Clin. exp. Immunol. (1992) 88, 112- 118
Modulation of the cellular immune response during Plasmodiumfalciparum infections in sickle cell trait individuals Y. A. ABU-ZEID, T. G. THEANDER*t, N. H. ABDULHADI, L. HVIID*t, B. 0. SAEED, S. JEPSENt, J. B. JENSEN§ & R. A. BAYOUMI Department of Biochemistry, Faculty of Medicine, University of Khartoum, Sudan, *Department of Infectious Diseases, University Hospital, Copenhagen, Denmark, tInstitute for Medical Microbiology, University of Copenhagen, Copenhagen, Denmark, I WHO National Malaria Diagnostic Centre, State Serum Institute, Copenhagen, Denmark, §Department of Microbiology, Brigham Young University, Provo, UT, USA
(Acceptedfor publication 18 November 1991)
SUMMARY Plasma and peripheral blood mononuclear cells (PBMC) were obtained from P.falciparum-infected individuals with and without the sickle cell trait at diagnosis and 7 days after treatment. HbAA and HbAS patients were compared for levels of plasma soluble IL-2 receptors (IL-2R) and the in vitro cellular reactivity to affinity-purified soluble P. falciparum antigens (SPAg), PPD and phytohaemagglutinin (PHA). At diagnosis, HbAS patients with clinical disease had lower plasma-soluble IL2R levels and parasite counts than the corresponding HbAA patients, whereas HbAS and HbAA patients with asymptomatic infections had comparable soluble IL-2R levels and parasite counts. PBMC from HbAS patients had higher proliferation and IFN-y production in response to SPAg than PBMC from HbAA patients. The difference in the lymphoproliferative responses to SPAg between the two groups was evident in patients with asymptomatic infections. In all patients, the clinical severity, the soluble IL-2R levels and the parasite counts were directly related. The former two were inversely related to the proliferative responses to SPAg. After treatment, all the studied parameters were comparable in the two groups. The study indicates that during P. falciparum infection, HbAS compared with HbAA patients had lower in vivo cellular activation and higher in vitro cellular reactivity in response to soluble malaria antigens. Keywords Plasmodium antigens interferon-gamma lymphoproliferative soluble IL-2 receptors sickle cell trait
responses
have demonstrated that healthy HbAS individuals from areas of unstable seasonal P. falciparum transmission had higher proliferative responses to soluble P.falciparum antigens (SPAg) and to PPD than HbAA individuals [9,10]. The difference between the two groups was more evident in children than in adults [10]. Furthermore, the lymphoproliferative responses to SPAg in HbAA children were suppressed during the malaria season compared with the responses before the season, whereas in HbAS children the responses to SPAg were higher during the malaria season than before the season [11]. Lymphocytes obtained from HbAA individuals during malarial infections were unable to proliferate in vitro in response to specific malarial antigens, whereas such responses were usually found after treatment [12-151. The present study was designed to investigate the possible differences in cellular immune reactivity of HbAA and HbAS patients with P. falciparum malaria. The two groups were compared for the in vitro cellular reactivity to soluble P.falciparum antigens (SPAg), to non-specific antigen (PPD) and to a mitogen, phytohaemagglutinin (PHA); and the degree of in vivo cellular activation, as indicated by the plasma soluble IL-2 receptor (IL-2R) levels.
INTRODUCTION The in vivo mechanisms which protect HbAS individuals from the severe manifestations of P. falciparum remain unclear. Several investigators have demonstrated that under low oxygen tension the in vitro growth of P.falciparum is retarded in HbAS erythrocytes [1-3]. Furthermore, other mechanisms such as toxic haem and generation of active oxygen radicals have been implicated in the resistance of HbAS erythrocytes to falciparum malaria [4,5]. Allison [6] proposed that the protective effect of the sickle haemoglobin is acting in synergy with specific acquired immunity, and Bayoumi [7] suggested that the selective advantage of HbAS individuals is due to earlier acquisition of clinical immunity. HbAS children have been shown to have high antibody titres against parasite-derived neoantigens, which are expressed on infected erythrocytes and which have been implicated in the protection against clinical falciparum malaria [8]. Recently, we Correspondence: Yousif A. Abu-Zeid, Lymphocyte Laboratory, Department of Infectious Diseases, M7641, University Hospital, Tagensvej 20, DK-2200 Copenhagen N, Denmark.
