Role of Th1 and Th2 Cytokines in Immune Response to Uncomplicated Plasmodium falciparum Malaria

The relative balance between Th1 and Th2 cytokines appears crucial,since the role of cytokines has been evaluated in several studiesby comparison of clinically heterogeneous groups of patients.The aim of this study is to determine the role of proinflammatoryTh1 cytokines, interleukin-12 (IL-12) and gamma interferon (IFN- ${gamma}$ ),and anti-inflammatory Th2 cytokines, IL-4 and IL-10, in a homogeneousgroup of patients with uncomplicated Plasmodium falciparum malaria.Levels of IL-12, IFN- ${gamma}$ , Il-4, and IL-10 in serum for 20 adultpatients and 15 healthy control subjects were determined byan immunoenzymatic assay. Serum levels of Th1 cytokines, IL-12(8.6 ± 2.8 pg/ml; controls, 3.2 ± 0.7 pg/ml) andIFN- ${gamma}$ (39.2 ± 67.6 pg/ml; controls, 8.4 ± 6.3 pg/ml),were significantly increased at admission; 3 days later, levelsof IL-12 in serum remained significantly high (8.8 ±2.6 pg/ml), whereas IFN- ${gamma}$ levels returned to control values.The anti-inflammatory response of Th2 cytokines (IL-10 and IL-4)was distinct. Levels of IL-10 in serum were not significantlyincreased at day 0 and day 3 (306.6 ± 200.4 pg/ml and56.6 ± 38.4 pg/ml, respectively; controls, 17.4 ±9.0 pg/ml). In contrast, levels of IL-4 in serum were not increasedon admission (3.4 ± 1.2 pg/ml; controls, 2.4 ±0.8 pg/ml), but at day 3 a moderate and significant increaseof IL-4 levels was observed (4.5 ± 1.7 pg/ml). In conclusion,the increase of Th1 cytokine IL-12 and IFN- ${gamma}$ levels during theacute phase of uncomplicated P. falciparum malaria may reflectan early and effective immune response regulated by proinflammatoryTh1 cytokines, and in particular IFN- ${gamma}$ may play a role in limitingprogression from uncomplicated malaria to severe and life-threateningcomplications.

INTRODUCTION

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Introduction

Impairment of cell-mediated immunity with specificity for bothparasite and nonparasite antigens is a peculiar feature of acutePlasmodium falciparum infection, the causative agent of uncomplicatedand severe malaria (1, 5, 15). The CD4 T-cell subset is of majorimportance for the induction of blood-stage immunity, whilethe CD8 subset has been shown to be cytolytic against liverstages of the parasite. Stevenson and Tam (7) have shown thatthe preferential activation of a Th1 response in mice is relatedto resistance to blood-stage Plasmodium chabaudi AS infection.

High gamma interferon (IFN- ${gamma}$ ) production as part of a Th1-drivenimmune response has been associated with a more favorable outcomein most animal models of malaria (3, 6). This effect has beenattributed to the monocyte-macrophage-activating capacity ofIFN- ${gamma}$ , with rapid killing of the malarial blood-stage parasitesby reactive oxygen and nitrogen intermediates (9). Winkler etal. (14) have shown in patients with uncomplicated P. falciparummalaria the role of IFN- ${gamma}$ as a key molecule in human antimalarialhost defense, and they do not support a direct involvement ofinterleukin-4 (IL-4) in the clearance of P. falciparum parasites.

Several studies have shown that administration of IL-12 beforeinfection of mice with Plasmodium yoelii or of rhesus monkeyswith Plasmodium cynomolgi provides full protection in both animalmodels through an IFN- ${gamma}$ -dependent antiplasmodial mechanism (2,6). In addition, Stevenson et al. (8) demonstrated that IL-12-inducedprotection against the blood stage of P. chabaudi AS is mediatedby upregulation of IFN- ${gamma}$ and tumor necrosis factor, at leastin part by generating high levels of nitric oxide. Thus, thesestudies suggest that the protective immunity in malaria is mediatedby activation of Th1 cytokines, including IL-12, IFN- ${gamma}$ , and tumornecrosis factor.

To confirm experimental and clinical results, particularly inpatients with uncomplicated P. falciparum malaria, we attemptedto identify the cytokine pattern displayed by these patientsduring the course of disease, as Th1 cytokines, such as IL-12and IFN- ${gamma}$ , as well as Th2 cytokines, such as IL-4 and IL-10.

