Elevated levels of nitric oxide and low levels of haptoglobin are associated with severe malarial anaemia in African children

Acta Tropica 83 (2002) 133– 140 www.parasitology-online.com

Elevated levels of nitric oxide and low levels of haptoglobin are associated with severe malarial anaemia in African children Ben Gyan a,e,*, Jorgen A.L. Kurtzhals a,b,c, Bartholomew D. Akanmori a, Michael Ofori a, Bamenla Q. Goka b, Lars Hviid c, Charlotte Behr d a

Immunology Unit, Noguchi Memorial Institute for Medical Research, Uni6ersity of Ghana, Legon, Ghana b Department of Child Health, Korle Bu Teaching Hospital, Accra, Ghana c Centre for Medical Parasitology at Department of Infectious Diseases and Department of Clinical Microbiology, Copenhagen Uni6ersity Hospital and Institute of Medical Microbiolgy and Immunology, Uni6ersity of Copenhagen, Copenhagen, Denmark d United’Immunologie Molecularies des Parasites, CNRS URA 1960, Institute Pasteur, Paris, France e Department of Immunology, Stockholm Uni6ersity, SE-106 91 Stockholm, Sweden Received 23 November 2001; received in revised form 14 February 2002; accepted 7 March 2002

Abstract Severe malarial anaemia (SA) is a major complication of malaria and an important cause of child mortality and morbidity. However, the pathogenesis behind SA is poorly understood. Nitric oxide (NO) is known to play a protective role against clinical malaria but is also suggested to have a pathogenic role in cerebral malaria (CM). Erythrophagocytosis by splenic macrophages has been implicated in the pathogenesis of SA. In this study, plasma levels of NO, neopterin, haptoglobin and C-reactive protein (CRP) were measured in paediatric patients with CM, n= 77, SA (n=28) and uncomplicated malaria (UM n= 53). Haptoglobin levels were significantly lower in SA (median (interquartile range) 25 (17–59) mg/l) than in both CM and UM (40 (24– 80) mg/l and 110 (60– 160) mg/l, respectively, P B0.001). In contrast, NO levels were higher in SA (38 (28– 51) mmol/l) than in CM and UM (21 (15–32) mmol/l and 10.3 (5.6–17) mmol/l, respectively, PB 0.001). A significant negative correlation between haptoglobin and NO was seen in the SA group. No such correlation was observed within the UM or CM groups. No significant differences in neopterin levels were observed between any of the three groups, neither was there any correlation between parasitaemias and neopterin levels. The low haptoglobin and high levels of NO in this SA group may contribute to haemolysis. Taken together our results support the hypothesis that immune-mediated erythrocyte destruction is involved in the pathogenesis of malarial anaemia. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Malaria; Severe anaemia; Nitric oxide; Neopterin; C-reactive protein; Haptoglobin

* Corresponding author. Tel.: + 46-8-164-175; fax: + 46-8-157-356. E-mail address: [email protected] (B. Gyan). 0001-706X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S0001-706X(02)00109-2

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1. Introduction Severe anaemia (SA) due to Plasmodium falciparum infections is a common and important complication of malaria. Together with cerebral malaria (CM), SA accounts for the majority of deaths in malaria in Africa (Greenwood et al., 1991). Although the precise mechanism underlying the pathophysiology of SA is poorly understood, it is believed to be complex and multifactorial. It is known that Plasmodium parasite invasion of host erythrocytes and release of merozoites during schizogony result in intravascular haemolysis. However, the destruction of parasitised-erythrocytes is not sufficient to explain the reduced haemoglobin levels seen in SA (Jakeman et al., 2000). Thus, destruction of uninfected erythrocytes may also play a central role. This destruction may be due to macrophage activation in the spleen (Clark and Chaudri, 1988), immunemediated haemolysis (Facer et al., 1979; Goka et al., 2001) or oxidative stress to erythocytes (Das and Nanda, 1999). In addition, malaria is associated with dyserythropoesis due to cytokines and other factors (Clark and Chaudri, 1988; Kurtzhals et al., 1997, 1998). Nitric oxide (NO) seems to play an important role in the protection and the pathogenesis of infectious diseases (Nussler and Billiar, 1993). We have previously shown that interferon-gamma stimulated human monocytes inhibit plasmodial growth in vitro through an L-arginine pathway (Gyan et al., 1994). Other studies have suggested that the sequestration of parasites within the cerebral blood vessels in CM is associated with inflammatory responses, with higher levels of circulating tumor necrosis factor (TNF-a) and NO (Clark et al., 1991). While NO has been demonstrated to be involved in the pathogenensis of CM, this has not been shown for SA. Reactive oxygen intermediates which have relatively low antiplasmodial effects by themselves have been shown to cause haemolysis and hence anaemia in Plasmodium 6inckei infected mice (Clark and Hunt, 1983). Cytokine-induced NO is known to decrease human erythropoesis and hence NO may be a possible causative effect of anaemia (Maciejewski et al., 1995; Domachowske, 1997).

