INFECTION AND IMMUNITY, OCt. 1991, p. 3846-3848
Vol. 59, No. 10
0019-9567/91/103846-03$02.00/0 Copyright © 1991, American Society for Microbiology
Prevention of Murine Cerebral Malaria by Stable Prostacyclin Analog K.
a
SLIWA,1 H. J. GRUNDMANN,l S. NEIFER,1 M. FERREIRA CHAVES,' G. SAHLMULLER,l E. BLITSTEIN-WILLINGER,2 U. BIENZLE,1* AND P. G. KREMSNER'
Landesinstitut fur Tropenmedizin Berlin,
1000
Berlin 19,1 and Schering AG Berlin, Berlin,2 Germany
Received 27 February 1991/Accepted 13 July 1991
Iloprost, a synthetic prostacyclin analog, successfully prevents the development of cerebral malaria in mice. Malaria antigen-induced tumor necrosis factor (TNF) production could be inhibited by iloprost in vitro and in vivo. Northern analysis of TNF mRNA revealed that malaria antigen-induced TNF expression was suppressed at the transcription level. In humans, cerebral malaria (CM) is a serious and often lethal complication of Plasmodium falciparum infection. Although differing in some respects, murine CM, in which approximately 80% of infected mice develop neurological symptoms and die within 1 to 5 h, is a useful model for the study of human CM. Plasmodium berghei ANKA infection in the genetically susceptible CBA/CA mouse strain is considered a valuable model for the study of the pathogenesis of CM and of the role of the immune system in this disease. Especially tumor necrosis factor (TNF) but also various other cytokines, such as interleukin-1, interleukin-3, granulocyte-macrophage colony-stimulating factor, gamma interferon, platelet-activating factor, and interleukin-6, have been investigated for their influence on CM (8, 9, 12). TNF, a secretory product of activated macrophages, is known to be closely associated with many clinical symptoms in human malaria, such as fever, rigor, vomiting, diarrhea, myalgia, pulmonary edema, and cerebral alterations (4). TNF levels in serum have been shown to be raised in humans with malaria and are extremely high in fatal CM (12). In mice, TNF appears to be an essential factor in the development of CM. Treatment with anti-TNF antibodies has been shown to suppress the development of CM in susceptible mice (7). An anecdotal report on the amelioration of the condition of a patient suffering from CM after prostacyclin treatment was published as early as 1982 (16). Iloprost as a stable prostacyclin analog would be more suitable than prostacyclin for any therapeutic purposes. Iloprost has been reported to inhibit neutrophil activation in vitro and to reduce neutrophil accumulation in inflammatory lesions (15). In malaria-infected mice, the accumulation of leukocytes such as monocytes, macrophages, and lymphocytes in brain vessels is the dominant picture. Such vascular plugging by mononuclear cells is also seen in the lungs of patients with severe P. falciparum malaria. In this context we decided to investigate the effect of iloprost on the development of CM in CBA/CA mice infected with P. berghei ANKA and on the TNF production of murine macrophages both in vitro and in vivo.
Six-week-old female CBA/CA mice (Shaws Farm, Hull, United Kingdom) were infected intraperitoneally with 106 erythrocytes parasitized with P. berghei ANKA. Para*
Corresponding author.