MATERIALS AND METHODS

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Materials and Methods

The study was carried out at the Division of Infectious Diseases,Varese, Italy, and at the Institute of Infectious Diseases,University of Brescia.

Twenty adult patients (17 male, 3 female; mean age ±standard deviation, 35.1 ± 5.0 years) were admitted atthe hospitals of Varese and Brescia with a diagnosis of uncomplicatedP. falciparum malaria. All patients were African immigrantsand had resided in Italy for at least 1 year. Malaria was diagnosedby examination of thick and thin blood films. All patients receivedadequate antimalarial treatment; most of them received therapywith quinine, given intravenously or orally. Uncomplicated malariawas defined as P. falciparum parasitemia (1,000 to 10,000 parasites/µl)with a hemoglobin level of >8.0 g/dl, glycemia of >60mg/dl, and no signs of severe malaria. Fifteen Caucasian healthysubjects (10 male, 5 female; mean age, 32.1 ± 5.9 years)served as a negative control group.

Blood samples were taken on admission (before start of the treatment)and on day 3 of the disease. After centrifugation, sera wereimmediately aliquoted and stored at -80°C until tested.

Samples, taken from all patients on admission, were evaluatedfor IL-12, IFN- ${gamma}$ , IL-4, and IL-10 by a sandwich enzyme-linkedimmunosorbent assay.

The assays use a monoclonal antibody specific for human IL-12,IFN- ${gamma}$ , IL-4, and IL-10 (Euroclone, Devon, United Kingdom). Theoptical density was read at 450 nm.

All cytokine concentrations (in picograms per milliliter) weredetermined by comparison with the standard curve. The lowerdetection limits for the IL-12 and the IFN- ${gamma}$ immunoassays were<20 and <5 pg/ml, respectively, whereas those for theIL-4 and the IL-10 immunoassays were <0.5 and <5 pg/ml,respectively.

Data are expressed as means and standard deviations. Statisticalanalysis was performed using an unpaired t test with Welch'scorrection. For multiple comparison, the Kruskal-Wallis nonparametricanalysis of variance test was used. Coefficients of correlationswere calculated by the Spearman rank test. P values are consideredsignificant when P is <0.05.

RESULTS

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Results

To evaluate the role of Th1 and Th2 cytokines in the immuneresponse of uncomplicated P. falciparum malaria, levels of Th1cytokines, IL-12 and IFN- ${gamma}$ , and Th2 cytokines, IL-4 and IL-10,in serum were tested in 20 adult patients and were comparedwith those detected in 15 seronegative healthy donors. Theirdemographic, clinical, and laboratory features are shown inTable 1. Our patients quickly improved after antimalarial treatment,including quinine or mefloquine, was started at admission, andall patients completely recovered from the infection within7 to 10 days.

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TABLE 1. Clinical, parasitological, and laboratory features of study participants

Figure 1 shows levels of Th1 cytokines, IL-12 and IFN- ${gamma}$ , in serum.Levels of IL-12 in the sera of our patients on admission weresignificantly increased (8.6 ± 2.8 pg/ml; controls, 3.2± 0.7 pg/ml; P < 0.001), with a persistent increaseof IL-12 levels after 3 days (8.8 ± 2.6 pg/ml).

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FIG. 1. Levels of Th1 cytokines, IL-12 and IFN- ${gamma}$ , in sera from 20 patients with uncomplicated P. falciparum malaria and 15 healthy control subjects at day 0 and day 3. The horizontal line within the box stands for the median value; the box includes the 25th and 75th percentiles and the extended T-line stands for standard deviation.

Furthermore, a significant increase of IFN- ${gamma}$ levels was onlyobserved on admission (39.2 ± 67.6 pg/ml; controls, 8.4±6.3 pg/ml; P = 0.0285), whereas IFN- ${gamma}$ levels returnedto normal levels after 3 days (7.0 ± 5.6 pg/ml).

Figure 2 illustrates levels in serum of Th2 cytokines, IL-4and IL-10. As shown, IL-4 levels were not increased on admission(3.4 ± 1.2 pg/ml; controls, 2.4 ± 0.8 pg/ml),but 3 days later, a moderate and significant increase was observed(4.5 ± 1.7 pg/ml; P = 0.0006). In addition, a nonsignificantincrease of IL-10 was observed at days 0 and 3 (306.6 ±200.4 and 56.6 ± 38.4 pg/ml, respectively; controls,17.4 ± 3.0 pg/ml).