Haptoglobin is an acute phase a-2-sialoglycoprotein and its polymorphism has been shown to influence immune responses as well as the prevalence and the expression of inflammatory responses (Langlois and Delanghe, 1996). Recent studies have suggested possible mechanisms by which haptoglobin may modulate the clinical course of malaria infections. The haptoglobin– haemoglobin complex reduces the generation of haemoglobin-related reactive-oxygen species (ROS) involved in signal transduction, enabling the production of inflammatory mediators (Palmer and Paulson, 1997). Haptoglobin is known to take up free haemoglobin in circulation. Thus, elucidation of the role of NO and haptoglobin during haemolysis will provide a better understanding of the pathogenesis of SA. In this study we have measured the plasma concentrations of NO, haptoglobin, C-reactive protein (CRP) and neopterin, a secretory product of activated macrophages, in Ghanaian children with CM, SA or uncomplicated malaria (UM).

2. Patients and methods

2.1. Patients characteristics Patients between the ages of 0.5 and 12 years were recruited, during the malaria transmission season (July and August) in 1997, at the Korle Bu Teaching Hospital in Accra, Ghana, as part of a prospective study (Akanmori et al., 2000). Selection criteria for inclusion into the study were asexual P. falciparum parasitaemia of more than 10000 per ml and axillary temperature of more than 37.5 °C. Children with diseases other than malaria or with positive sickling test (metabisulphite method) were excluded from the study. Patients with confirmed cases of CM, SA or UM were enrolled into the study. The criteria used for severe SA were haemoglobin (Hb) B 50 g/l, no other cause of anaemia and full consciousness (score 5 on the Blantyre coma scale, Molyneux et al., 1989); UM, as for severe anaemia but with Hb \ 80 g/l and no other complications. For CM, the criteria were unarousable coma, with a score of three or less for more than 60 min and no sign

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of meningitis or encephalitis. Parents or guardians signed consent forms after receiving simple standard information in the local language. The Ethics and Protocol Review committee of the University of Ghana Medical School and the Ministry of Health approved of the study. Enrolled patients were routinely treated with antimalarials and full supportive measures. Venous blood was collected on ice in test tubes containing heparin or ethylamine tetra-acetic acid (EDTA) and plasma was obtained by centrifuging at 4 °C within 30 min of blood collection and stored at − 80 °C. Measurements of Hb levels were done in an automated haematology analyser (Coulter). From each patient, thick and thin blood films were made and stained with Giemsa for microscopic parasite detection and identification.

2.2. Nitric oxide assay The assay was based on that described by Rockett et al. (1994) using a commercial test kit (Boehringer Mannheim, Germany). Briefly, 30 ml of serum were dispensed in duplicate into 1.5 ml Eppendorf tubes for nitrite and nitrate measurements, respectively. Nitrate reductase that enzymatically reduces the nitrate to nitrite, was added to the samples for nitrate determination. NADPH was then added to each tube and the resultant mixture was assayed with Griess reagent (Sigma, St. Louis, USA), with absorbance at OD 540 nm measured in a Microplate reader (MTP-32). The results were expressed as the sum of nitrite and nitrate (NO) which are the end products in the NO degradation.

2.3. CRP and haptoglobin determinations Plasma levels of both CRP and haptoglobin were determined by an in-house enzyme-linked immunosorbent assay (ELISA). For CRP, rabbit anti-human CRP polyclonal antibodies (DAKO, Denmark) were used for coating and horse-radish peroxidase (HRP) conjugated anti-human CRP antibodies (DAKO, Denmark) were used as a detector antibody. For haptoglobin determinations, plates were coated with polyclonal sheep

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anti-haptoglobin antibodies (the Binding Site, UK), with rabbit anti-human haptoglobin (DAKO, Denmark) as the secondary antibody and HRP-conjugated anti-rabbit IgG as detector. In both assays, o-phenylenediamine dihydrochloride was added after incubation with the conjugated antibody. The reaction was stopped with 1M HCL and the optical density was measured at OD 492 nm.

2.4. Neopterin Plasma concentrations of neopterin were determined using a commercial enzyme immunoassay kit (Immunotech, Marseilles France).

2.5. Statistical analysis Statistical analysis was carried out using Sigma Stat 2.0 (Jandell Scientific, San Rafael, CA, USA). Data were logarithmically transformed to achieve normal distribution before analysis by one-way ANOVA. Correlations were analysed by Spearman’s rank correlation. P values B 0.05 were considered as significant.