determined daily on Giemsa-stained blood In two different sets of experiments, mice were divided into two groups of six. Each mouse received a subcutaneous injection of either iloprost (1 ,ug in 100 RI of 0.9% NaCl) or saline (0.9% NaCl) as a control daily for 10 consecutive days, beginning on the day of infection. The survival time and neurological signs such as paralysis, ataxia, deviations of the head, and convulsions, which serve as common parameters of CM, were noted. Death occurred 1 to 5 h after the onset of symptoms. In another experiment, three mice from each group were sacrificed on days 6 and 12, and the TNF concentrations in the sera were measured. Statistical analysis was performed with the chi-square test. Parasite soluble antigen was prepared as described previously (1). It was used undiluted and diluted 1:10 and 1:100 with RPMI 1640 (GIBCO). Immune serum was obtained from parasitized mice treated with chloroquine. Mice were bled 4 weeks after infection. Peritoneal cells were recovered from CBA/CA mice through peritoneal lavage with RPMI 1640 3 to 5 days after intraperitoneal injection of 2 ml of 2% amylose. Washed cells were suspended in RPMI 1640 containing 10% fetal calf serum and 5 ,ug of polymyxin B (Sigma, Deisenhofen, Germany) per ml and counted after staining with acridine orange. After adjustment to 3 x 106 macrophages per ml, 0.1-ml volumes were aliquoted into wells of 96-well microtiter plates (Nunclon delta, Wiesbaden, Germany). These cells were then incubated for 2 to 3 h at 37°C in an atmosphere of 5% CO2 to allow macrophages to adhere. Nonadherent cells were washed off with RPMI 1640 without polymyxin B. The medium was replaced 24 h later with 0.2 ml of RPMI 1640 containing the stimulant to be tested. Cultures were incubated for 18 h. The stimulants used were 5 ,ug of lipopolysaccharide (from Escherichia coli 055:b5; Sigma) per ml, parasite soluble antigen, and parasite soluble antigen plus 10 or 100 ng of iloprost per ml. All probes were tested in triplicate. The prostacyclin derivative iloprost {ZK 36374; 5-(E)-(lS,5S,6R)-7-hydroxy-6[(E)-(3S, 4RS)-3-hydroxy-4-methyl-1-octen-6-inyl] bicyclo [3.3.0]octen-3-ylidene pentanoicacid} was provided by Schering, Berlin, Germany. Supernatants were collected and assayed for TNF bioactivity. Each supernatant was diluted 1:2.5 in medium containing 10% fetal calf serum and 1 ,ug of emetine (Sigma) per ml and stored at -20°C for further use. Cultures incubated with 5 ,ug of lipopolysaccharide per ml and with medium alone were included in every experiment as positive and negative controls, respectively. TNF was assayed colorimetrically for its cytotoxicity to sitemia
smears.
3846
was
NOTES
VOL. 59, 1991 160
1
2
3 4
3847
5
140 120 E \; 100~ z
8060 40-
20spontaneous
LPS
H
antigen
n
28S -
antigen + Iloprost 18S -
FIG. 1. Peritoneal macrophages of CBA/CA mice were incubated with 5 ,ug of lipopolysaccharide (LPS) per ml, soluble P. berghei ANKA antigen diluted 1:10, and antigen plus 10 ng of iloprost per ml. The TNF bioactivity of the supernatants was measured after an 18-h culture period.
