Plasmodium vinckei Infection - Infection and Immunity - American ...

3 downloads 0 Views 875KB Size Report
and blackwater fever. Blackwell Scientific Publications. Ox- ford. 19. Makimura, S., V. Brinkmann, H. Mossmann, and H. Fischer. 1982. Chemiluminescence ...
INFECTION AND IMMUNITY, Sept. 1984, p. 708-712 0019-9567/84/090708-05$02.00/0 Copyright C) 1984, American Society for Microbiology

Vol. 45, No. 3

Production of Luminol-Reactive Oxygen Radicals During Plasmodium vinckei Infection R.

STOCKER,'*

N. H. HUNT,2 I. A. CLARK,3 AND M. J. WEIDEMANN'

Departments of Biochemistry1 and Zoology,3 Faculty of Science, and John Curtin School of Medical Research,2 Australian National University, Canberra, A.C.T. 2601, Australia Received 3 February 1984/Accepted 24 May 1984

We tested the ability of whole blood and enriched fractions of peripheral blood polymorphonuclear leukocytes obtained from mice during the course of infection with Plasmodium vinckei to produce luminolmediated chemiluminescence in response to phagocytic and nonphagocytic stimuli. The chemiluminescence response of whole blood to all stimuli increased dramatically and nonlinearly as the infection progressed, and there was a concomitant increase (80%) and decrease (70%) in the total numbers of leukocytes and erythrocytes, respectively. The proportion of polymorphonuclear leukocytes in the total leukocyte population increased threefold. On a per cell basis and at a constant hematocrit, the chemiluminescence response of peripheral leukocytes from infected animals to phorbol myristate acetate or opsonized zymosan was only slightly greater than that of cells from uninfected animals. Polymorphonuclear leukocytes isolated from the blood of infected animals also showed no large increase per cell in chemiluminescence responsiveness. Thus, although leukocyte numbers increase during a murine malarial infection, there appears to be no major change in the capacity of individual peripheral blood leukocytes to produce activated species of oxygen. However, the physiological reduction in the total concentration of hemoglobin at high parasitemia, due to hemolysis and hemoglobin digestion by the parasites, increases the possibility of oxygen radical-mediated damage to tissues and intraerythrocytic parasites as a result of decreased antioxidant protection.

During the course of malarial infection, the humoral and cellular immune systems of the host become activated. Since the early in vivo observations of Taliaferro et al. (25, 26), phagocytosis of parasitized erythrocytes (PRBC) has been regarded as a part of the host's defence mechanism against this parasite. In vitro experiments with Plasmodium falciparum have shown that free merozoites and PRBC are phagocytosed by peripheral blood polymorphonuclear leukocytes (PMNs) (5, 28) and monocytes (15, 29), particularly when associated with immune serum. In mice, PRBC are also ingested in vitro by peritoneal (20, 24) and splenic (24) macrophages in the presence of immune serum. It has been suggested that macrophages release a soluble factor(s) that causes intraerythrocytic death of rodent malarial parasites (7, 10), and this principle has been argued to extend to clinical immunity against P. falciparum in humans (14, 14a). The possible involvement of oxygen radicals in the processes leading to intraerythrocytic death of malarial parasites is supported by the observations that oxygen radical-generating compounds such as alloxan (9), hydrogen peroxide (9, 12), and t-butyl hydroperoxide (8) reduce parasitemia significantly when injected into malaria-infected mice and that P. falciparum is susceptible to low in vitro concentrations of the latter agent (8). The ability of leukocytes (WBC), especially PMNs, monocytes, and macrophages, to produce activated species of oxygen upon stimulation is well known (2, 3, 6). The production of hydrogen peroxide by stimulated PMNs is initiated at the plasma membrane (21), thus releasing H202 extracellularly at points of contact between the cell surface and other cells (22). However, the extent and physiological significance of the production of activated species of oxygen by WBC in malaria-infected animals or humans is not *

