Background
The most characteristic presentations of acute
Plasmodium vivax infections are the periodic episodes of fever with chills and rigors, which follow the rupture of schizont-infected erythrocytes in the patients' circulation. Previous investigations have been made on events associated with paroxysms in
P. vivax infections including the inactivation of sexual stages of the parasites (gametocytes) in the presence of plasma taken at the time of a paroxysm [
1,
2]. These studies have demonstrated the roles in this process of the human cytokines TNF-α, GM-CSF and IL-2 together with parasite products. Previous studies also demonstrated the prominent rise and fall of TNFα – levels which corresponds very closely to the rise and fall of fever during the paroxysms [
3] and of the transient appearance of elevated numbers of gamma/delta T cells in the peripheral circulation at the time of these events [
4].
In the present study, the aggregation of peripheral blood white cells was investigated in the presence of plasmas taken at the time of a P. vivax paroxysm. These cell aggregations were formed only in the presence of paroxysm plasma and not in the presence of plasma from before or after a paroxysm, or from a healthy donor. Further, this paroxysm plasma-mediated cell aggregation phenomenon was exploited as an assay to identify and characterize biologically active mediators of host and parasite origin during the paroxysms of P. vivax malaria. This assay enabled preliminary chemical and physical-chemical characterization of parasite-derived products present and active during a P. vivax paroxysm.
Methods
Patients
Two study groups were included. They were patients with P. vivax infections from two localities in Sri Lanka. Those from the first were adult residents of malaria-non-endemic regions attending the National Hospital of Sri Lanka, in Colombo. These patients were residents of Colombo and its suburbs where there is no local transmission of malaria; they had acquired their infections following travel to a malaria-endemic area of Sri Lanka. These patients (except six patients who had one past infection) had no previous recorded malarial infections and were immunologically naïve (non-immune) with respect to malaria.
The second group of patients comprised adults in a
P. vivax-endemic region of Sri Lanka at Kataragama [
5]. The patients were similarly diagnosed as having
P. vivax infections by blood smear examination. All these individuals had experienced several previous
P. vivax infections (8–28 infections; median 18), as revealed during verbal interview and also based on the case records. The clinical symptoms of paroxysms were invariably milder in these patients from Kataragama (most did not experience rigors) than in non-immune patients from Colombo; these patients were considered as clinically semi-immune with respect to malaria [
6].
Control groups
Two age and sex matched control groups were included. One group consisted of healthy volunteers residing in areas with no local malaria transmission and with no past history of malaria infections. The second control group included healthy individuals resident in Kataragama (a malaria-endemic area). All these individuals were blood film-negative for malaria parasites.
Cell samples
Five ml of blood samples were collected from apparently healthy individuals residing in Colombo who were blood film-negative for malarial parasites and who did not give a past history of malaria. Blood was collected (and diluted) into a solution containing Tris-1.2 mg/ml (Biorad), NaCl – 8.2 mg/ml (Sigma) and Glucose – 1.8 mg/ml (Sigma) at pH 7.4 at 10% concentration [
2]. Whole blood cells were separated by centrifugation at a speed of 800 × G for 10 minutes. The supernatant containing the diluted plasma was discarded. The upper 40% of the cell pellet, mainly consisting of white blood cells, was pipetted out. This was mixed gently to make a homogenous cell suspension.
Plasma samples
Following identification of the infection by blood smear, informed volunteers completed a single paroxysm without drug treatment. The time of paroxysms was predetermined by the increasing oral temperature (above 37.8°C) and the presence of chills in most cases and supported by examination of the blood smears made from these patients. From the onset to the end of fever paroxysms lasted 4 – 5 hours. Two milliliters of blood were collected for plasma prior to the onset of paroxysm (pre-paroxysm plasma samples) into 0.1% EDTA containing a protease inhibitor (aprotinin; 0.6 trypsin inhibitor U/ml). A second set of samples (paroxysm plasma) was collected at, or within one hour of, the peak of a rigor and a third set, four to six hours after the fever peak (post-paroxysm plasma). Serum samples were collected from P.vivax- infected patients, four to six weeks after drug cure (convalescent serum).
Another set of plasma samples was collected from fever patients admitted to the National Hospital of Sri Lanka with oral temperatures above 37.8°C and blood film-negative for malarial parasites when tested at least twice on consecutive days (non-malarial fever plasma).
Control plasma samples were collected from healthy volunteers (normal human plasma).
