Abstract
Background/objective: The relative balance between Th1 and Th2 cytokines appears crucial in the outcome of infections. We assayed the levels of some proinflammatory Th1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and anti-inflammatory Th2 cytokines, IL-4 and IL-10 in homozygous haemoglobin (Hb) AA, heterozygous HbAS-genotyped and sickle cell (HbSS) individuals with uncomplicated Plasmodium falciparum malaria.
Methods: Levels of Th1 and Th2 cytokines of 111 children aged 1–5 years with uncomplicated malaria and 89 healthy controls were determined by enzyme linked immunosorbent assay and haematological parameters were estimated using the automated Swelab counter (Boule Medical, Stockholm, Sweden).
Results: Th1 and Th2 cytokine levels were significantly higher in HbAA and HbAS-genotyped patients compared to their respective healthy controls (P<0·05). IFN-gamma, IL-2, and IL-10 were significantly elevated in HbAA compared to HbAS and HbSS subjects (P<0·05). The mean haematological parameters (total white cell count and monocytes) of HbSS-infected children were significantly higher compared to those of HbAA and HbAS subjects (P<0·05); however, their mean packed cell volume was significantly lower compared to others (P<0·05).
Conclusion: Our results showed a stronger Th1 cytokine response in HbAA than HbAS and HbSS individuals; this may suggest an immunocompetence of the HbAA individuals in early infection.
Introduction
After the first description of sickle cell disease in a Grenadian dental student in Chicago in 1910, subsequent reports indicated that the disease was confined to people of African origin. Over the next 40 years, the clinical features were recorded in Americans, but it was not until the late 1940s that reports began to appear from Africa itself. These documented the high prevalence of the sickle cell trait throughout equatorial Africa and its geographical coincidence with the distribution of Plasmodium falciparum malaria. Studies from west, east, and central Africa showed that individuals with the sickle cell trait had a relative protection against malaria during a critical period of early childhood.Citation1
A number of factors are likely to be involved and to contribute in varying degrees to the defence against malaria in these individuals. Red cells from people with sickle trait do not sickle to any significant degree at normal venous oxygen tension. Very low oxygen tension will cause the cells to sickle.Citation2 In malarious areas, the high frequency of haemoglobinopathies, such as sickle cell disease, supports their protective role against P. falciparum malaria.Citation3–Citation5
However, in patients homozygous for sickle haemoglobin (SS), the persistence of undetectable P. falciparum infection could trigger acute haemolytic events.Citation6
Reports in the literature suggest a central role for inflammation in the disease process of sickle cell patients. Elevated basal leukocyte countsCitation7 are typical, including activated monocytes.Citation8 Sickle cell children with the highest white blood cell counts are more likely to develop disease complications such as frequent pain and stroke.Citation9 Some investigators report elevated plasma levels of certain proinflammatory cytokines, for example, tumour necrosis factor-alpha,Citation10,Citation11 supporting a role for cytokine-driven inflammation. Others report normal levels of some proinflammatory cytokinesCitation12–Citation14 and reduced levels of others such as interferon-gamma (IFN-gamma)Citation15 in sickle cell disease with uncomplicated malaria.
Furthermore, some studies report high circulating levels of anti-inflammatory cytokines in the asymptomatic state.Citation15 Haemoglobin genotype is believed to contribute to malaria resistance alongside several other factors. Some cytokines have been reported to have antiplasmodial activities. In this study, we assay some Th1 and Th2 cytokine response of HbAA, HbAS, and HbSS individuals.
Materials and Methods
Study design
A total of 111 venous blood samples from children (with P. falciparum slide positive) aged 1–5 years (37 HbAA, 39 HbAS, and 35 HbSS) who presented with symptoms of uncomplicated malaria (i.e. temperature ⩽39°C, bitterness of mouth, joint pains, and lack of nervous system involvement) as well as 89 healthy controls (asymptomatic Plasmodium sp slide negative) were collected during March–June 2008 in Medical Centres in the Benin metropolis (Lahor Medical Centre, Suyi Hospital and Milestone Hospital). Benin City is a cosmopolitan town located in the southern region of Nigeria where malaria infection is endemic. Informed consent of the participant and ethical clearance from the institution were obtained before blood samples were collected.
Malaria parasites screening
The 111 blood samples from symptomatic subjects and 89 from asymptomatic control subjects were used for the study. Thick blood films were made on a clean glass slide and stained with Giemsa stain for 15 minutes. The slides were air-dried and examined under ×100 objective microscope lens for characteristic features of malaria parasites.Citation16
Haemoglobin electrophoresis
An aliquot of washed blood cells was transferred into a clean test tube and lysed by adding few drops of distilled water. Each sample was spotted on a cellulose acetate paper alongside with control with an applicator stick. Then the cellulose acetate paper was then placed in an electrophoretic tank. Electrophoresis was run at 60 V for 10 minutes. The haemoglobin types were determined by distance compared to standard.Citation17
Cytokine assay
Serum samples from P. falciparum slide positive and negative (control) were used for cytokines assay. IFN-gamma, IL-2, IL-4, and IL-10 cytokines levels were assayed using enzyme linked immunosorbent assays (ELISA or EIA)Citation18 reagents from MABTECH Inc. Company (Stockholm, Sweden).
