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Inducible nitric oxide synthase (iNOS) is one of the three distinct isoenzymes responsible for the catalytic oxidation of the terminal guanidino nitrogen atom of L-arginine, yielding citrulline and the short-lived reactive free radical NO in various nucleated cells. iNOS, which was originally identified in macrophages, is a cytosolic Ca²⁺-independent enzyme whose transcription is induced by microbial products or inflammatory cytokines (23, 24, 38). NO produced from monocytes/macrophages by the enzymatic action of iNOS has been implicated in mediating cytotoxicity during host defense reactions. A unique feature of iNOS is that when it is triggered by an immunologic or inflammatory stimulus, a sustained production of NO results. Since the initial observation by James and Glaven (20) that NO has schistosomicidal activity, its antimicrobial functions in a variety of microorganisms have been confirmed with both humans and animal models (11, 18, 19, 33).

The past decade has witnessed an explosion of interest in the role of NO in falciparum malaria; however, there is currently no consensus on the clinical significance of NO in falciparum malaria. Some authors have associated NO with severe and complicated malaria, particularly cerebral malaria (1, 3, 6, 9, 10, 12, 13, 29), whereas other authors have argued that NO has a protective role (2, 4, 21, 22, 26, 34-37). An interesting contradiction concerning the role of NO in malaria emerged from two recent studies. Kun et al. (22) demonstrated an association between polymorphism in the promoter region of the iNOS gene and protection from severe malaria in a Gabonese population. However, observations by Burgner et al. (6) of a Gambian population are somewhat different. Data from this study suggest that similar regions of iNOS genes encode a susceptibility determinant for fatal cerebral malaria.

To investigate these apparently contradictory reports, we studied the expression of iNOS in whole-blood RNA samples of patients with falciparum malaria and attempted to relate the transcription of this gene to the outcome of the disease. Since NO is a short-lived free radical, its measurement is difficult (5). To circumvent this obstacle, we indirectly assessed its role in malaria by measuring iNOS, an enzyme responsible for the sustained synthesis of NO in large amounts.

Characteristics of the study subjects. The patients included in this study were admitted to the Medical Department of the Bernhard Nocht Institute for Tropical Medicine with a confirmed diagnosis of falciparum malaria. This diagnosis was based on identification of asexual forms of the parasite in peripheral blood smears subjected to thin or thick staining. The percentage of parasitemia was calculated from the number of infected erythrocytes per 1,000 erythrocytes in thinly stained film. When parasites were found only in the thickly stained film, parasitemia was defined as below 1% (less than 50 parasites per thickly stained film field). The study population was comprised of 24 patients (age, 40 ± 18 years). After written informed consent was obtained from the patients and/or their immediate relatives, peripheral blood samples were drawn before treatment from (i) 12 patients with severe and complicated falciparum malaria, (ii) 12 patients with uncomplicated falciparum malaria, and (iii) 12 healthy medication-free donors (age, 32 ± 12 years) with no previous history of malaria, who were evaluated as controls. All patients who had severe and complicated falciparum malaria were of European origin. Of the 12 patients with uncomplicated falciparum malaria, 4 were of European origin and 8 were African immigrants from either western or eastern Africa who had been living in Germany for at least 2 years. All patients presented with fever lasting between 1 and 5 days and had contracted their malarial infection in Africa. All patients included in this study had not spent more than 4 weeks in Africa when they contracted their malarial infection and had no evidence of concomitant infections. None of the patients had received antimalarial treatment before our administration. Patients were defined as semi-immune if they were raised in a malarious area and had lived in a nonmalarious area for a period not exceeding 5 years. Nonimmune patients were patients who were raised in a nonmalarious area and had lived in a malarious area for a period not exceeding 2 years. Nonimmune patients included in this study had no previous history of malaria. Patients were described as having severe and complicated malaria if they met one of the following criteria: (i) impaired cerebral function (disorientation, drowsiness, unconsciousness), (ii) pathological global clotting tests (prothrombin time activity, <50%, and partial thromboplastin time, >45 s), (iii) impaired renal function (creatinine in serum, >2.0 mg/dl), (iv) respiratory insufficiency (partial pressure of oxygen, <60 mm Hg), (v) hepatic damage (alanine aminotransferase and aspartate aminotransferase levels, 100 U/liter), and (vi) parasitemia (5%) (7).