mouse WEHI 164 cells (13). Serial dilutions of samples (0.1 ml per well) were tested in duplicate. One unit of TNF activity was defined as the amount causing 50% cell destruc-
tion. Total RNA from adherent peritoneal cells was isolated by the method of Chomzcynski and Sacchi (2). Briefly, 4 h after the addition of stimulants, cells were washed in ice-cold phosphate-buffered saline and homogenized in a lysis buffer containing 4 M guanidiumisothiocyanate, 25 mM sodium citrate (pH 7), 0.5% Sarkosyl, and 0.1 M 2-mercaptoethanol. After the sequential addition of 0.1 volume of 2 M sodium acetate (pH 4), 1 volume of phenol (water saturated), and 0.2 volume of chloroform-isoamyl alcohol (24:1), RNA was extracted and precipitated in 1 volume of 2-propanol. A final suspension in lysis buffer followed by a second 2-propanol precipitation yielded RNA that was sufficiently pure for electrophoresis in a formaldehyde-denaturing 1% agarose gel (50 ,ug per lane). Fractionated nucleic acids were blotted on nylon membrane (Hybond N; Amersharn & Buchler, Braunschweig, Germany) and cross-linked by UV irradiation. Blot hybridization and washing were performed as described by Sambrook et al. (14). Blots were prehybridized overnight at 680C in 6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 10 mM EDTA (pH 8), 200 ,ug of denaturated salmon sperm DNA per ml, and 0.1% (wt/vol) (each) Ficoll 400, polyvinylpyrrolidone, bovine serum albumin, and sodium dodecyl sulfate (SDS). Hybridization was carried out for 2 days at 680C in the above solution containing heat-denatured 32P-labeled murine TNF-cDNA (2.0 x 106 cpm/ml). The blots were washed at room temperature twice in 2x SSC-0.1% (wt/vol) SDS for 10 min and then in lx SSC-0. 1% (wt/vol) SDS at 68°C for 2 h. Autoradiography was performed at -80°C with Kodak XAR5 film and Quanta III intensifying screens (DuPont, Dreieich, Germany). Peritoneal macrophages produced nearly 80 U of TNF per ml after stimulation with soluble P. berghei ANKA antigen diluted 1:10. Iloprost in a concentration of 10 ng/ml inhibited the TNF release of antigen-stimulated cells by more than 75% (Fig. 1). Stimulation with 5 jig of LPS per ml was used as a positive control. Iloprost alone neither inhibited nor stimulated TNF secretion as measured on macrophages and TNF-sensitive WEHI 164 cells. The effect of iloprost on parasite soluble antigen-induced TNF gene expression in
FIG. 2. Northern blot mRNA analysis of mouse peritoneal macrophages treated the following (lanes): 1, medium alone; 2, 1 ml of parasite soluble antigen diluted 1:10; 3, as in lane 2 plus 100 ng of iloprost per ml; 4, as in lane 2 plus 10 ng of iloprost per ml; 5, parasite soluble antigen preincubated with mouse immune serum diluted 1:10.
macrophages was investigated by Northern RNA blot analysis. At a concentration of 100 ng/ml, iloprost completely abrogated malaria antigen-induced TNF transcription, whereas 10 ng/ml showed incomplete suppression of TNF mRNA production (Fig. 2). Parasite soluble antigen preincubated for 30 min with immune serum was used as a control. Two groups of 12 mice infected with P. berghei ANKA received subcutaneously either 1 ,ug of iloprost or normal saline as a control. Around 15 min after the injection with iloprost, the test mice were markedly less active than mice treated with saline. No other changes could be observed. The course of parasitemia showed no differences between the two groups. Nine of 12 mice in the two control groups developed neurological symptoms and died between days 9 and 15 after infection. Only 1 of 12 mice in the groups treated with iloprost had died on day 14 (P < 0.001). The surviving mice all died between weeks 4 and 5 after infection. They showed parasite densities of more than 60% accompanied by severe anemia. Neurological abnormalities were not observed. In a further experiment involving two groups of six CBA/CA mice, each infected with P. berghei ANKA and treated with 1 ,g of iloprost or normal saline, the TNF levels in serum were determined. Three mice per group and date were bled on days 6 and 12 after infection. Day 12 was chosen because most of the untreated mice had developed neurological signs by this day. The TNF concentrations in serum on day 6 were between 2 and 4 U/ml in the control group and below 0.5 U/ml in all mice treated with iloprost. On day 12 the individual TNF levels in the sera of the control mice were 8, 9, and 13 U/ml in comparison with 1, 2, and 6 U/ml in the iloprost-treated group. The data about the inhibition of TNF concentration in serum do not permit a statistical analysis, because only 3 mice were examined per group per day. However these data support the in vitro results in which iloprost inhibited the TNF synthesis. Our results demonstrate that malaria antigen-induced