known. The measurement of luminol-mediated chemiluminescence (CL) is a very sensitive method for detecting the production of activated species of oxygen by WBC (1). To find out whether WBC from the peripheral blood of malariainfected animals have an enhanced ability to produce luminol-reactive species of oxygen that might contribute to parasite killing and tissue damage, we examined the CL response of whole blood to different exogenous stimuli. The present report demonstrates that, concurrently with the progressive increase in parasitemia that occurs in murine malaria, there is a dramatic (up to 24-fold) increase in the production of luminol-reactive oxygen radicals of whole blood in response to phagocytic and nonphagocytic stimuli. This increased CL response can be largely explained by malaria-associated changes in the total numbers of erythrocytes (RBC) and WBC and in the proportion of the latter which are PMNs.

MATERIALS AND METHODS Animals and parasites. CBA/CaH mice of either sex, 6 to 10 weeks old, were used. The Plasmodium vinckei subsp. vinckei, strain V52, originally obtained from F. E. G. Cox, King's College, London, was stored at -196°C. The parasites had been passaged several times in CBA/CaH mice before use. Infections were initiated by intraperitoneal injection of 106 PRBC contained in 200 RI of blood diluted with phosphate-buffered saline (PBS). Blood samples. Heparinized blood (20 U/ml) from control or infected animals was collected from the subclavian arteries of anesthetized mice. Samples of whole blood were used for (i) monitoring parasitemia and differential counting of WBC, using thin smears; and (ii) determining the total RBC and WBC counts. Thin smears were stained with Harleco Diff-Quik Stain Set (AHS, Sydney, Australia), and 500 cells were counted to determine the differential WBC count. A sample of whole blood (10 p[l), diluted in 0.01% gentian violet

Corresponding author. 708

VOL. 45, 1984

LUMINOL-REACTIVE OXYGEN RADICALS

0

.1.

18

A23187

-

0

E

02

mg/ml), A218

th

4

5

o

.EPMA

MA(

10ito2 /f zyoaOSm/l,osnzdzymosan(P)(. A

20

40

80

60

100

% Parasitemia

FIG.

1.

CL response of whole blood from infected animals after

the addition of zymosan (0.5

mg/ml),

A23187

(5

p.M),

mg/ml), opsonized

or

PMA

(1

zymosan

(OPZ) (0.5

~LgIml). Experiments

were

performed as described in the text. CL signals obtained from infected blood are compared with the corresponding signal from control blood.

in 2% acetic acid (190 RI1),

was

counted with a hemacytome-

ter to determine the total WBC count.

PMNs. A PMN-enriched fraction was prepared as follows. Heparinized blood (0.8 to 1.0 ml) was layered over a preformed Percoll gradient (10 ml of Percoll; starting density, p = 1.100 g/ml; centrifuged for 15 min at 20,000 x g in a Sorvall SS-34 rotor) and centrifuged for 20 min at 500 x g. The PMN-enriched fraction (60 to 80% PMNs, 20 to 40% lymphocytes, 0% monocytes and macrophages) was removed, and the cells were washed once with PBS before counting. CL measurement. CL was measured at 37°C in a Packard Tricarb liquid scintillation counter operating in the out-ofcoincidence mode. All experiments were carried out in plastic scintillation vials containing a final volume of 2 ml. In a typical experiment, vials containing 1.8 ml of medium and luminol (1 mM) were dark adapted for at least 1 h at 37°C before a sample (100 Rl) of whole blood or PMN-enriched cell suspension was added. This produced negligible changes