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Cellular responses in HbAS individuals with P. falciparum infections Data were obtained from samples collected from P. falciparum patients with and without the sickle cell trait at diagnosis and during convalescence. MATERIALS AND METHODS
Study area The study was done in Darawish village (Sudan) which has a total population of approximately 350 individuals. The village is situated 420 km southeast of Khartoum. The area is endemic for malaria and characterized by unstable seasonal transmission. The rains fall mainly during July-September, and the rest of the year is dry. Positive blood films are encountered from September to December with the highest incidence rates during October-November [16]. The predominant malaria species is P. falciparum; rarely P. vivax is seen. Darawish is inhabited by 42 families; the gene for the sickle cell haemoglobin (Hb S) is present in 14. Individuals with the sickle cell trait amount to 11% of the total village population. Patients The study was carried out during October-November. Thirteen HbAS and 15 HbAA P. falciparum patients were grouped according to the severity of the disease at the time of bleeding. Five HbAS and seven HbAA patients with asymptomatic infections were identified during screenings of thick blood films collected from the village population; which were subsequently Giemsa stained and examined for the presence of malaria parasites. These donors had no physical complaints, their body temperature was < 38 C and they reported not to have had fever for at least 2 weeks prior to bleeding. The others had clinical disease of moderate severity; temperature >38 C, and complained of fever, headache, body or joint pains and can be described as cases of acute non-complicated falciparum malaria. In order to match the two groups, severely ill HbAA patients were not included in the study as HbAS patients had asymptomatic infections or presented with malaria of moderate severity. The median age for HbAS and HbAA patients was 13 years (range 8-35) and 12 years (range 8-36), respectively. There was no significant age difference neither between HbAA and HbAS patients with asymptomatic infections nor between patients with clinical disease. Informed consent was obtained from the patients and/or their parents before blood samples were drawn. A heparinized blood sample was obtained from each patient at diagnosis (Day 0) and chloroquine treatment was initiated. A second blood sample was obtained from all 13 HbAS patients and from 13 out of the 15 HbAA patients 7 days after the initiation of treatment (Day 7). Two HbAA patients were found parasitaemic at Day 7, they were treated successfully with Fansidar and their Day 7 samples were excluded from the study. Two HbAA patients were unwilling to donate a Day 7 sample. Malaria parasites were counted on Giemsa-stained thick smears as number of parasites per 300 leucocytes (n), and the parasite count per pl of blood was calculated according to the formula [n x (number of leucocytes per pl/300)]. The number of leucocytes was normalized as 5 x 103 per M1 in all patients. Thin smears were used for identification of the parasite species. All patients had microscopically pure P. falciparum infections. Haemoglobin phenotype was determined by the sickling test and electrophoresis.
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PBMC preparation Blood samples were collected by venepuncture into heparinized vacutainers (Becton-Dickinson, Rutherford, NJ). Following centrifugation, plasma was collected and stored frozen until use. The buffy coat and pellet were diluted to twice the original volume using RPMI 1640 medium containing 58-4 pg/ml L-
glutamine, 20 U/ml penicillin, 20 pg/ml streptomycin (all from Gibco, Paisley, UK). PBMC were isolated by density gradient centrifugation on Lymphoprep (Nyegaard, Oslo, Norway). The isolated cells were then washed three times in medium supplemented with 10% fetal calf serum (FCS), and frozen over liquid nitrogen, by a temperature gradient control device, using RPMI 1640 medium containing 10% dimethyl sulphoxide (DMSO) and 20% FCS as freezing medium. Cells were kept cryopreserved in liquid nitrogen until use. On the day of use, cells were quickly thawed at 37°C and washed three times in the above washing medium. Cell viability after thawing was always > 90% by Trypan blue exclusion. Mitogen and antigens PHA (Difco Laboratories, Detroit, MI) was added to cell cultures at a final concentration of 47 pg/ml; PPD (State Serum Institute, Copenhagen, Denmark) at 12 pg/ml. Soluble purified antigens (SPAg) were isolated from supernatants of P. falciparum in vitro cultures by affinity chromatography as previously described [17]. The resulting preparation contains 7-10 distinct single antigens detectable by crossed immuno-electrophoresis. The end product (SPAg) was diluted in RPMI 1640 medium and added to cell cultures at dilutions of 1:40, 1:80 and 1:160, previously shown to induce optimal proliferation. The purified malarial antigen preparation used in this study has previously been shown to be free of non-specific, polyclonal stimulating activity [18] and only T helper (CD4+) cells proliferate in response to these antigens when co-cultured with monocytes [19].
