INFECTION AND IMMUNITY, May 2004, p. 3054–3058 Vol. 72, No. 50019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.5.3054–3058.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cerebral Edema and Cerebral Hemorrhages inInterleukin-10-Deficient Mice Infected

with Plasmodium chabaudiLatifu A. Sanni,† William Jarra, Ching Li,‡ and Jean Langhorne*

Division of Parasitology, MRC National Institute for Medical Research,The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

Received 19 December 2003/Returned for modification 13 January 2004/Accepted 27 January 2004

During a Plasmodium chabaudi infection in interleukin-10 (IL-10) knockout mice, there is greater parasitesequestration, more severe cerebral edema, and a high frequency of cerebral hemorrhage compared withinfection of C57BL/6 mice. Anti-tumor necrosis factor alpha treatment ameliorated both cerebral edema andhemorrhages, suggesting that proinflammatory responses contributed to cerebral complications in infectedIL-10�/� mice.

It is well established that pro-inflammatory cytokines, suchas gamma interferon (IFN-�), tumor necrosis factor alpha(TNF-�), and lymphotoxin, are involved in some of the com-plications of malaria infection in humans and mice (3, 4, 6, 10).Lower interleukin-10 (IL-10) plasma concentration (9) andIL-10/TNF-� ratios (17) in children with malarial anemia sug-gest that an imbalance in pro- and anti-inflammatory cyto-kines, such as IL-10, may be related to the development ofdisease. In support of this, IL-10 knockout mice (IL-10�/�) aremore susceptible to a lethal infection with Plasmodiumchabaudi, develop a more severe anemia and hypoglycemia,and have higher concentrations of IFN-� and TNF-� in plasmathan infected C57BL/6 mice (11–13). Treatment of infectedIL-10�/� mice with antibodies to IFN-� and TNF-� eliminatesmortality and some of the pathological sequelae (11, 12), sug-gesting that proinflammatory cytokines play a role in thepathogenesis of malaria in P. chabaudi infections of mice.

One of the severe complications of malaria infection that hasnot been described for P. chabaudi infection is cerebral in-volvement. It is possible that under conditions where proin-flammatory cytokines are upregulated, P. chabaudi infectionmay induce responses that result in cerebral complications.Therefore, we have examined P. chabaudi infections in IL-10�/� mice for evidence of cerebral involvement.

Female IL-10�/� mice (8), back-crossed at least eight timesonto the C57BL/6 background, and control C57BL/6 mice (6 to12 weeks), bred and maintained as described previously (11),were infected with P. chabaudi chabaudi (AS) (P. chabaudi)and monitored as described previously (21). The courses ofinfection, associated anemia, hypoglycemia, loss in body

weight, and mortality were all as described previously for IL-10�/� and C57BL/6 mice (11–13). Mice were checked for signsof neurological impairment, such as gait disturbance, convul-sions, hemiplegia, and coma; 3 out of 27 of the IL-10�/� miceshowed signs of a wobbling gait suggestive of limb paralysis.Convulsions, tonic or clonic, were not observed in any of themice.

Sequestration of P. chabaudi (AS) was determined by mea-suring total and differential peripheral blood parasitemia overa 24-h period on days 7 to 8 postinfection (p.i.) for IL-10�/�

and C57BL/6 mice. Parasitemias of IL-10�/� and C57BL/6over this time period are shown in Fig. 1a and b. We observeda transient reduction in the total parasitemia and in the per-centage of trophozoite-infected red blood cells (RBC) in theblood of both groups of mice. Mean reduction of peripheralparasitemia was significantly greater for IL-10�/� mice thanfor IL-10�/� mice (87% compared with 61% for trophozoite-infected RBC; 24% compared with 9% in the total parasitemiafor IL-10�/� and IL-10�/� mice, respectively [P � 0.05 foreach; n � 5; Mann-Whitney test]).