3848
NOTES
TNF production in vitro and in vivo (orientative data) is inhibited by iloprost, a stable prostacyclin, and that this drug successfully and significantly prevents the development of CM in genetically susceptible CBA/CA mice if the treatment is started on the same day as the inoculation of the parasites. At the molecular level, we show that iloprost impedes TNF gene transcription in a dose-dependent manner. This inhibition is brought about by an interference at the transcription level and not by posttranslational events, since no accumulation of TNF mRNA molecules could be demonstrated in vitro after incubation with a stimulant together with iloprost. Iloprost is a chemically and metabolically stable prostacyclin analog. The biological effects of prostacyclin and iloprost are believed to be mediated via binding to specific prostacyclin receptors on the membrane of target cells (mainly platelets, vascular smooth muscle, and vascular endothelium) followed by an increase of intracellular cyclic AMP (5). Prostacyclin and the stable analog iloprost are strong vasodilators and potent endogenous inhibitors of platelet aggregation and show moderate fibrinolytic activity (10, 11). Endothelial cells act as powerful generators of prostacyclin and also are known to play an important role in the development and amplification of inflammatory responses. More than one of these properties of iloprost may be involved in the prevention of murine CM. However, in our study the protective effect of iloprost is probably due to the inhibition of TNF, which is known to enhance neutrophil accumulation and activation, stimulate leukocyte adherence to endothelium, potentiate coagulation, and induce production of oxygen radicals (3). By inhibiting TNF and inducing vasodilation, iloprost prevents the development of this process. It was already reported by Grau et al. (7) that CBA/CA mice infected with P. berghei ANKA were protected against CM by treatment with antibodies directed to TNF. They described the morphopathological changes in the murine CM as an accumulation of macrophages containing infected erythrocytes in the cerebral vessels. They also observed ultrastructural signs of endothelial damage. These lesions were not seen in animals treated with antibodies to TNF. As in our model, inhibition of TNF did not suppress parasitemia
(7).
Iloprost has already been safely and successfully tested in several clinical trials with patients suffering from peripheral arterial occlusive disease (6). The results of our current work suggest that the therapeutic benefits of the drug should be taken into consideration and that iloprost should be tested in clinical trials in patients with severe P. falciparum malaria, for which there is no available drug to prevent or ameliorate the course of CM. We gratefully acknowledge the expert technical support of C. Klindworth and S. Schermuck. The TNF cDNA sample was gener-
INFECT. IMMUN.
ously provided by A. Mantovani, Instituto Mario Negri, Milano, Italy. REFERENCES 1. Bate, C. A., J. Taverne, and H. Playfair. 1988. Malarial parasites induce TNF production of macrophages. Immunology 64:227231. 2. Chomzcynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159. 3. Clark, I. A. 1987. Monokines and lymphokines in malaria pathology. Ann. Trop. Med. Parasitol. 81:577-585. 4. Clark, I. A., G. Chaudhri, and W. B. Cowden. 1989. Roles of tumor necrosis factor in the illness and pathology of malaria. Trans. R. Soc. Trop. Med. Hyg. 83:436-440. 5. Dembinska-Kiec, A., W. Rucker, and P. S. Schonhofer. 1980. Effects of PGI2 and PGI2 analogs on cAMP levels in cultured endothelial and smooth muscle cells derived from bovine arteries. Naunyn-Schmiedebergs Arch. Pharmacol. 311:67-72. 6. Diehm, C. H. 1988. Effect of a stable prostacyclin analog in peripheral arterial occlusive disease (PAOD) stage IV: a controlled double blind multicenter trial. Circulation 4:234-238. 7. Grau, G. E., L. F. Fajardo, P. F. Piguet, B. Allet, P. H. Lambert, and P. Vassali. 1987. Tumor necrosis factor (cachectin) as essential mediator in murine cerebral malaria. Science 237: 1210-1212. 8. Grau, G. E., H. Heremans, P. F. Piguet, P. Pointaire, P. H. Lambert, A. Billiau, and P. Vassalli. 