Proliferation assay RPMI 1640 supplemented with 15% pooled human serum, L-glutamine (58-4 pg/ml), penicillin (20 U/ml) and streptomycin (20 ,g/ml) was used as culture medium. Cells were cultured in triplicate in round-bottomed microtitre plates (Nunc, Roskilde, Denmark). Twenty microlitres of mitogen or antigen and 150 P1 of PBMC containing 1 x 105 cells were added to each well. Cultures were incubated for 3 days for mitogen and 7 days for antigens in a humidified 5% CO2-95% air mixture at 37°C and pulsed by 20 pl/well (1.85 mBq/ml) 3H-thymidine (New England Nuclear, Boston, MA) 24 h before cultures were harvested onto glass-fibre filters. 3H-thymidine incorporation was measured in a liquid scintillation counter (Tri-Carb; Packard Instrument Co. Inc., Rockville, MD, USA). For each triplicate the median ct/min was recorded. Unstimulated cultures were always included as controls. The results were expressed both as A kct/min (kct/mins,,m. - kct/minunstim.) and as a stimulation index (kct/minstim./kct/minunstim.). A response was considered to be positive when the A kct/min > 1-0 and the stimulation index was > 2-0. Determination of IFN-y in culture supernatants Eighty microlitres per well of cell supernatant (240 pl/triplicate) were carefully collected from the 7 days proliferation assay
Y. A. Ab&u-Zeid et al.
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cultures immediately before they were harvested. The supernatants were stored frozen until use. IFN-1y was determined by a sandwich ELISA employing polyclonal rabbit-antibody against human recombinant IFN-y (kindly provided by Professor K. Bendtzen, University Hospital, Copenhagen, Denmark). The IFN-y standard used was produced in our laboratory and was calibrated against two IFN-y reference standards (standard Gg23-901-530, NIH, Bethesda, Maryland, USA, and standard 82/587, NIBSC, Potters Bar, Hertfordshire, England). The IFN-y levels in the supernatants were determined from standard curves with the standard IFN-y prepared for each plate. The sensitivity limit of the assay was 1 5 U/ml. The antigen-induced IFN-y production was considered positive if the supernatant contained at least 1-5 U/ml (background subtracted) and at least twice as much as the corresponding unstimulated cultures.
30
0 S
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Modulation of the cellular immune response during Plasmodiumfalciparum infections in sickle cell trait individuals Y. A. ABU-ZEID, T. G. THEANDER*t, N. H. ABDULHADI, L. HVIID*t, B. 0. SAEED, S. JEPSENt, J. B. JENSEN§ & R. A. BAYOUMI Department of Biochemistry, Faculty of Medicine, University of Khartoum, Sudan, *Department of Infectious Diseases, University Hospital, Copenhagen, Denmark, tInstitute for Medical Microbiology, University of Copenhagen, Copenhagen, Denmark, I WHO National Malaria Diagnostic Centre, State Serum Institute, Copenhagen, Denmark, §Department of Microbiology, Brigham Young University, Provo, UT, USA
(Acceptedfor publication 18 November 1991)
SUMMARY Plasma and peripheral blood mononuclear cells (PBMC) were obtained from P.falciparum-infected individuals with and without the sickle cell trait at diagnosis and 7 days after treatment. HbAA and HbAS patients were compared for levels of plasma soluble IL-2 receptors (IL-2R) and the in vitro cellular reactivity to affinity-purified soluble P. falciparum antigens (SPAg), PPD and phytohaemagglutinin (PHA). At diagnosis, HbAS patients with clinical disease had lower plasma-soluble IL2R levels and parasite counts than the corresponding HbAA patients, whereas HbAS and HbAA patients with asymptomatic infections had comparable soluble IL-2R levels and parasite counts. PBMC from HbAS patients had higher proliferation and IFN-y production in response to SPAg than PBMC from HbAA patients. The difference in the lymphoproliferative responses to SPAg between the two groups was evident in patients with asymptomatic infections. In all patients, the clinical severity, the soluble IL-2R levels and the parasite counts were directly related. The former two were inversely related to the proliferative responses to SPAg. After treatment, all the studied parameters were comparable in the two groups. The study indicates that during P. falciparum infection, HbAS compared with HbAA patients had lower in vivo cellular activation and higher in vitro cellular reactivity in response to soluble malaria antigens. Keywords Plasmodium antigens interferon-gamma lymphoproliferative soluble IL-2 receptors sickle cell trait
responses
have demonstrated that healthy HbAS individuals from areas of unstable seasonal P. falciparum transmission had higher proliferative responses to soluble P.falciparum antigens (SPAg) and to PPD than HbAA individuals [9,10]. The difference between the two groups was more evident in children than in adults [10]. Furthermore, the lymphoproliferative responses to SPAg in HbAA children were suppressed during the malaria season compared with the responses before the season, whereas in HbAS children the responses to SPAg were higher during the malaria season than before the season [11]. Lymphocytes obtained from HbAA individuals during malarial infections were unable to proliferate in vitro in response to specific malarial antigens, whereas such responses were usually found after treatment [12-151. The present study was designed to investigate the possible differences in cellular immune reactivity of HbAA and HbAS patients with P. falciparum malaria. The two groups were compared for the in vitro cellular reactivity to soluble P.falciparum antigens (SPAg), to non-specific antigen (PPD) and to a mitogen, phytohaemagglutinin (PHA); and the degree of in vivo cellular activation, as indicated by the plasma soluble IL-2 receptor (IL-2R) levels.