To determine if blood-brain barrier breakdown and cerebraledema occur during P. chabaudi infection, the extent of leak-age of Evans Blue dye into the brain was determined as de-scribed previously (22). Leakage of Evans Blue dye into thebrains of IL-10�/� and IL-10�/� mice was observed between 6and 10 days p.i. (Fig. 2a). In two independent experiments, thelevel of dye leakage was significantly higher for the IL-10�/�

mice than for the IL-10�/� mice (Fig. 2b): in experiment 1 ondays 6, 8, and 10 p.i. (P � 0.05, Mann-Whitney test) andexperiment 2 on day 8 p.i. (P � 0.05).

To investigate the role of TNF-� in the development ofcerebral edema in infected IL-10�/� mice, groups of mice wereinjected intraperitoneally with 1 mg of either anti-TNF-�(Mp6-XT22) monoclonal antibody (Alexis Corporation, Not-tingham, United Kingdom) (16) or immunoglobulin G1 iso-type control antibody (GL113) on day 0 and day 5 or 6 duringinfection. At intervals in the infection, mice were injected withEvans Blue as described above and dye leakage into the brainwas quantified. The degree of cerebral edema in the brain was

* Corresponding author. Mailing address: Division of Parasitology,National Institute for Medical Research, The Ridgeway, Mill Hill,London NW7 1AA, United Kingdom. Phone: 44 (0)20 8816 2558. Fax:44 (0)20 8816 2638. E-mail: jlangho@nimr.mrc.ac.uk.

† Present address: Department of Histopathology, The General In-firmary at Leeds, Leeds LS2 9JT, United Kingdom.

‡ Present address: Kennedy Institute of Rheumatology Division,Imperial College London, Faculty of Medicine, London W6 8LH,United Kingdom.

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determined on day 8 p.i. Treatment with anti-TNF-� antibodysignificantly decreased the amount of cerebral edema in the P.chabaudi-infected IL-10�/� mice (Fig. 2c [P � 0.05, Mann-Whitney test]).

Cerebral hemorrhages were observed in 60% of IL-10�/�

mice between days 8 and day 12 of infection, whereas nohemorrhages were observed in IL-10�/� mice by either grossor microscopic examination at any time of infection (Fig. 3aand 4). Light microscopy confirmed the gross anatomical find-ings of brain hemorrhage. Hemorrhages ranged from micro-scopic to petechial and larger-size hemorrhages (Fig. 4c to f)and were mainly in the cerebrum, but any region of the braincould be affected (data not shown). Uninfected and saline-injected IL-10�/� or infected IL-10�/� mice sacrificed beforeday 8 had no evidence of cerebral hemorrhages, suggestingthat this manifestation is related to infection with P. chabaudiand is not the result of spontaneous hemorrhaging in IL-10�/�

mice.Treatment of infected IL-10�/� mice with anti-TNF-� anti-

body reduced the frequency of hemorrhages by approximately50%; 5 of 21 anti-TNF-�-treated mice had cerebral hemor-rhages compared with 11 of 22 control immunoglobulin�treated mice at days 8 and 9 p.i. (Fig. 3b).

This study shows that in addition to the higher mortality,greater body weight loss, anemia, and hypoglycemia observedpreviously (11–13), acute P. chabaudi infection in IL-10�/�

mice is also accompanied by increased sequestration of para-sites, cerebral hemorrhages, and cerebral edema. Similar to themortality and other features of malarial disease (11, 12) inIL-10 �/� mice, both cerebral edema and cerebral hemor-rhages are related to the increased production of TNF-�. Theobservation of cerebral involvement during P. chabaudi infec-tion is consistent with a previous report that IL-10 is protectiveagainst the development of cerebral malaria (CM) in Plasmo-dium berghei ANKA infection (7) and supports the view thatregulation of inflammatory cytokines may be an importantfactor in the pathogenesis of cerebral complications (2, 3, 18,20).