1989. Monoclonal antibody against interferon gamma can prevent experimental cerebral malaria and its associated overproduction of tumor necrosis factor. Proc. Natl. Acad. Sci. USA 86:5572-5574. 9. Grau, G. E., P. F. Piguet, V. Kindler, P. Vassali, and P. H. Lambert. 1989. Tumor-necrosis factor and other cytokines in cerebral malaria: experimental and clinical data. Immunol. Rev. 112:49-70. 10. Gryglwski, R. J., and G. Stock. 1987. Prostacyclin and its stable analogue Iloprost. Springer Verlag, Berlin. 11. Korbut, R., A. Byrska-Danek, and R. J. Gryglwski. 1983. Fibrinolytic activity of 6-keto-prostaglandin El. Thromb. Haemostasis 50:893-899. 12. Kwiatkowski, D., A. V. S. Hill, I. Sambou, P. Twumasi, J. Castrane, K. R. Manogue, A. Cerami, D. R. Brewster, and B. M. Greenwood. 1990. TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet 336:1201-1204. 13. Rollinghoff, M. O., and N. I. Warner. 1973. Specificity of in vivo tumor rejection assessed by mixing immune spleen cells with target and unrelated tumor cells. Proc. Soc. Exp. Biol. Med. 144:813-818. 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Simpson, P., J. C. Mickelson, J. C. Fantone, K. P. Gallagher, and B. R. Luccesi. 1987. Iloprost inhibits neutrophil function in vitro and in vivo and limits experimental infarct size in canine heart. Circ. Res. 5:660-673. 16. Weston, M. J., N. Jachman, and C. Rudge. 1982. Prostacycline in falciparum malaria. Lancet ii:609. (Letter.)
Vol. 59, No. 10
0019-9567/91/103846-03$02.00/0 Copyright © 1991, American Society for Microbiology
Prevention of Murine Cerebral Malaria by Stable Prostacyclin Analog K.
a
SLIWA,1 H. J. GRUNDMANN,l S. NEIFER,1 M. FERREIRA CHAVES,' G. SAHLMULLER,l E. BLITSTEIN-WILLINGER,2 U. BIENZLE,1* AND P. G. KREMSNER'
Landesinstitut fur Tropenmedizin Berlin,
1000
Berlin 19,1 and Schering AG Berlin, Berlin,2 Germany
Received 27 February 1991/Accepted 13 July 1991
Iloprost, a synthetic prostacyclin analog, successfully prevents the development of cerebral malaria in mice. Malaria antigen-induced tumor necrosis factor (TNF) production could be inhibited by iloprost in vitro and in vivo. Northern analysis of TNF mRNA revealed that malaria antigen-induced TNF expression was suppressed at the transcription level. In humans, cerebral malaria (CM) is a serious and often lethal complication of Plasmodium falciparum infection. Although differing in some respects, murine CM, in which approximately 80% of infected mice develop neurological symptoms and die within 1 to 5 h, is a useful model for the study of human CM. Plasmodium berghei ANKA infection in the genetically susceptible CBA/CA mouse strain is considered a valuable model for the study of the pathogenesis of CM and of the role of the immune system in this disease. Especially tumor necrosis factor (TNF) but also various other cytokines, such as interleukin-1, interleukin-3, granulocyte-macrophage colony-stimulating factor, gamma interferon, platelet-activating factor, and interleukin-6, have been investigated for their influence on CM (8, 9, 12). TNF, a secretory product of activated macrophages, is known to be closely associated with many clinical symptoms in human malaria, such as fever, rigor, vomiting, diarrhea, myalgia, pulmonary edema, and cerebral alterations (4). TNF levels in serum have been shown to be raised in humans with malaria and are extremely high in fatal CM (12). In mice, TNF appears to be an essential factor in the development of CM. Treatment with anti-TNF antibodies has been shown to suppress the development of CM in susceptible mice (7). An anecdotal report on the amelioration of the condition of a patient suffering from CM after prostacyclin treatment was published as early as 1982 (16). Iloprost as a stable prostacyclin analog would be more suitable than prostacyclin for any therapeutic purposes. Iloprost has been reported to inhibit neutrophil activation in vitro and to reduce neutrophil accumulation in inflammatory lesions (15). In malaria-infected mice, the accumulation of leukocytes such as monocytes, macrophages, and lymphocytes in brain vessels is the dominant picture. Such vascular plugging by mononuclear cells is also seen in the lungs of patients with severe P. falciparum malaria. In this context we decided to investigate the effect of iloprost on the development of CM in CBA/CA mice infected with P. berghei ANKA and on the TNF production of murine macrophages both in vitro and in vivo.