INTRODUCTION The in vivo mechanisms which protect HbAS individuals from the severe manifestations of P. falciparum remain unclear. Several investigators have demonstrated that under low oxygen tension the in vitro growth of P.falciparum is retarded in HbAS erythrocytes [1-3]. Furthermore, other mechanisms such as toxic haem and generation of active oxygen radicals have been implicated in the resistance of HbAS erythrocytes to falciparum malaria [4,5]. Allison [6] proposed that the protective effect of the sickle haemoglobin is acting in synergy with specific acquired immunity, and Bayoumi [7] suggested that the selective advantage of HbAS individuals is due to earlier acquisition of clinical immunity. HbAS children have been shown to have high antibody titres against parasite-derived neoantigens, which are expressed on infected erythrocytes and which have been implicated in the protection against clinical falciparum malaria [8]. Recently, we Correspondence: Yousif A. Abu-Zeid, Lymphocyte Laboratory, Department of Infectious Diseases, M7641, University Hospital, Tagensvej 20, DK-2200 Copenhagen N, Denmark.
112
Cellular responses in HbAS individuals with P. falciparum infections Data were obtained from samples collected from P. falciparum patients with and without the sickle cell trait at diagnosis and during convalescence. MATERIALS AND METHODS
Study area The study was done in Darawish village (Sudan) which has a total population of approximately 350 individuals. The village is situated 420 km southeast of Khartoum. The area is endemic for malaria and characterized by unstable seasonal transmission. The rains fall mainly during July-September, and the rest of the year is dry. Positive blood films are encountered from September to December with the highest incidence rates during October-November [16]. The predominant malaria species is P. falciparum; rarely P. vivax is seen. Darawish is inhabited by 42 families; the gene for the sickle cell haemoglobin (Hb S) is present in 14. Individuals with the sickle cell trait amount to 11% of the total village population. Patients The study was carried out during October-November. Thirteen HbAS and 15 HbAA P. falciparum patients were grouped according to the severity of the disease at the time of bleeding. Five HbAS and seven HbAA patients with asymptomatic infections were identified during screenings of thick blood films collected from the village population; which were subsequently Giemsa stained and examined for the presence of malaria parasites. These donors had no physical complaints, their body temperature was < 38 C and they reported not to have had fever for at least 2 weeks prior to bleeding. The others had clinical disease of moderate severity; temperature >38 C, and complained of fever, headache, body or joint pains and can be described as cases of acute non-complicated falciparum malaria. In order to match the two groups, severely ill HbAA patients were not included in the study as HbAS patients had asymptomatic infections or presented with malaria of moderate severity. The median age for HbAS and HbAA patients was 13 years (range 8-35) and 12 years (range 8-36), respectively. There was no significant age difference neither between HbAA and HbAS patients with asymptomatic infections nor between patients with clinical disease. Informed consent was obtained from the patients and/or their parents before blood samples were drawn. A heparinized blood sample was obtained from each patient at diagnosis (Day 0) and chloroquine treatment was initiated. A second blood sample was obtained from all 13 HbAS patients and from 13 out of the 15 HbAA patients 7 days after the initiation of treatment (Day 7). Two HbAA patients were found parasitaemic at Day 7, they were treated successfully with Fansidar and their Day 7 samples were excluded from the study. Two HbAA patients were unwilling to donate a Day 7 sample. Malaria parasites were counted on Giemsa-stained thick smears as number of parasites per 300 leucocytes (n), and the parasite count per pl of blood was calculated according to the formula [n x (number of leucocytes per pl/300)]. The number of leucocytes was normalized as 5 x 103 per M1 in all patients. Thin smears were used for identification of the parasite species. All patients had microscopically pure P. falciparum infections. Haemoglobin phenotype was determined by the sickling test and electrophoresis.