The role of cerebral edema in the pathogenesis of CM iscontroversial. Several publications support such a role in hu-man (1, 14) and mouse (19, 20) CM. However, others (23)argue against the permeability hypothesis. We show here thatcerebral edema occurs in IL-10�/� mice as early as day 6 p.i.,earlier than any clinical signs of illness or cerebral hemor-rhages in these mice. The exact cause of increased cerebralmicrovascular permeability during P. chabaudi infection in IL-

FIG. 1. Sequestration of parasitized RBC in IL-10�/� and IL-10�/� mice infected with P. chabaudi. (a and b) Parasitemias, total (�),trophozoites (F), schizonts (Œ), and rings (�), in a representative C57BL/6 (a) and IL-10�/� (b) mouse over a 24-h period on day 8 of infection.Percentage parasitemias were determined on Giemsa-stained blood films taken hourly for 24 h. Schizont rupture generally took place atapproximately midnight. A parasite was considered to be at the schizont stage when at least two nuclei could be identified clearly. (c and d) Dropin parasitemia of maturing trophozoites prior to schizont rupture, expressed as the mean percentage reduction of the total parasitemia (c) (filledbars) and trophozoites (d) (hatched bars). The reduction was calculated as percentage reduction of parasitemia: [average parasitemia before drop� (lowest parasitemia/average parasitemia before drop)] � 100. An asterisk indicates that the differences in pathology or reduction in parasitemiaare significant between IL-10�/� and IL-10�/� mice (P � 0.05 [Mann-Whitney test; n � 5 mice]).

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10�/� mice is unknown, although its amelioration by treatmentof mice with anti-TNF-� antibody supports the idea that in-flammatory mediators might contribute.

Development of brain hemorrhages is a hallmark of humanCM. However, despite the occurrence of brain hemorrhages ininfected IL-10�/� mice, they did not show overt clinical signsof cerebral involvement, apart from occasional signs of hindlimb paralysis. Therefore, although, IL-10�/� mice show someof the pathological features of CM in humans, they do notprovide the complete model of the disease spectrum seen inhumans. This host-parasite combination, however, might proveuseful in dissecting the contributory roles of the inflammatorycytokines, IFN-� and TNF-�, in the development of CM.

Although we observed increased sequestration of P.chabaudi in IL-10�/� mice, gross histological examination ofbrain sections did not reveal large numbers of parasitized RBCon brain endothelium (data not shown). However, studies byMota et al. (15) using electron microscopy have clearly dem-onstrated P. chabaudi-infected RBC in the brains of infectedCBA/ca mice. It would therefore be of great importance todetermine the relative extent of parasite sequestration in IL-10�/� and C57BL/6 mice by this method, and by more sensitiveand quantitative PCR approaches for the measurement of totalparasite load (5) in the brain and other organs. Such studiesare under way.

FIG. 2. Cerebral edema in P. chabaudi-infected IL-10�/� mice. (a)Photograph showing a bluish discoloration of the brain of a P.chabaudi-infected IL-10�/� mouse on day 8 p.i. caused by leakage ofEvans Blue into the extravascular brain tissue due to a breakdown ofthe blood-brain barrier. The brain of an IL-10�/� wild-type mouse onday 8 p.i. is shown for comparison. (b) The histograms represent twoindependent experiments in which Evans Blue dye leakage into thebrains of IL-10�/� (filled bars) and IL-10�/� (open bars) mice ismeasured by spectrophotometry on days 6, 8, and 10 p.i. The valuesshown are the means � standard errors of the means for four to fivemice per group at each time point. (c) Effect of anti-TNF-� treatmenton Evans Blue dye leakage into the brain of IL-10�/� mice infectedwith P. chabaudi. The values are means � standard errors of the means(n � 5) of Evans Blue dye (micrograms of dye per gram of wet braintissue) for anti-TNF-�-treated and control antibody-treated IL-10�/�

mice on day 8 p.i. Brains of uninfected C57BL/6 mice not injected withEvans Blue were used as a reference, and the amount of dye presentin the supernatants is calculated from a standard curve of Evans Bluedye. An asterisk indicates that the difference in dye leakage into thebrains of IL-10�/� mice treated with anti-TNF-� is significant com-pared with results for IL-10�/� mice treated with control antibody (P� 0.05 [Mann-Whitney test]).