Six-week-old female CBA/CA mice (Shaws Farm, Hull, United Kingdom) were infected intraperitoneally with 106 erythrocytes parasitized with P. berghei ANKA. Para*
Corresponding author.
determined daily on Giemsa-stained blood In two different sets of experiments, mice were divided into two groups of six. Each mouse received a subcutaneous injection of either iloprost (1 ,ug in 100 RI of 0.9% NaCl) or saline (0.9% NaCl) as a control daily for 10 consecutive days, beginning on the day of infection. The survival time and neurological signs such as paralysis, ataxia, deviations of the head, and convulsions, which serve as common parameters of CM, were noted. Death occurred 1 to 5 h after the onset of symptoms. In another experiment, three mice from each group were sacrificed on days 6 and 12, and the TNF concentrations in the sera were measured. Statistical analysis was performed with the chi-square test. Parasite soluble antigen was prepared as described previously (1). It was used undiluted and diluted 1:10 and 1:100 with RPMI 1640 (GIBCO). Immune serum was obtained from parasitized mice treated with chloroquine. Mice were bled 4 weeks after infection. Peritoneal cells were recovered from CBA/CA mice through peritoneal lavage with RPMI 1640 3 to 5 days after intraperitoneal injection of 2 ml of 2% amylose. Washed cells were suspended in RPMI 1640 containing 10% fetal calf serum and 5 ,ug of polymyxin B (Sigma, Deisenhofen, Germany) per ml and counted after staining with acridine orange. After adjustment to 3 x 106 macrophages per ml, 0.1-ml volumes were aliquoted into wells of 96-well microtiter plates (Nunclon delta, Wiesbaden, Germany). These cells were then incubated for 2 to 3 h at 37°C in an atmosphere of 5% CO2 to allow macrophages to adhere. Nonadherent cells were washed off with RPMI 1640 without polymyxin B. The medium was replaced 24 h later with 0.2 ml of RPMI 1640 containing the stimulant to be tested. Cultures were incubated for 18 h. The stimulants used were 5 ,ug of lipopolysaccharide (from Escherichia coli 055:b5; Sigma) per ml, parasite soluble antigen, and parasite soluble antigen plus 10 or 100 ng of iloprost per ml. All probes were tested in triplicate. The prostacyclin derivative iloprost {ZK 36374; 5-(E)-(lS,5S,6R)-7-hydroxy-6[(E)-(3S, 4RS)-3-hydroxy-4-methyl-1-octen-6-inyl] bicyclo [3.3.0]octen-3-ylidene pentanoicacid} was provided by Schering, Berlin, Germany. Supernatants were collected and assayed for TNF bioactivity. Each supernatant was diluted 1:2.5 in medium containing 10% fetal calf serum and 1 ,ug of emetine (Sigma) per ml and stored at -20°C for further use. Cultures incubated with 5 ,ug of lipopolysaccharide per ml and with medium alone were included in every experiment as positive and negative controls, respectively. TNF was assayed colorimetrically for its cytotoxicity to sitemia
smears.