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FIG. 2. Levels of Th2 cytokines, IL-4 and IL-10, in sera from 20 patients with uncomplicated P. falciparum malaria and in 15 healthy control subjects at day 0 and day 3. The horizontal line within the box stands for the median value; the box includes the 25th and 75th percentiles and the extended T-line stands for the standard deviation.

We also determined a correlation between levels of Th1 cytokines,IL-12 and IFN- ${gamma}$ , in serum; levels of Th2 cytokines, IL-4 andIL-10; and parasitemia. As shown in Fig. 3, parasitemia showedan association only with levels of IL-12 or IFN- ${gamma}$ levels (r =0.60; P < 0.001).

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FIG. 3. Correlation between Th1 cytokines, IL-12 and IFN- ${gamma}$ , and Th2 cytokines, IL-4 and IL-10, and parasitemia on admission in 20 patients with uncomplicated P. falciparum malaria. Correlation coefficients (r²) and their statistical significance (P) are also indicated.

DISCUSSION

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Discussion

The present study demonstrates a Th1-driven immune responseby IL-12 and IFN- ${gamma}$ in adult patients with uncomplicated P. falciparummalaria, whereas the response of Th2 cytokines, IL-4 and IL-10,seems to be not involved during the acute phase of the disease,and these decrease in accordance with improvement and recoveryof the disease. More recently, we have demonstrated elevatedlevels of IL-18 in adult patients with uncomplicated P. falciparummalaria, and these levels decrease in accordance with improvementof and recovery from disease (12). Walker et al. (13) have shownthat IL-18 in combination with IL-12 enhanced the rate of transcriptionof the IFN- ${gamma}$ gene. Increased levels of IL-18, a proinflammatoryTh1 cytokine, along with IL-12 in uncomplicated malaria maybe crucial to stimulate effective production of IFN- ${gamma}$ .

Thus, IFN- ${gamma}$ represents a key molecule in human antimalarial hostdefense, as recently demonstrated by Winkler et al. (14) inpatients with uncomplicated P. falciparum malaria. These investigatorshave shown a more pronounced Th2-driven immune response duringacute untreated and uncomplicated malaria caused by P. falciparum,with a shift towards Th1 responsiveness induced by parasiteclearance.

It is interesting that patients with uncomplicated P. falciparummalaria and higher production of IL-18 at days 0 and 3 had increasedlevels of IFN- ${gamma}$ (12). It is conceivable to think that an earlyproduction of Th1 cytokines such as IL-12, IFN- ${gamma}$ , and IL-18 iscrucial in a better response and resolution of malaria infection.

In the murine model of infection with P. chabaudi AS, a Th2-dominatedimmune response has been demonstrated to be crucial in preventingrecrudescent malaria during the course of disease (10). Successively,Taylor-Robinson and Phillips (11) have shown that in the samemurine model of infection, the dose of antigen may affect thebalance between Th1- and Th2-mediated immune functions; increasingthe infective dose in a susceptible mouse strain led to up-regulationof Th2 cytokine (IL-4) production, whereas that in a resistantmouse strain enhanced the Th1 cytokine (IFN- ${gamma}$ ). Our study demonstratesup-regulation of the Th1 response through increase of IL-12and more importantly through increase of IFN- ${gamma}$ .

A more favorable outcome in animal models of malaria has beenassociated with increased production of IFN- ${gamma}$ (3, 6), and treatmentof lethal murine P. vinckei infection with IFN- ${gamma}$ enhanced theeffect of antimalarial treatment (4). It is intriguing to postulatethat inability to mount a Th1 response, and to maintain an adequatelybalanced immune response may underlie the progression from uncomplicatedP. falciparum malaria to severe, life-threatening complications.

In conclusion, the results of this study suggest a role of Th1cytokines, IL-12 and IFN- ${gamma}$ , in uncomplicated P. falciparum malariaas a proinflammatory Th1 cytokine, since the elimination ofintracellular pathogens, including P. falciparum, requires activationof Th1 cells with consequent cytokines, mainly IFN- ${gamma}$ , to initiatean early and prompt cell-mediated immune response.

FOOTNOTES

* Corresponding author. Mailing address: Department of Infectious Diseases, Regional Hospital, Viale Borri 57, 21100 Varese, Italy. Phone: 039-0332-278447. Fax: 039-0332-278580. E-mail: eiwle{at}tin.it.

REFERENCES

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