FIG. 3. Frequency of brain hemorrhage in P. chabaudi-infectedIL-10�/� mice. (a) The frequency of brain hemorrhages determined bygross anatomical examination or light-microscopic examination in IL-10�/� mice. Brain hemorrhages were not seen in IL-10�/� mice. Thedata are representative of 15 mice per group. (b) Reduction in thefrequency of brain hemorrhages as determined by gross anatomicalobservation in IL-10�/� mice treated with anti-TNF-� or control an-tibody on day 0 and day 5 or 6 as described in Materials and Methods.The values shown represent the frequency of brain hemorrhages in 21anti-TNF-�-treated mice and 22 control immunoglobulin-treatedmice.

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We thank Alec Gallagher and Tracey Lamb for their help with theexamination of cerebral hemorrhages, Frank Albano and Robin Ste-phens for their critical comments and careful reading of the manu-script, and Anne O’Garra for helpful advice.

This work was supported by the Medical Research Council, UnitedKingdom.

REFERENCES

1. Brown, H., S. Rogerson, T. Taylor, M. Tembo, J. Mwenechanya, M. E.Molyneux, and G. Turner. 2001. Blood-brain barrier function in cerebralmalaria in Malawian children. Am. J. Trop. Med. Hyg. 64:207–213.

2. Grau, G. E., L. F. Fajardo, P. F. Piguet, B. Allet, P. H. Lambert, and P.Vassalli. 1987. Tumor necrosis factor (cachectin) as an essential mediator inmurine cerebral malaria. Science 237:1210–1212.

3. Grau, G. E., H. Heremans, P. F. Piguet, P. Pointaire, P. H. Lambert, A.Billiau, and P. Vassalli. 1989. Monoclonal antibody against interferongamma can prevent experimental cerebral malaria and its associated over-production of tumor necrosis factor. Proc. Natl. Acad. Sci. USA 86:5572–5574.

4. Grau, G. E., P. F. Piguet, P. Vassalli, and P. H. Lambert. 1989. Tumor-necrosis factor and other cytokines in cerebral malaria: experimental andclinical data. Immunol. Rev. 112:49–70.

5. Hulier, E., P. Petour, G. Snounou, M.-P. Nivez, D. Mazier, and L. Renia.1996. A method for the quantitative assessment of malaria parasite devel-opment in organs of the mammalian host. Mol. Biochem. Parasitol. 77:127–135.

6. Knight, J. C., I. Udalova, A. V. Hill, B. M. Greenwood, N. Peshu, K. Marsh,and D. Kwiatkowski. 1999. A polymorphism that affects OCT-1 binding to

FIG. 4. Hemorrhages in the brain of P. chabaudi-infected IL-10�/� mice. (a and b) Hematoxylin and eosin-stained sections of the brain of anIL-10�/� mouse on day 8 p.i. without hemorrhage (a) and the brain of an infected IL-10�/� mouse on day 8 p.i. (b). There is hemorrhage in theright cerebral hemisphere (arrow) in the region of the motor cortex above the corpus callosum. (c) A normal meningeal vessel (magnification,�400) from an IL-10�/� mouse on day 8 p.i. (d) Hemorrhage next to a collapsed meningeal vessel (magnification, �400) from an IL-10�/� mouseon day 8 p.i. (e) Hemorrhage in the region of the white matter (magnification, �200) in an IL-10�/� mouse on day 8 p.i. (f) Hemorrhage into thethird ventricle (magnification, �200) in another IL-10�/� mouse on day 8 p.i.

VOL. 72, 2004 NOTES 3057

on June 9, 2020 by guesthttp://iai.asm

.org/D

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the TNF promoter region is associated with severe malaria. Nat. Genet.22:145–150.

7. Kossodo, S., C. Monso, P. Juillard, T. Velu, M. Goldman, and G. E. Grau.1997. Interleukin-10 modulates susceptibility in experimental cerebral ma-laria. Immunology 91:536–540.

8. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, and W. Muller. 1993. Inter-leukin-10-deficient mice develop chronic enterocolitis. Cell 75:263–274.