709

in background CL. After 2 to 5 min, medium (100 Rl) containing various stimulants (at concentrations shown in preliminary experiments to give the maximal response) was added as detailed in the legends to the figures. Signals were recorded, and counts, printed at 1-min intervals, were integrated for 10 min. After background CL was subtracted, the results were expressed as integrated counts per minute. Medium. PBS (pH = 7.2; Ca2' and Mg2+ supplemented [Dulbecco]; containing glucose [5 mM]) was used for washing and resuspending cells and for CL experiments. Luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione). A stock solution of 10 mM luminol was prepared as follows: triethylamine (25 1.d) was added to PBS (10 ml) containing luminol (17.72 mg), and the suspension was sonicated with an MSE 100-W Ultrasonic Disintegrator until rendered clear. Opsonized zymosan. Zymosan was opsonized by incubating 50 mg with PBS (1 ml) and fresh serum from noninfected CBA/CaH mice (3 ml) for 30 min at 37°C. It was then washed twice with PBS and finally resuspended in 5 ml of PBS. Chemicals. Percoll was obtained from Pharmacia (Uppsala, Sweden), phorbol myristate acetate (PMA) from Sigma Chemical Co. (St. Louis, Mo.), zymosan from ICN Nutritional Biochemicals (Cleveland, Ohio), and A23187 from Calbiochem-Behring (La Jolla, Calif.). RESULTS Exposure of whole blood containing 1 mM luminol to zymosan, opsonized zymosan, the divalent-ion ionophore A23187, or PMA resulted in light emission that varied in intensity depending on the stimulus used (data not shown). With progressively increasing parasitemia, addition of any one of these exogenous stimuli to whole blood induced a correspondingly larger CL response (Fig. 1). Opsonized zymosan was the most potent stimulus used, evoking a 24fold-greater production of luminol-reactive oxygen radicals in parasitized than in control blood. The enhanced CL responses observed with parasitized blood could be partly explained by the changes in the total number of WBC per unit volume of blood associated with malarial infection (Table 1). The total number of WBC decreased during the first 5 days after infection, after which there was an 80% increase at high levels of parasitemia. As infection progressed, hemolysis occurred, resulting in decreased total numbers of RBC. In addition, the differential WBC count changed during the course of infection. The percentage of circulating PMNs increased up to threefold, whereas the numbers of lymphocytes decreased proportionally. The numbers of monocytes remained more or less constant (Table 1). Luminol-mediated CL is a rather nonspecific method for

TABLE 1. Changes in WBC and RBC during the course of malarial infection Cell type (mean + SD) WBC

% Parasitemia

Control blood 0-25 25-50 50-75 Above 75

RBC (x 109)a

8.8 10.2 7.6 6.2 2.5

± 1.4 (21) ± 1.7 (4) ± 1.7 (4) ± 0.5 ± 1.2

(5) (5)

Total (x 106)a

6.2 3.2 4.3 7.0 10.9

%

PMNs"

b

± 3.5 ± 1.4 ± 1.0

(15) (13) (4)

21.2 ± 0.8 (10) 24.5 ± 8.4 (6) 29.4 ± 5.7 (4)

± 3.7 ± 3.7

(7)

(7)

38.3 ± 22.0 (6) 57.7 ± 12.0 (9)

%

Monocytes and

macrophagesb 18.9 ± 1.0 (10) 29.4 ± 3.3 (6) 33.1 ± 8.0 (4) 22.3 ± 7.6 (6) 17.1 + 4.7 (7)

%

Lymphocytes"

58.4 ± 1.1 (10) 52.4 ± 5.8 (5) 36.5 ± 10.6 (4) 30.8 ± 10.2 (6) 24.0 ± 12.6 (10)

a Arithmetic mean ± standard deviation of total number of cells per milliliter of blood; number of determinations in parentheses. b Arithmetic mean + standard deviation of individual WBC subpopulations expressed as a percentage of the total number of cells; number of determinations in parentheses.

710

STOCKER ET AL.