3846
was
NOTES
VOL. 59, 1991 160
1
2
3 4
3847
5
140 120 E \; 100~ z
8060 40-
20spontaneous
LPS
H
antigen
n
28S -
antigen + Iloprost 18S -
FIG. 1. Peritoneal macrophages of CBA/CA mice were incubated with 5 ,ug of lipopolysaccharide (LPS) per ml, soluble P. berghei ANKA antigen diluted 1:10, and antigen plus 10 ng of iloprost per ml. The TNF bioactivity of the supernatants was measured after an 18-h culture period.
mouse WEHI 164 cells (13). Serial dilutions of samples (0.1 ml per well) were tested in duplicate. One unit of TNF activity was defined as the amount causing 50% cell destruc-
tion. Total RNA from adherent peritoneal cells was isolated by the method of Chomzcynski and Sacchi (2). Briefly, 4 h after the addition of stimulants, cells were washed in ice-cold phosphate-buffered saline and homogenized in a lysis buffer containing 4 M guanidiumisothiocyanate, 25 mM sodium citrate (pH 7), 0.5% Sarkosyl, and 0.1 M 2-mercaptoethanol. After the sequential addition of 0.1 volume of 2 M sodium acetate (pH 4), 1 volume of phenol (water saturated), and 0.2 volume of chloroform-isoamyl alcohol (24:1), RNA was extracted and precipitated in 1 volume of 2-propanol. A final suspension in lysis buffer followed by a second 2-propanol precipitation yielded RNA that was sufficiently pure for electrophoresis in a formaldehyde-denaturing 1% agarose gel (50 ,ug per lane). Fractionated nucleic acids were blotted on nylon membrane (Hybond N; Amersharn & Buchler, Braunschweig, Germany) and cross-linked by UV irradiation. Blot hybridization and washing were performed as described by Sambrook et al. (14). Blots were prehybridized overnight at 680C in 6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 10 mM EDTA (pH 8), 200 ,ug of denaturated salmon sperm DNA per ml, and 0.1% (wt/vol) (each) Ficoll 400, polyvinylpyrrolidone, bovine serum albumin, and sodium dodecyl sulfate (SDS). Hybridization was carried out for 2 days at 680C in the above solution containing heat-denatured 32P-labeled murine TNF-cDNA (2.0 x 106 cpm/ml). The blots were washed at room temperature twice in 2x SSC-0.1% (wt/vol) SDS for 10 min and then in lx SSC-0. 1% (wt/vol) SDS at 68°C for 2 h. Autoradiography was performed at -80°C with Kodak XAR5 film and Quanta III intensifying screens (DuPont, Dreieich, Germany). Peritoneal macrophages produced nearly 80 U of TNF per ml after stimulation with soluble P. berghei ANKA antigen diluted 1:10. Iloprost in a concentration of 10 ng/ml inhibited the TNF release of antigen-stimulated cells by more than 75% (Fig. 1). Stimulation with 5 jig of LPS per ml was used as a positive control. Iloprost alone neither inhibited nor stimulated TNF secretion as measured on macrophages and TNF-sensitive WEHI 164 cells. The effect of iloprost on parasite soluble antigen-induced TNF gene expression in
FIG. 2. Northern blot mRNA analysis of mouse peritoneal macrophages treated the following (lanes): 1, medium alone; 2, 1 ml of parasite soluble antigen diluted 1:10; 3, as in lane 2 plus 100 ng of iloprost per ml; 4, as in lane 2 plus 10 ng of iloprost per ml; 5, parasite soluble antigen preincubated with mouse immune serum diluted 1:10.
