very in both lines of mice.

RESUMI~

P A R A S I T 1 E M I E P A L U D I ~ E N N E

C H E Z LES S O U R I S B I O Z Z I B O N N E S OU M A U V A I S E S R t ~ P O N D E U S E S :

I M P O R T A N C E DE S A N T I C O R P S E T DE S M A C R O P H A G E S D A N S L ' I M M U N I T I ~

Des souris de souches Biozzi H e t L (bonnes et mauvaises productrices d'anticorps) ont ~t~ infect~es par le parasite malarien Plasmodium goelii non-l~tal, ou par une variante normalement l~tale du mfime parasite, ou enfin par P. chabaudi (non-l~tal). Les souris H ont bien ma~tris~ les deux infections P. goelii tandis que les souris L sont mortes. Cependant, les sou- ris L ont mieux ma[trisfi les trois infections au stade initial. La r~duction de la parasit~mie ~ P. chabaudi, apr~s avoir atteint le niveau de 40-50%, a pr~sent~ une ~volution identique pour les deux souehes, mais les souris L n'ont pu ~liminer complgtement l'infection. Les souris H ont produit beaucoup plus d'anticorps, surtout au moment de la gu~rison. L'~volution des parasit~mies sugg~re que les facteurs diff~rents des anticorps aident j uguler l'infection pendant les premiers j ours, et que les anticorps sont impor- rants pour la protection contre P. goelii. Bien que les anticorps soient appa- remment n~cessaires pour la gufirison complete de l'infection ~ P. chabaudi,

MALARIA IN HIGH AND LOW Ig RESPONDER MICE 239

la premibre chute rapide de la parasit6mie ne parait pas associ6e au niveau des anticorps.

MOTS-CL~S : R6ponse immuni ta ire ,Plasmodium, Parasit6mie; Anticorps, Macrophage, Souris Biozzi.

ACKNOWLEDGMENTS

H. D. and B. de S. are supported by the Medical Research Council of Great Britain and I. N. B. by WHO.

REFERENCES

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[2] Blozzi, G., STIFFEL, C., MOUTON, D. & BOUTHILLIER, Y., Selection of lines of mice with high and low antibody responses to complex immunogens, in ~( Immunogenetics and immunodeficiency 7) (B. Benacerraf) (pp. 179-227). MTP, Lancaster, 1975.

[3] BnowN, K. N. & BROWN, I. N., Immunity to malaria: antigenic variation in chronic infections of Plasmodium knowlesi. Nature (Lond.), 1965, 208, 1286-1288.

[4] CLARK, I. A. & HUNT, N. H., Evidence for reactive oxygen intermediates causing haemolysis and parasite death in malaria. Infect. Immun., 1983, 39, 1-6.

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[7] HALE, C. & HOWAnD, J. G., Immunological regulation of experimental cuta- neous leishmaniasis. - - 2. Studies with Biozzi high and low responder lines of mice. Parasite Immunol., 1981, 3, 45-55.

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[9] HEUMANN, A. M., BuccI, A., STIFFEL, C., CASAGLIA, 0., ZENOBI, P. & BtozzI, G., Inheritance of post-vaecinal resistance against Plasmodium berghei infection in mice. Parasite Immunol., 1983, 5, 143-150.

[10] JENKINS, D. C. & CARRINGTON, T. S., Nemalospiroides dubius: the course of primary, secondary and tertiary infections in high and low responder Biozzi mice. Parasitology, 1981, 82, 311-318.

[11] KIERSZENBAUM, F. & HOWARD, J. G., Mechanisms of resistance against experimental Trypanosoma eruzi infection: the importance of antibodies and antibody-forming capacity in the Biozzi high and low responder mice. J. Immunol., 1976, 116, 1208-1211.

[12] PLANT, J. E. & GLYNN, A. A., Genetic control of resistance to Salmonella lyphimurium in high and low antibody responder mice. Clin. exp. Immu- nol., 1982, 50, 283-290.

[13] PLAYEAIR, J. H. L., Immunity to malaria. Brit. reed. Bull., 1982, 38, 153-159.

240 I. N. BROWN AND COLL.

[14] PLAYFAIR, J. H. L., DE SOUZA, J. B. & COTTRELL, B. J., Protection of mice against malaria by a killed vaccine: differences in effectiveness against P. yoelii and P. berghei. Immunology, 1977, 33, 507-515.

[15] RUFF, M. R. & GIFFORD, G. E., Tumour necrosis factor, in (( Lymphokines )) (E. Pick), 2, (pp. 235-272). Academic Press, London, New York, 1981.

[16] STEVENSON, M. M., LYANGA, J. J. & SKAMENE, E., Murine malaria: genetic control of resistance to Plasmodium chabaudi. In/ect. Immun., 1982, 38, 80-88.

[17] TAVERNE, J., DEPLEDGE, P. & PLAYFAIR, J. H. L., Differential sensitivity in vivo of lethal and non-lethal malaria parasites to endotoxin-induced serum factor. In/ect. Immun., 1982, 37, 927-934.

[18] VOLLER, A. & O'NEILL, P., Immunofluorescence method suitable for large- scale application to malaria. Bull. Org. mond. Sanld, 1971, 45, 524-529.

[19] WIENEn, E. & BANDIERI, A., Differences in antigen handling by peritoneal macrophages from the Biozzi high and low responder lines of mice. Europ. J. Immunol., 1974, 4, 457-463.