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PBMC preparation Blood samples were collected by venepuncture into heparinized vacutainers (Becton-Dickinson, Rutherford, NJ). Following centrifugation, plasma was collected and stored frozen until use. The buffy coat and pellet were diluted to twice the original volume using RPMI 1640 medium containing 58-4 pg/ml L-
glutamine, 20 U/ml penicillin, 20 pg/ml streptomycin (all from Gibco, Paisley, UK). PBMC were isolated by density gradient centrifugation on Lymphoprep (Nyegaard, Oslo, Norway). The isolated cells were then washed three times in medium supplemented with 10% fetal calf serum (FCS), and frozen over liquid nitrogen, by a temperature gradient control device, using RPMI 1640 medium containing 10% dimethyl sulphoxide (DMSO) and 20% FCS as freezing medium. Cells were kept cryopreserved in liquid nitrogen until use. On the day of use, cells were quickly thawed at 37°C and washed three times in the above washing medium. Cell viability after thawing was always > 90% by Trypan blue exclusion. Mitogen and antigens PHA (Difco Laboratories, Detroit, MI) was added to cell cultures at a final concentration of 47 pg/ml; PPD (State Serum Institute, Copenhagen, Denmark) at 12 pg/ml. Soluble purified antigens (SPAg) were isolated from supernatants of P. falciparum in vitro cultures by affinity chromatography as previously described [17]. The resulting preparation contains 7-10 distinct single antigens detectable by crossed immuno-electrophoresis. The end product (SPAg) was diluted in RPMI 1640 medium and added to cell cultures at dilutions of 1:40, 1:80 and 1:160, previously shown to induce optimal proliferation. The purified malarial antigen preparation used in this study has previously been shown to be free of non-specific, polyclonal stimulating activity [18] and only T helper (CD4+) cells proliferate in response to these antigens when co-cultured with monocytes [19].
Proliferation assay RPMI 1640 supplemented with 15% pooled human serum, L-glutamine (58-4 pg/ml), penicillin (20 U/ml) and streptomycin (20 ,g/ml) was used as culture medium. Cells were cultured in triplicate in round-bottomed microtitre plates (Nunc, Roskilde, Denmark). Twenty microlitres of mitogen or antigen and 150 P1 of PBMC containing 1 x 105 cells were added to each well. Cultures were incubated for 3 days for mitogen and 7 days for antigens in a humidified 5% CO2-95% air mixture at 37°C and pulsed by 20 pl/well (1.85 mBq/ml) 3H-thymidine (New England Nuclear, Boston, MA) 24 h before cultures were harvested onto glass-fibre filters. 3H-thymidine incorporation was measured in a liquid scintillation counter (Tri-Carb; Packard Instrument Co. Inc., Rockville, MD, USA). For each triplicate the median ct/min was recorded. Unstimulated cultures were always included as controls. The results were expressed both as A kct/min (kct/mins,,m. - kct/minunstim.) and as a stimulation index (kct/minstim./kct/minunstim.). A response was considered to be positive when the A kct/min > 1-0 and the stimulation index was > 2-0. Determination of IFN-y in culture supernatants Eighty microlitres per well of cell supernatant (240 pl/triplicate) were carefully collected from the 7 days proliferation assay
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cultures immediately before they were harvested. The supernatants were stored frozen until use. IFN-1y was determined by a sandwich ELISA employing polyclonal rabbit-antibody against human recombinant IFN-y (kindly provided by Professor K. Bendtzen, University Hospital, Copenhagen, Denmark). The IFN-y standard used was produced in our laboratory and was calibrated against two IFN-y reference standards (standard Gg23-901-530, NIH, Bethesda, Maryland, USA, and standard 82/587, NIBSC, Potters Bar, Hertfordshire, England). The IFN-y levels in the supernatants were determined from standard curves with the standard IFN-y prepared for each plate. The sensitivity limit of the assay was 1 5 U/ml. The antigen-induced IFN-y production was considered positive if the supernatant contained at least 1-5 U/ml (background subtracted) and at least twice as much as the corresponding unstimulated cultures.
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