macrophages was investigated by Northern RNA blot analysis. At a concentration of 100 ng/ml, iloprost completely abrogated malaria antigen-induced TNF transcription, whereas 10 ng/ml showed incomplete suppression of TNF mRNA production (Fig. 2). Parasite soluble antigen preincubated for 30 min with immune serum was used as a control. Two groups of 12 mice infected with P. berghei ANKA received subcutaneously either 1 ,ug of iloprost or normal saline as a control. Around 15 min after the injection with iloprost, the test mice were markedly less active than mice treated with saline. No other changes could be observed. The course of parasitemia showed no differences between the two groups. Nine of 12 mice in the two control groups developed neurological symptoms and died between days 9 and 15 after infection. Only 1 of 12 mice in the groups treated with iloprost had died on day 14 (P < 0.001). The surviving mice all died between weeks 4 and 5 after infection. They showed parasite densities of more than 60% accompanied by severe anemia. Neurological abnormalities were not observed. In a further experiment involving two groups of six CBA/CA mice, each infected with P. berghei ANKA and treated with 1 ,g of iloprost or normal saline, the TNF levels in serum were determined. Three mice per group and date were bled on days 6 and 12 after infection. Day 12 was chosen because most of the untreated mice had developed neurological signs by this day. The TNF concentrations in serum on day 6 were between 2 and 4 U/ml in the control group and below 0.5 U/ml in all mice treated with iloprost. On day 12 the individual TNF levels in the sera of the control mice were 8, 9, and 13 U/ml in comparison with 1, 2, and 6 U/ml in the iloprost-treated group. The data about the inhibition of TNF concentration in serum do not permit a statistical analysis, because only 3 mice were examined per group per day. However these data support the in vitro results in which iloprost inhibited the TNF synthesis. Our results demonstrate that malaria antigen-induced
3848
NOTES
TNF production in vitro and in vivo (orientative data) is inhibited by iloprost, a stable prostacyclin, and that this drug successfully and significantly prevents the development of CM in genetically susceptible CBA/CA mice if the treatment is started on the same day as the inoculation of the parasites. At the molecular level, we show that iloprost impedes TNF gene transcription in a dose-dependent manner. This inhibition is brought about by an interference at the transcription level and not by posttranslational events, since no accumulation of TNF mRNA molecules could be demonstrated in vitro after incubation with a stimulant together with iloprost. Iloprost is a chemically and metabolically stable prostacyclin analog. The biological effects of prostacyclin and iloprost are believed to be mediated via binding to specific prostacyclin receptors on the membrane of target cells (mainly platelets, vascular smooth muscle, and vascular endothelium) followed by an increase of intracellular cyclic AMP (5). Prostacyclin and the stable analog iloprost are strong vasodilators and potent endogenous inhibitors of platelet aggregation and show moderate fibrinolytic activity (10, 11). Endothelial cells act as powerful generators of prostacyclin and also are known to play an important role in the development and amplification of inflammatory responses. More than one of these properties of iloprost may be involved in the prevention of murine CM. However, in our study the protective effect of iloprost is probably due to the inhibition of TNF, which is known to enhance neutrophil accumulation and activation, stimulate leukocyte adherence to endothelium, potentiate coagulation, and induce production of oxygen radicals (3). By inhibiting TNF and inducing vasodilation, iloprost prevents the development of this process. It was already reported by Grau et al. (7) that CBA/CA mice infected with P. berghei ANKA were protected against CM by treatment with antibodies directed to TNF. They described the morphopathological changes in the murine CM as an accumulation of macrophages containing infected erythrocytes in the cerebral vessels. They also observed ultrastructural signs of endothelial damage. These lesions were not seen in animals treated with antibodies to TNF. As in our model, inhibition of TNF did not suppress parasitemia
(7).
Iloprost has already been safely and successfully tested in several clinical trials with patients suffering from peripheral arterial occlusive disease (6). The results of our current work suggest that the therapeutic benefits of the drug should be taken into consideration and that iloprost should be tested in clinical trials in patients with severe P. falciparum malaria, for which there is no available drug to prevent or ameliorate the course of CM. We gratefully acknowledge the expert technical support of C. Klindworth and S. Schermuck. The TNF cDNA sample was gener-
INFECT. IMMUN.
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