Antiparasitic therapy. Patients with severe and complicated malaria received the standard regimen of quinine (20 mg/kg of body weight/day) and doxycyline (100 mg/day) for 10 days. Patients with uncomplicated falciparum malaria received mefloquine (20 mg/kg/day) in three doses taken 6 h apart). Therapy was initiated immediately after confirming the diagnosis of malaria with blood smears on admission.

RNA isolation from whole blood. Heparinized blood was drawn with minimum venostasis, and RNA was isolated with TRI Reagent BD (Molecular Research Center, Inc., Cincinnati, Ohio) according to the manufacturer's specifications.

Isolation of monocytes, lymphocytes, neutrophils, and platelets. Peripheral blood mononuclear cells were isolated by Ficoll-Paque (Pharmacia, Freiburg, Germany) density gradient centrifugation. Monocytes were isolated by adherence to 75-ml plastic culture flasks (Costar). The nonadherent lymphocytes were removed by aspiration. Neutrophils were isolated by the method of Ross and Densen (30) as modified by Estabrook et al. (16). Briefly, heparinized blood from patients was sedimented in an equal volume of 3% dextran T-500 (Pharmacia) in 0.85% NaCl at room temperature for 25 min. The leukocyte-rich supernatant was removed by centrifugation at 200 × g for 10 min at 4°C. The supernatant was layered on Ficoll-Paque (Pharmacia) and centrifuged at 250 × g for 20 min. Heparinized blood drawn with minimum venostasis at times of admission was collected into a 1/10 volume of 3.8% sodium citrate and then centrifuged at 400 × g for 20 min at room temperature. Platelet-rich plasma was collected and centrifuged for 20 min at 1,000 × g to sediment platelets. The resulting pellet was resuspended in modified Tyrode's buffer (140 mM NaCl, 5 mM KCl, 10 mM glucose, 15 mM HEPES, 0.38 g of bovine serum albumin [pH 7.4] per 100 ml), and the platelets were subsequently washed three times with the buffer. The levels of purity of cells were 96% for monocytes, 96% for platelets, 98% for neutrophils, and 98% for lymphocytes as determined by differential cell counting.

RNA isolation from monocytes, lymphocytes, and neutrophils. Total cellular RNA was prepared with TRIZol reagent (Life Technologies) based on the acid guanidinium thiocynate-phenol-chloroform method of Chomcyznski and Sacchi (8) as modified by the manufacturers.