Leukocyte aggregation
Whole blood cells were obtained from blood samples collected from apparently healthy individuals. 80 μl of cell suspension rich in white blood cells (obtained as described under cell samples), was pipetted into 800 μl of RPMI 1640 [
1] in a 24-well tissue culture plate (Flow Laboratories, UK) and 200 μl of test (paroxysm plasma, pre-paroxysm plasma, post-paroxysm plasma, non-malarial fever plasma or plasma from clinically semi-immune endemic patients) or control plasma samples (normal human plasma) were added into the cell suspension. The culture plate was incubated for 3 hours at 37°C in an incubator. The supernatant was discarded and from the remaining cell pellet 40 μl was pipetted out and dispersed evenly on a microscopic slide, in a circle of 1.5 cm diameter using a template. These blood smears were air dried, stained with Giemsa stain and examined under 400× magnification.
Cell aggregation index
Blood smears were prepared from cultures containing cells from healthy individuals incubated with test or control plasma samples as described above. Nucleated cell aggregates containing five or more cells and the number of un-clumped cells (that included single nucleated cells and clumps of less than five cells) were counted in 100 microscopic fields and the total number of the nucleated cells in these fields were also noted.
Based on these counts, a 'cell aggregation index' (CAI), indicative of the ability of a particular plasma sample to induce aggregation of white blood cells, was calculated as follows:
CAI = ([Total no. of white blood cells/(No. of clumps + no. of un-clumped white cells)] - 1), in 100 microscopic fields at 400× magnification
% Relative Cell Aggregation Index (RCAI) = [CAI of test sample/CAI of paroxysm plasma] × 100
Characterization of cell types involved in inducing cell aggregation
Monocytes
Monocyte depletion was done using two methods:
Method A
80 μl of cell suspension was diluted with 80 μl of a buffer solution (containing Phosphate Buffered Solution/Foetal Calf Serum at 1:1 ratio). This cell suspension was added (at1:1 ratio) to a suspension of magnetic polystyrene beads coated with a primary monoclonal antibody specific for the CD14 membrane antigen (Dynabeads M-450-CD14, Dynal A.S., Oslo, Norway). This mixture was then incubated for twenty minutes at 4°C. After incubation, the tube containing the cell suspension was placed in the magnetic particle concentrator (Dynal, MPC-1, Dynal A.S. Oslo, Norway) which has a magnetic field to attract the antibody coated Dynabeads together with the attached cells. The remaining cells were pipetted out and this step was repeated five times. The remaining cell suspension (depleted of CD14 monocytes) was used for the relevant experiments. Absence of monocytes from the cell suspension was confirmed using blood smears which were made on glass slides, stained and examined under the microscope.
Method B
80 μl of cell suspension was depleted of monocytes using the plate-adherent method described previously [
7]. Briefly, the white cell-rich cell suspension was incubated at 37°C for 30 minutes in a polystyrene petri dish (size: 35 × 10 mm, Becton Dickinson and Co.) at 10% cell to liquid volume in RPMI 1640. Then the supernatant containing the non-adherent cells was carefully removed and the cells were spun down.
Monocyte-depleted cells thus obtained by each method were resuspended and used in the cell aggregation assay as described above.
Monocyte reconstitution
For reconstitution experiments, a preparation of mononuclear cells was obtained from whole blood by sodium metrizoate density gradient centrifugation (Lymphoprep, Norway) according to standard procedure [
1]. The mononuclear cells were resuspended in RPMI 1640 pH 7.4 at 10% concentration and introduced into polystyrene petri dishes (size: 35 × 10 mm, Becton Dickinson and Co.). 1 ml volumes at 2 × 10
7/ml concentrations were incubated at 37°C for half an hour [
7]. After incubation, the cell suspension was pipetted out. The cells adhered on the surface of the plate were scraped out with the rubber end of a 1 ml syringe plunger and a cell count was taken using a haemocytometer. Cells thus obtained (monocytes) were used for reconstitution experiments at a concentration of 6 × 10
6 cells per well.
T cells
T cell depletion:T cells were depleted from 80 μl of cell suspension by following the same procedures as adopted in the case of macrophages, using Dynabeads coated with antibodies specific for CD2 membrane antigen (Dynabeads-CD2, Dynal A.S., Oslo, Norway). The remaining (T cell-depleted) cell suspension was used for relevant experiments.
Identification of factors mediating cell aggregation
(i) Parasite factors
Effect of neutralization of parasite factors on paroxysm plasma-induced cell aggregation
Serum containing anti-parasite antibodies i.e. hyper-immune rabbit serum raised against freeze-thawed extracts of either
P. vivax or
P. falciparum [
2] or with human convalescent serum was pre-incubated with paroxysm plasma at 1:1 of concentration for 30 minutes before incubating with un-primed whole blood cells collected from healthy individuals.