INFECT. IMMUN.

the detection of oxidizing species and is influenced by a number of factors. We therefore examined the relationship between the amount of whole blood used and the CL produced upon stimulation with opsonized zymosan. Figure 2 shows that there was a linear relationship between the intensity of light emitted and the number of WBC in whole blood if the total number of RBC and the total volume of serum present were kept constant. It was possible that the 24-fold increase in CL produced by highly infected whole blood in response to opsonized zymosan was associated with an increased responsiveness of individual cells within the WBC population as a result of their "'activation." In an attempt to answer this question, we examined the CL response of whole blood from infected animals to opsonized zymosan or PMA under conditions of constant hematocrit by titrating RBC back to a constant number with RBC from a noninfected mouse (Table 2). Under these conditions, peripheral WBC from infected animals showed little tendency to produce increased amounts of luminol-reactive species of oxygen on a per cell basis. Since whole blood CL is mainly dependent on the PMNs present in blood (27), we attempted to isolate these cells from malaria-infected mice to see if they were the source of the activity we were detecting. Using a separation method based on the different densities of the WBC, it was possible to obtain fractions that, although contaminated with small lymphocytes and RBC, were enriched 60 to 80% in PMNs. These PMN-enriched cell suspensions isolated from infected mice at different levels of parasitemia were examined for their CL response to opsonized zymosan. Figure 3 shows that, in accordance with the results obtained with whole blood when the hematocrit was constant (Table 2), there was no large increase in the CL responsiveness of PMNs from infected animals during malarial infection. Essentially the same result was obtained with monocyte-enriched fractions

oe

TABLE 2. CL response of whole blood from infected animals to opsonized zymosan (0.5 mg/ml) or PMA (1 p.g/ml) under conditions of constant hematocrit CL (test control)" with the following amt Stimulus of parasitemia (%): -

tested

0-20

20-40

40-60

Above 60

Opsonized zymosan

1.7

0.7 (5)

1.7 ± 0.9 (4)

2.1 ± 0.6 (3)

1.3 ± 0.6

PMA

2.1 ± 1.2 (3)

3.1 ± 0.9 (4)

2.6 ± 0.1 (3)

2.3 ± 0.8 (4)

+

(4)

"CL was calculated as cpm/106 WBC. Values are arithmetic means ± standard deviation of the ratio of CL signals of blood from infected mice to CL signals of blood from control mice. Numbers of experiments are in parentheses.

prepared from blood from the same infected animals (data not shown). DISCUSSION The results presented in this report show that stimulation of WBC from malaria-infected mice by phagocytic or nonphagocytic stimuli leads to an increased production of luminol-mediated CL compared with the results obtained with their counterparts from control mice (Fig. 1). The significant difference between the stimulatory effects of opsonized and unopsonized zymosan is in agreement with the observations of others (5, 15, 28, 29) that an increased rate of phagocytosis of free merozoites and PRBC is dependent upon the presence of immune serum. Specific antibodies might be responsible, by their opsonizing capacity, for triggering the Fcreceptor-mediated phagocytosis and its associated release of destructive radicals from the cell. Malarial infection in mice is accompanied by changes in the total number and proportion of WBC. However, the 80% increase in total WBC number and the threefold increase in the proportion of PMNs (Table 1) cannot account entirely for the observed 8- to 12-fold-higher CL obtained with zymosan, PMA, and A23187 nor for the 24-fold increase in light emission when opsonized zymosan was used (Fig. 1).

1.0

z

I,

o

If

LUI

0

z u 0.2 -A FI.-.Reainhpbtentecnetaiono

hllo Z2 1.

0

I.

0

Un

z

20

60 WHOLE BLOOD

100

0

(IAI)

FIG. 2. Relationship between the concentration of whole blood used and the CL response obtained after the addition of opsonized zymosan (0.5 mg/ml). CL responses obtained with 100 ,ul of whole blood are referred to as 100%. Control blood without further additions (A), and control (0) and infected (A) blood each supplemented with a constant volume of serum (50 ,ul) and maintained at a constant hematocrit (1.2 x 10'( RBC per ml). Results represent mean values ± standard deviation of four independent experiments, each performed in duplicate.

20

0~~~~

40 60 PARASITEMIA (%)

FIG. 3. CL response of PMN-enriched fractions (60 to 80% PMNs, 20 to 40% lymphocytes, 0% monocytes and macrophages) from the blood of infected animals after the addition of opsonized zymosan (0.5 mg/ml). Experiments were performed as described in the text.