3. Results In all, a total of 158 patients were enrolled in the study, comprising 77 children with CM, 28 with SA and 53 with UM. As shown in Table 1, the levels of parasitaemia were generally high in all three groups, but the CM group showed the highest median parasitaemia (PB0.005). Comparing the levels of NO between the groups revealed that the children with SA had the highest concentrations of NO as compared with the CM and UM groups (PB 0.05). There was no correlation between NO and parasite density (r= 0.08, P\0.05), when all patients were grouped together. However, there was a positive correlation between NO and parasite densities in the CM group (r=0.28, PB0.05) but not in the SA or the UM groups (data not shown). The concentration of CRP was twice as high in the CM and SA groups as compared with the UM. Haptoglobin levels were significantly higher in the UM as

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compared with SA and CM groups (Table 1). The levels of haptoglobin were significantly lower in the SA than in the CM group. Haptoglobin levels in the SA group correlated inversely with NO (r = − 0.6, P= 0.05 Fig. 1B). Adjustment for age and sex did not alter the association. No correlations between haptoglobin and NO were observed in the CM and the UM groups (Fig. 1A, C). Haemoglobin levels showed a significant positive correlation with haptoglobin concentration but not with neopterin or CRP (data not shown). No significant differences were observed between any of the groups in the neopterin concentrations. Neither were there any correlations between neopterin and parasitaemias.

4. Discussion The primary objective of this study was to assess the importance of NO, the acute phase proteins and neopterin as possible pro-inflammatory markers for severity of malaria as well as their possible role in the pathogenesis of severe malaria. The most significant findings in the study were that the severe anaemia group exhibited elevated levels of NO and low levels of haptoglobin. Previous studies have reported an association between low plasma concentrations of interleukin-10 (IL-10) and severe anaemia (Kurtzhals et al., 1998; Othoro et al., 1999). In

those studies, the pathogenic effect of low levels of IL-10 was suggested to be due to its imbalance with TNF-a production, a cytokine known to be associated with severe malaria. However, IL-10 also plays a down-regulatory role on NO by inhibiting its secretion (Oswald et al., 1992). Thus, low levels of IL-10 could result in increased NO levels. This hypothesis is supported by our previously reported study using the same patients that low levels of IL-10 were correlated with SA (Akanmori et al., 2000). Excessive production of NO has been implicated in the pathogenesis of severe malaria (Clark et al., 1991). The erythrocyte membrane contains proteins and carbohydrates which when modified are capable of initiating an immune response. One of these responses may involve the production of NO. A recent report from Tanzania demonstrates an inverse correlation between haemoglobin levels and NO in malaria patients. Although the low levels of haemoglobin were attributed to the overproduction of NO it was mainly found in asymptomatic children rather than in children with severe anaemia (Anstey et al., 1999). An important role of high concentrations of NO is its downregulatory effect on the development of haematopoetic cells (Mannick et al., 1994; Conrad et al., 1993). Little is known about the effects of NO in anaemia, but its role in the pathogenesis of severe anaemia could perhaps be attributed to interference with the haem groups in the electron-

Table 1 Mean and median levels of clinical and laboratory characteristics of 158 children with cerebral malaria, severe anaemia and uncomplicated malaria

Number of subjects Age (years)a Temperature ( °C)a Hemoglobin (g/l)a Parasite density(g/ml)b Nitric oxide (mmol/l)b Haptoglobin(mg/l)b CRP (mmol/l)b Neopterin (mg/l)b a

Cerebral malaria

Severe anaemia

Uncomplicated malaria

P valuec

77 4.2 (2.7) 38.5(0.7) 7.6 (2.2) 82 437 (35,866–171 928) 21 (15–32) 40 (24–80) 240 (150–340) 93 (64–110)

28 2.7 (2.6) 38.5 (0.7) 4.5 (0.6) 41,474 (17 230–110 181) 38 (28–51) 25 (17–59) 230 (172–295) 85 (75–110)

53 4.6 (3.3) 38.5 (0.6) 10.7 (17.2) 46 327 (21 500–92 064) 10.3 (5.6–17) 110 (60–160) 110 (77–200) 80 (67–90)

0.005 B0.001 B0.001 B0.001 0.6

Mean (S.D.). Median (25–75 percentiles). c P values by one-way ANOVA on ranks. b

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Fig. 1. Relationship between haptoglobin and nitric oxide concentrations in patients with (A) cerebral malaria, (B) severe anaemia and (C) uncomplicated malaria.

transfer chain of the red blood cells which has been shown to result in haemolysis of red blood cells (Kolb and Kolb-Bachofen, 1998). One reason for the destruction of erythrocytes in children with severe P. falciparum anaemia could be that