Effect of re-constitution of parasite factors in normal human plasma treated with freeze-thawed P. vivax schizont extracts
Normal human plasma obtained from healthy individuals were reconstituted with freeze-thawed extracts of 5 × 10
6 P. vivax schizonts per ml or 5 × 10
6 of uninfected red blood cells per ml as a control prepared as previously described [
1]. This re-constituted plasma was incubated with unprimed whole blood cells of healthy individuals.
As a control, normal human plasma was reconstituted with
E. coli lipopolysaccharide (LPS) at a final concentration of 2 μg/ml and tested in cell aggregation assay as previously described [
1].
(ii) Host factors
Cytokines
Involvement of cytokines in the mediation of cell aggregation by paroxysm plasma was investigated by their neutralisation with anti-cytokine antibodies and reconstitution with the addition of recombinant human cytokines. The optimum concentrations of anti-cytokine antibodies and recombinant cytokines required to overcome the neutralization imposed by the antibodies were determined based on experiments using dilution series of these, using the same principles and methodologies adopted in previous studies [
8].
Depletion and reconstitution of plasmas with specific components
Depletion experiments
Paroxysm plasmas were pre-incubated at 37°C for 30 minutes with the following immune reagents, singly or in combination, before assessing the effect on paroxysm plasma-induced cell aggregation.
(i) Rabbit polyclonal antibodies (IgG) against human IL1-α [0.014 μg/ml], IL1-β [0.075 μg/ml], IL-2 [1.5 μg/ml], IL-3 [5 μg/ml], IL-4 [0.125 μg/ml], IL-6 [0.1 μg/ml], IL-10 [7.5 μg/ml], IFNγ [5 μg/ml], TNF-α [0.04 μg/ml], TNF-α [0.05 μg/ml] and GM-CSF [5 μg/ml] (R&D systems, UK).
Reconstitution experiments
Recombinant human cytokines (IL1-α [5 pg/ml ], IL1-β [7.5 pg/ml], IL-2 [0.375 ng/ml], IL-3 [0.25 ng/ml], IL-4 [0.125 ng/ml], IL-6 [0.5 ng/ml], IL-10 [0.75 ng/ml]), IFNγ [1.15 ng/ml ], TNF-α [0.05 ng/ml], TNF-α [0.03 ng/ml] and GM-CSF [0.05 ng/ml] were added to cytokine-depleted paroxysm plasma to confirm the involvement of cytokines in paroxysm plasma-induced cell aggregation. Recombinant human cytokines were also added to normal human plasma or post paroxysm plasma in other experiments. Freeze-thawed extracts of 5 × 10
6/ml
P. vivax schizonts or 5 × 10
6/ml of uninfected red blood cells as a control (prepared as previously described, [
1]) were added to plasmas in relevant experiments.
The physical/chemical characterization of plasma factors of putative parasite origin that mediate leukocyte aggregation.
Heating
Paroxysm plasma and normal human plasma, as a control, were heated in 1 ml volumes in a water bath at 60°C, 80°C or 100°C for five minutes before testing in the cell aggregation assay. The heat-treated fraction of paroxysm plasma, was reconstituted either with an extract of P. vivax schizonts or with recombinant human cytokines (rh TNF-α, rh GM-CSF, rh IL-10, rh IL-6) and tested for cell aggregation.
Ultra-centrifugation
Paroxysm plasma and normal human plasma were centrifuged in 1 ml volumes at 180,000 × G for 15 minutes at 4°C. Two layers were formed. The thin opaque, whitish upper layer was removed in 100 to 200 ul. The remaining liquid was yellowish but clear and was retained as a single fraction. The two fractions were tested separately and reconstituted with recombinant human cytokines (rh TNF-α, GM-CSF, IL-10, IL-6) or with
P. vivax schizont extract and re-tested in the cell aggregation assay. The separated fractions were also tested for lipids (standard Sudan III test) [
9].
Filtration
1 ml samples of paroxysm plasma or normal human plasma were passed through 0.45 μm millipore filters (Flow laboratories, UK) and the filtrates were tested in the cell aggregation assay. The filtrates were re-tested in the cell aggregation assay following reconstitution with recombinant human cytokines (rh TNF-α, GM-CSF, IL-6, IL-10) or P. vivax schizont extracts.