RT-PCR, slot blot analysis, and cloning and sequencing of RT-PCR products. The expression of iNOS was measured by amplification by reverse transcription-PCR (RT-PCR). This approach was necessary, as the available amounts of RNA were too small for analysis by either Northern blotting or ribonuclease protection assays. Prior to RT, possible chromosomal DNA contaminants were degraded by treating the total RNA samples with 10 U of DNase I (GenHunter Corp., Nashville, Tenn.) for 30 min at 37°C, followed by phenol extraction, ethanol precipitation, and finally dilution in RNase-free water. In batched and parallel reactions, equal amounts of total cellular RNA from each control were subjected to RT with SuperScript II RNase H reverse transcriptase (Gibco BRL Life Technologies) according to manufacturer's specifications. In a 12-µl reaction mixture, 1 µg of RNA and 1 µg of oligo(dT) (0.5 g/µl) were heated for 10 min at 70°C and immediately chilled on ice for 5 min. The reaction mixture was preincubated for 5 min at 42°C. The mixture contained 4 µl of 5× First Strand Buffer (250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM MgCl₂), 1 µl of a deoxynucleoside triphosphate mix (10 mM), 2 µl of 0.1 M dithiothreitol, and 200 U of Superscript reverse transcriptase. After preincubation, the mixture was incubated at 42°C for 50 min and the reaction was terminated by heating the mixture at 95°C for 5 min and chilling it on ice. cDNAs were subjected to enzymatic amplification with primers specific for human iNOS (Clontech) and reagents from a GeneAmp kit (Perkin-Elmer). The 35 cycles of PCR were performed under the following conditions as recommended by the manufacturer: denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 2 min; the last cycle extension was performed at 72°C for 7 min in a Biometra UNO-Thermoblock. To assess the integrity of the starting RNA as well as to control for variability in RNA or cDNA handling during the RT-PCR method and as the standard for semiquantitative comparisons, the levels of -actin RNA transcripts were determined simultaneously for every PCR sample with primers specific for -actin (Clontech) according to the manufacturer's instructions. After completion of the PCR, the amplified DNA was recovered with QIAquick (Qiagen, Hilden, Germany). Eight microliters of each of the recovered iNOSs and 8 µl of the respective coamplified -actin PCR products were denatured by heating at 95°C for 4 min in 190 µl of 0.4 M NaOH containing 25 mM EDTA. Samples were rapidly cooled on ice and then blotted directly onto a Nylon membrane (Hybond-N⁺) (Amersham, Braunschweig, Germany) presoaked in 20× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA [pH 7.7]) in a slot blot apparatus (Minifold II; Schleicher & Schuell, Dassel, Germany). Each well was washed with 400 µl of 20× SSPE. After transfer, the DNA was covalently linked to the nylon membrane by exposure to UV light. Human iNOS and -actin cDNA probes obtained by PCR were radiolabeled by random primer extension with [-³²P]dCTP. The blots were prehybridized for 4 h in hybridization solution at 42°C, hybridized overnight with labeled cDNA, and washed as described previously (31). The level of hybridization for ³²P-labeled cDNA probes was visualized by autoradiography with Kodak XAR film and quantitated with a PhosphorImager and the ImageQuant program (Molecular Dynamics, Sunnyvale, Calif.). To confirm that the amplified PCR product represented the iNOS mRNA, we eluted the PCR product and cloned it using a TA cloning kit (Invitrogen) according to the manufacturer's instructions and determined the nucleotide sequence by the dideoxynucleotide chain-termination method (32) using a Sequenase kit (version 2.0; United States Biochemicals). Analysis of the sequence of the PCR-amplified DNA fragment revealed that bases 568 through 3459 were identical to the previously described sequence (17).

Statistical analysis. Statistical analysis was performed by Student's t test with the STAT software package from SAS Institute Inc., Cary, N.C. All test results were judged to be significant at a P of <0.05.

View larger version (7631K): [in this window] [in a new window]   FIG. 1.   (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to Hybond-N+ membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to -actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of -actin). Samples are shown in the same order as in panel a.

View larger version (6223K): [in this window] [in a new window]   FIG. 2.   (a) Comparison of levels of expression of iNOS mRNAs in different peripheral blood cells. Total RNA (1 µg) from lymphocytes, neutrophils, platelets, monocytes, and whole blood was reverse transcribed with an oligo(dT) primer. Five percent of the product was amplified for 35 cycles with primer pairs specific for human iNOS or -actin iNOS. Eight microliters of each PCR product was slot blotted, hybridized, and visualized by autoradiography. (b) Ratios of iNOS gene expression to -actin gene expression. Densitometric values were determined from the blot shown in panel a and are presented as iNOS mRNA levels (percentages of -actin). Samples are shown in the same order as in panel a.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Comparison of absolute monocyte counts from patients with severe and complicated malaria, nonimmune patients with uncomplicated malaria, semi-immune patients with uncomplicated malaria, and healthy donors. Solid lines indicate the median absolute monocyte count for each group. The mean absolute monocyte count for patients with uncomplicated malaria reflected a P of <0.001.