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the erythrocyte surface is modified as a result of exposure to reactive oxygen species. Indeed, in a recent study it was shown that IgG bound to erythrocytes and the expression of surface CR1 and CD55 accelerated the destruction of red blood cells through phagocytic or complementmediated lysis of the cells in patients with SA (Waitumbi et al., 2000). The way NO modifies the RBC surface could be through peroxynitrite, a strong oxidant derived from NO which is known to chemically modify polypeptide proteins and subsequently alter their biological activity (Kuo et al., 1999). Haptoglobin is an a-2 sialoglycoprotein and an acute phase protein with haemoglobin-binding capacity. The production of haptoglobin is increased during inflammatory reactions (Buchanan et al., 1990) but depleted due to its binding to haemoglobin. Low levels of haptoglobin are found in ineffective erythropoesis (Dobryszycka, 1997) although the reason for this is not well understood. Haptoglobin binds to cells that express the CD11b/CD18 (El Ghmati et al., 1996) molecules involved in transducing signals generating inflammatory mediators (Palmer and Paulson, 1997). Thus the low levels of haptoglobin seen could be due to the fact that haptoglobin– haemoglobin in the form of complexes is internalised by cells expressing the CD11b/CD18 molecules. Expression of CD11b/CD18 is shown to be downregulated by NO (Kubes et al., 1991). In addition, the haptoglobin-haemoglobin complexes present within the phagolysosomes of phagocytic cells have been found to display inhibitory effects on NO production (Edwards et al., 1989). The function of NO in intracellularparasitic diseases like malaria is rather complex as this depends on many immunological molecules in the microenvironment. Haemoglobin is reported to have high affinity for NO (Stamler et al., 1997) and in malaria this scavenging property depends on the oxygen tension in the microenvironment (Taylor-Robinson and Looker, 1998). The elevated levels of NO seen in the severe anaemia group could be due to that more nitrate or nitrite are generated when the free haemoglobin levels are reduced.

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Different genetic polymorphisms of the inducible nitric-oxide synthase (iNOS) promoter region have been associated with increased risk of death from cerebral malaria in Gambian children (Burgner et al., 1998) and to protect Gabonese children from severe malaria in (Kun et al., 1998). These differences suggest that complex interactions and factors may determine the production of iNOS and thereby influence the clinical outcome. Also, functional differences in the different haptoglobin phenotypes have been shown to differently affect disease conditions (Langlois and Delanghe, 1996). A study conducted in Sudan demonstrated an association of the Hp 1-1 phenotype with malaria disease severity (Elagib et al., 1998). We have recently shown that this phenotype is associated with susceptibility to P. falciparum malaria in general and to the development of severe disease in particular (Quaye et al., 2000). Thus, the beneficial or pathogenic role of haptoglobin may be specific for a particular phenotype or reflect the expression of varying concentrations of haptoglobin. One of the possible causes of severe anaemia is increased erythrophagocytosis by splenic macrophages. We thus expected to find high levels of neopterin, an indicator of macrophage activation and which have been previously shown to be elevated in patients with severe anaemia (Biemba et al., 2000) and in Coombs’ positive anaemia patients (Goka et al., 2001). However, in the present study we found that the neopterin levels were similar in all three patient categories. C-reactive protein has been proposed as a surrogate marker of malaria morbidity (Rougemont et al., 1988; Trape and Fribourg-Blanc, 1988). The high CRP levels in SA and CM seen in our study agree with previous reports in which CRP was elevated in severe as compared with uncomplicated malaria and hence proposed as a useful predictor of malaria severity (Kremsner et al., 1996). Although the mean parasitaemia was higher in CM than SA, this was neither correlated with the levels of NO nor with the haptoglobin levels. This may imply that parasitaemias are independent of the induction of NO and the decrease in haptoglobin and in support of earlier findings regarding haptoglobin (McGuire et al., 1996).

Overall, our study emphasises the importance of a dual role of NO as a cytotoxic molecule and as an immunoregulatory agent. However, further investigations are required to elucidate the functional roles of NO and haptoglobin in the pathogenesis of severe malaria anaemia.

Acknowledgements Abdulraham Hammond and John Tetteh of NMIMR, Ben Quashie, John Tsakpo, Isaac Antwi, and Caleb Boye-Nortey of CTCPT, Ghana are thanked for their invaluable technical assistance. The staff of the Deparment of Child Health, Korle-Bu Teaching Hospital, Accra Ghana are also thanked for carrying out clinical investigations. We are also grateful to Professor Marita Troye-Blomberg of Department of Immunology, Stockholm University for critical reviewing of manuscript. This work was supported by the ENRECA programme of the Danish International Development Assistance, DANIDA, UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) MIM grant No. 900037 and the INCODC programme of the European Union. L.H. is supported by grants from the Danish Medical Research Council and the Danish Research Council for Development Research.

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