Extraction and fractionation of lipids
1 ml samples of paroxysm plasma, or 1 ml of normal human plasma (NHP) as a control, were heated at 100°C for five minutes in a water bath. Freeze-thawed extracts of
P. vivax schizonts at 5 × 10
6 schizonts in 1 ml of NHP were also heat-treated in the same way. The following extractions were then carried out, all at room temperature, using standard biochemical techniques [
10‐
12]. Briefly, each of the heat-treated plasma samples was extracted first with 1 ml of acetone, centrifuged for 10 seconds at × 15,000 × G and the supernatant retained as fraction 1 (cholesterol, triglycerides); the pellet was extracted with 1 ml of 95% ethanol, spun as before and the supernatant retained as fraction 2 (lecithin); the pellet was extracted in 1 ml of petroleum-ether, spun as before and the supernatant retained as fraction 3 (composition unidentified). The pellet obtained, and half of the retained fraction 3, were added together and extracted with 1 ml of diethyl-ether, spun as before, and the supernatant retained as fraction 4 (phospholipids) and the pellet as fraction 5 (sphingolipids). Each of these fractions was evaporated to dryness by flushing with nitrogen gas. The residues were dissolved in 1 ml either of normal human plasma or of normal human plasma reconstituted with recombinant human cytokines (rh TNF-α, GM-CSF, IL-6 & IL-10) as described above and then tested in the cell aggregation assay.
Statistical analysis
As the data dispersion was found to be not normal, Mann-Whitney U test was applied to compare two groups and Kruskal – Wallis test was applied when there were more than two groups to be compared and the post hoc significance was analysed. Median values were given with inter-quartiles (as 25%–75% percentiles). (statistical software package – SPSS 10.0 for Windows).
Ethical clearance
All aspects of the study were approved by the Ethical review committee of Faculty of Medicine, University of Colombo, Sri Lanka. Informed-written consent was obtained from all participants.
Discussion
The present study describes an
in vitro phenomenon, the aggregation of white blood cells, which is mediated by the activity of factors present in plasma taken at the time of a paroxysm of
P. vivax malaria. In the presence of paroxysm plasma from non-immune
P. vivax-infected patients, aggregates were formed by white blood cells collected from healthy uninfected and malariologically-naive individuals. Similar aggregates were also seen when cells were obtained from
P. vivax-infected patients and similarly incubated with paroxysm plasma. Although the biological relevance of such aggregate formation remains speculative, it might be a scavenging mechanism used by the host to get rid of the parasite debris released following schizont rupture [
13].
Leukocyte activation associated with fever episodes has been shown in
P. vivax malaria [
14]. Peripheral leukopenia in
P. vivax infections has been observed to be at its maximum during paroxysms (authors' unpublished observations). Pulmonary accumulation and sequestration of neutrophils and monocytes have been seen in murine models [
15,
16]. Furthermore, pulmonary oedema has been reported in
P. vivax malaria [
17], which may be due to accumulation of leukocytes. In
P. falciparum malaria, cyto-adherent infected erythrocytes that bind to leukocytes enhance antibody-independent phagocytosis and induce cellular aggregation [
18]. Pancytopaenia, especially thrombocytopaenia is common in malaria patients [
19,
20]. These findings provide indirect evidence as to the possible
in vivo significance of such aggregate formation in malarial infections.
The active plasma factors, which mediated the cell aggregation included heat-labile substances consisting of mainly the monocyte-derived cytokines TNFα, GM-CSF and IL-6 and a T cell-derived cytokine IL-10. However, the activity of these cytokines in mediating leukocyte aggregation was absolutely dependent upon the simultaneous presence of heat-stable, and presumably parasite-derived, material released into the plasma at the time of a
P. vivax paroxysm. It has been reported that aggregation of neutrophils may develop in EDTA – anti-coagulated blood [
21]. However, in the present study EDTA-dependent aggregation of white blood cells can be excluded based on the experiments carried out using normal human plasma samples obtained from healthy individuals into similar EDTA concentrations, which did not show aggregation. Moreover, immune serum raised against
P. vivax parasites could reverse this effect.
Ever since the description, at the end of the 19
th century, of the synchronous rupture of blood stage schizonts during human malarial infections and the association of these events with the periodic fevers of malaria, it has been clear that parasite products released into the plasma by the rupturing schizonts must be crucial to the initiation of a malarial paroxysm [
22]. Evidence for the existence of malaria paroxysm-associated pyrogens, or "malaria toxins" as they were known, was provided by demonstrating that plasma from an individual undergoing a paroxysm of
P. vivax malaria could rapidly induce equivalent symptoms when the plasma was injected into the circulation of a healthy volunteer [
23]. Thus the concept of a malaria toxin is not novel [
2,
13,
22‐
26]. It is now evident that the mediators of a malarial paroxysm, which include the parasite products, or "toxins", themselves, are involved in interactions with and between circulating white blood cells.