Haematological parameters
The haematological parameters of the subjects and controls (differential white blood cells, total white blood cells, and packed cell volume) were assayed using the Swelab Automatic Counter (Boule Medical, Stockholm, Sweden). Detection of white blood cells and the red blood cells is accomplished by measuring the impedance in the orifice of the transducer. The transducer is mounted in a conductive solution. Electrodes with opposite charges establish a weak current. As the blood cells pass through the orifice, they block the current, causing voltage pulses. The amplitude of the pulse is directly related to the size of the represented cell. The number of pulses is equivalent to the number of cells passing through the orifice during the counting period.
Statistical analysis
Data are expressed as means and standard deviations. Statistical analysis was performed using Kruskal–Wallis test (one-way analysis of variance). P values are considered significant when P is <0·05.
Results
Levels of Th1 cytokines, IL-2 and IFN-gamma, and Th2 cytokines, IL-4 and IL-10, in serum were tested in 111 children with uncomplicated malaria and were compared to those detected in healthy children controls. IFN-gamma, IL-2, IL-4, and IL-10 cytokine levels were significantly higher in HbAA, HbAS, and HbSS-genotyped patients than those obtained in their respective healthy controls (P<0·05). IFN-gamma, IL-2, and IL-10 levels were significantly elevated in HbAA than those in HbAS and HbSS subjects (P<0·05) ().
Table 1. A comparison of the mean±standard deviation Th1 and Th2 cytokine levels between children presenting with uncomplicated malaria symptoms and healthy control children (grouped by genotype)
The mean haematological parameters (total white cell count and monocytes) in HbSS-infected children were significantly higher than those in HbAA and HbAS subjects (P<0·05); however, their mean packed cell volume was significantly lower than others (P<0·05) ().
Table 2. A comparison of the mean±standard deviation haematological parameters between subjects and controls
Discussion and Conclusion
P. falciparum malaria is responsible for over 1 million deaths each year, mostly in children under the age of 5 years living in sub-saharan Africa. The pathogenesis of severe malaria is not fully understood.Citation19 As highlighted by Long et al.,Citation20 cytokines play an extremely significant role in both the prevention and exacerbation of severe diseases. There is ample evidence that higher levels of inflammatory cytokines are associated with cerebral malaria.
Our study revealed that IFN-gamma, IL-2, IL-4, and IL-10 cytokine levels were significantly higher in HbAA, HbAS, and HbSS-genotyped patients than their respective healthy controls (P<0·05). Furthermore, Th1 and Th2 cytokines considered in this study were significantly elevated in HbAA than in HbAS and HbSS subjects (P<0·05).
Higher levels of these cell mediators in HbAA individuals may translate to their immunocompetence. Surprisingly, clinical malaria is commonly reported in these individuals than in the heterozygote HbAS and in homozygous HbSS patients where it triggers haemolytic events.Citation6
The elevated cytokine levels in HbAA subjects could be as a result of higher parasitaemia that is usually observed in HbAA individuals.Citation16 Nevertheless, the outcome of malaria infection may not rest only on cytokine levels; several other factors have been identified and are found to induce resistance to malaria. Sickle cell trait (genotype HbAS) confers a high degree of resistance to severe and complicated malaria.Citation3 Certainly it is due to the peculiar physical or biochemical properties of HbAS red blood cells: invasion, growth, and development of P. falciparum parasites are all reduced in such cells under physiological conditions in vitro and parasite-infected HbAS red blood cells also tend to sickle a process that may result in their premature destruction by the spleen.Citation21
Th1 cytokines are important in controlling early parasitaemia, although they need to be counterbalanced later in the infection by a Th2 response which leads to antibody production. Both Th1 and Th2 cytokines are required for adequate protection, and their mechanisms of action are different and must be finely tuned in time and intensity. Pathogenesis of malaria is a complex process in which a common outcome might be reached by different routes. For example, various proinflammatory cytokines that clearly play a role in cerebral malaria may be redundant, making it difficult to unequivocally assign to them a pathogenic role in all clinical situations. Although animal models, most notably knockout mice, have been paramount to our understanding of the role of cytokines in malaria by providing much valuable information, it is still controversial whether they can reproduce all of the features of human malaria.Citation22 The haematological indices of our subjects revealed that total white cells and monocyte count of HbSS were significantly elevated. Proliferation of these cells is coordinated by cytokines, strikingly; individuals in this group had lower levels of Th1 and Th2 cytokines.Citation8 These findings are in line with that of West et al.,Citation7 who reported that elevated basal leukocyte counts are typical, including activated monocytesCitation8 and HbSS sickle cell children with the highest white blood cell counts are more likely to develop disease complications such as frequent pain and stroke.Citation9
In conclusion, this study showed stronger Th1 cytokine response in HbAA individuals with uncomplicated malaria. This may suggest an effective parasitaemia control in HbAA individuals in early infection.
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