9. Kurtzhals, J. A. L., V. Adabayeri, B. Q. Goka, B. D. Akanmori, J. O. Oliva-Commey, F. K. Nkrumah, C. Behr, and L. Hviid. 1998. Low plasma concen-tration of interleukin 10 in severe malarial anaemia compared with cerebraland uncomplicated malaria. Lancet 351:1768–1772.

10. Langhorne, J., S. J. Quin, and L. A. Sanni. 2002. Mouse models of blood-stage malaria infections: immune responses and cytokines involved in pro-tection and pathology. Chem. Immunol. 80:204–228.

11. Li, C., I. Corraliza, and J. Langhorne. 1999. A defect in interleukin-10 leadsto enhanced malarial disease in Plasmodium chabaudi chabaudi infection inmice. Infect. Immun. 67:4435–4442.

12. Li, C., L. A. Sanni, F. Omer, E. Riley, and J. Langhorne. 2003. Pathology ofPlasmodium chabaudi chabaudi infection and mortality in interleukin-10-deficient mice are ameliorated by anti-tumor necrosis factor alpha and ex-acerbated by anti-transforming growth factor antibodies. Infect. Immun.71:4850–4856.

13. Linke, A., R. Kuhn, W. Muller, N. Honarvar, C. Li, and J. Langhorne. 1996.Plasmodium chabaudi chabaudi: differential susceptibility of gene-targetedmice deficient in IL-10 to an erythrocytic-stage infection. Exp. Parasitol.84:253–263.

14. Maegraith, B., and A. Fletcher. 1972. The pathogenesis of mammalian ma-laria. Adv. Parasitol. 10:49–75.

15. Mota, M. M., W. Jarra, E. Hirst, P. K. Patnaik, and A. A. Holder. 2000.Plasmodium chabaudi-infected erythrocytes adhere to CD36 and bind to

microvascular endothelial cells in an organ-specific way. Infect. Immun.68:4135–4144.

16. Neighbors, M., X. Xu, F. J. Barrat, S. R. Ruuls, T. Churakova, R. Debets,J. F. Bazan, R. A. Kastelein, J. S. Abrams, and A. O’Garra. 2001. A criticalrole for interleukin 18 in primary and memory effector responses to Listeriamonocytogenes that extends beyond its effects on interferon gamma produc-tion. J. Exp. Med. 194:343–354.

17. Othoro, C., A. A. Lal, B. Nahlen, D. Koech, A. S. S. Orago, and V. Udhaya-kumar. 1999. A low interleukin-10 tumor necrosis factor-� ratio is associatedwith malaria anemia in children residing in a holoendemic malaria region inwestern Kenya. J. Infect. Dis. 179:279–282.

18. Rudin, W., N. Favre, G. Bordmann, and B. Ryffel. 1997. Interferon-gammais essential for the development of cerebral malaria. Eur. J. Immunol. 27:810–815.

19. Sanni, L. A. 2001. The role of cerebral oedema in the pathogenesis ofcerebral malaria. Redox Rep. 6:137–142.

20. Sanni, L. A., S. R. Thomas, B. N. Tattam, D. E. Moore, G. Chaudhri, R.Stocker, and N. H. Hunt. 1998. Dramatic changes in oxidative tryptophanmetabolism along the kynurenine pathway in experimental cerebral andnon-cerebral malaria. Am. J. Pathol. 152:611–619.

21. Slade, S. J., and J. Langhorne. 1989. Production of interferon-gamma duringinfection with Plasmodium chabaudi chabaudi. Immunobiology 179:353–365.

22. Thumwood, C. M., N. H. Hunt, I. A. Clark, and W. B. Cowden. 1988.Breakdown of the blood-brain barrier in murine cerebral malaria. Parasitol-ogy 96:579–589.

23. Warrell, D. A., S. Looareesuwan, R. E. Phillips, N. J. White, M. J. Warrell,H. M. Chapel, S. Areekul, and S. Tharavanij. 1986. Function of the blood-cerebrospinal fluid barrier in human cerebral malaria: rejection of the per-meability hypothesis. Am. J. Trop. Med. Hyg. 35:882–889.

Editor: W. A. Petri, Jr.

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