In addition to cytokines, the activated white blood cells release free radicals and other active molecular intermediates [
27,
28]. These mediators of inflammatory and pathogenic effects and also anti-parasitic effects [
29‐
33] are speculated to help control parasite densities at the initial phase of a malarial infection. Inhibition of the maturation of
P. falciparum schizonts
in vitro by paroxysm serum from a case of
P. vivax malaria has been demonstrated by other workers [
33]. Similar anti-parasitic effect of paroxysm plasma from acute
P. vivax-infected patients has been demonstrated to induce suppression of infectivity of malarial gametocytes to mosquitoes [
1]. Like the cell aggregation effect described here, paroxysm-associated inactivation of gametocytes is mediated by cytokines and parasite products released at the time of the paroxysm [
1,
2,
13]. In both the cell aggregation and the gametocyte inactivation phenomena, however, the parasite materials and moieties involved remain to be identified.
This paper describes the initial attempts made at characterization of the nature of these active parasite products. It is shown here that the heat-stable, cell-aggregating activity present in the paroxysm plasmas has the following properties. It is present in, and only in, plasmas taken at the time of the acute symptoms of a P. vivax paroxysm. No equivalent activity is present either before the acute symptoms begin or more than one or two hours after they cease. No equivalent activity is found in association with non-malarious fevers. The white cell-aggregating activity can be effectively substituted by extracts of schizonts of either P. vivax or P. falciparum when these are added to normal human plasmas in the presence of the cytokine combination identified above. Moreover, the activity in the paroxysm plasmas from acute P. vivax-infected patients is neutralised in the presence of immune sera raised against extracts of schizonts of either P. vivax or P. falciparum. The activity is more effectively neutralised by immune serum against P. vivax than that against P. falciparum, and is supportive evidence of parasite species-specificity.
These properties of the putative parasite-derived activity in
P. vivax paroxysm plasma in relation to white cell aggregation are indistinguishable from those identified in relation to paroxysm plasma-mediated gametocyte inactivation [
1,
2,
13,
34]. The difference between the mediators involved in these two paroxysm-associated phenomena lies in the combination of cytokines involved. Thus the necessary and sufficient cytokines involved in gametocyte inactivation are the monocyte-derived cytokines TNFα, GM-CSF and the T cell derived IL-2; those involved in the white cell aggregation are principally the monocyte-derived TNFα, GM-CSF and IL-6 and the mainly T cell derived cytokine IL-10. In both phenomena the immediate presence of monocytes was shown to be essential. On the other hand, the depletion of T lymphocytes did not affect cell aggregation. This does not, however, exclude a role for T cells at an earlier stage in the events of a paroxysm as, for example, during the induction of other cytokines involved in the process. This is probably because, the T cell-derived cytokines having already been induced by the time of the active paroxysm. Therefore, the continued immediate presence of the T cells is no longer essential for either gametocyte inactivation or white cell aggregation to take place. Though, T cells strongly respond to phosphoantigens from
Plasmodium parasites [
35], prolonged exposure to
P. vivax malaria infections in endemic areas are known to cause immuno-suppression of human T cells [
36].
The cell aggregation phenomenon was used as an assay to explore the physical and chemical nature of the heat stable, and presumably parasite-derived, activity in the paroxysm plasmas from acute
P. vivax-infected patients. Results indicate a prominent role of cholesterol and triglycerides containing fraction of paroxysm plasma in mediating cell aggregation even in the absence of added cytokines. Only one other fraction gave significant activity. This was the phospholipid fraction and this activity was largely dependent upon the presence of the added cytokines. Equivalent lipidic fractions were made from extracts of schizonts of
P. vivax but significant, and largely cytokine-dependent, activity was found only in the phospholipid fraction. Interestingly the characteristics of this fraction were congruent with those of the malaria parasite-derived TNF-inducing GPI moieties studied by other workers [
37‐
43]. GPI surface-anchored antigens including MSP-1 have been viewed as potential candidates for vaccine development [
44,
45]. Studies in other laboratories have demonstrated lipogenesis-inducing activity in the lipid fractions of boiled supernatant of
P. falciparum cultures [
46] with subsets of T lymphocytes being affected at an early stage during paroxysms of non-endemic malaria infections [
35].
Authors' contributions
NK conceptualized and designed the study, interpreted the data and drafted the manuscript. DW carried out the laboratory experiments and data tabulation. VC provided advice on design, analysis and interpretation of biochemical characterization experiments. RC gave considerable intellectual input in analyzing the results and helped draft the manuscript and KM made contributions for interpretation of data and drafting the manuscript. All authors have given final approval of the version to be published. The research work was carried out at Malaria Research Unit, Faculty of Medicine, University of Colombo, Sri Lanka.