VOL. 45, 1984

Peritoneal macrophages from normal mice can be activated in vitro by medium conditioned by antigen-stimulated spleen cells from malaria-immune mice (19). Furthermore, these macrophages, in the presence of immune serum, produce approximately twice as much parasite-induced CL as that produced by nonactivated macrophages (19). Thus, we examined whether circulating WBC, especially PMNs, become activated during malarial infection so that they produce greater CL per cell in response to several stimuli than do control cells. Initially, we examined the relationship between the concentration of whole blood used and the CL response obtained after stimulation, since the two parameters have been reported to be relatively independent of each other over a restricted range of blood concentration (11). RBCs and hemoglobin apparently reduce the photon counts measured in liquid scintillation counters (13), and RBC inhibit the CL signals of granulocytes in a dose-dependent manner (27). The results in Fig. 2 confirm these observations and show that, with whole blood CL, a linear relationship between the concentration of whole blood and the emitted light recorded is obtained only if the total number of RBC and the total volume of serum are kept constant. Under these conditions, whole blood CL can be used to measure large differences in the ability of WBC to produce activated species of oxygen. Small differences, however, cannot be discriminated readily. There is no major activation of WBC from the peripheral blood of mice undergoing malarial infection in terms of the production of luminol-reactive oxygen radicals per cell (Table 2). Nevertheless, our results do not exclude the possibility that subpopulations of WBC from infected mice might be modestly activated, as has been reported for peritoneal cavity cells (19). The results obtained with PMN-enriched fractions from peripheral blood (Fig. 3) do not clarify this point further. The characteristics of sequestered WBC populations, such as those in the liver and spleen, are yet to be studied. In addition to their role in the oxidative destruction of microorganisms which occurs within the phagocytes, oxygen radicals are released extracellularly (21, 22). Some of these radicals can traverse the RBC membrane by the anion channel (17) and can attack both the plasma membrane (16) and the intracellular hemoglobin (30). Hemoglobin acts as a preferential reactant for hydrogen peroxide and superoxide anion and thus provides an antioxidant buffer for the membrane (4) and, possibly, for intraerythrocytic protozoa such as the malarial parasite. As the parasite grows and consumes up to 80% of the hemoglobin (R. Stocker, unpublished observations), the buffering capacity of the hemoglobin pool within the PRBC is diminished (23). The dramatic increase in the production of stimulus-induced CL in whole blood from mice with a high parasitemia indicates the potential effectiveness of the activated species of oxygen released by WBC to cause damage at this stage of infection. This increase in activated oxygen production appears to be due to both the increased number of circulating WBC, mainly PMNs, present during the later stages of infection and also to a decrease in the protective buffering capacity afforded by RBC, in terms of both their number and their hemoglobin content. We suggest that these factors alone might be sufficient to contribute to the increased tissue damage seen in malarial infection (18). Injection of 400 to 500 ,ul of washed RBC into mice sick from infection with P. vinckei, a normally lethal parasite, allows recovery (unpublished observations). Increased antioxidant buffering capacity, as well as oxygen carriage, may contribute to this survival. Thus, at high parasitemia the decreased buffering capacity within infected

LUMINOL-REACTIVE OXYGEN RADICALS

711

RBC due to hemoglobin digestion by the parasite might favor oxygen radical-mediated damage to intraerythrocytic parasites and RBC membranes. ACKNOWLEDGMENTS This investigation received support from the malaria component of the United Nations Development Programme/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, the National Health and Medical Research Council of Australia, and the Australian Research Grants Scheme (grant no. D27915664 to M.J.W.). We thank Liz Bruce for typing the manuscript. LITERATURE CITED 1. Allen, R. C., and L. D. Loose. 1976. Phagocytic activation of a luminol-dependent chemiluminescence in rabbit alveolar and peritoneal macrophages. Biochem. Biophys. Res. Commun. 69:245-252. 2. Babior, B. M. 1978. Oxygen-dependent microbial killing by phagocytes. N. Engl. J. Med. 298:659-668. 3. Badwey, J. M., and M. L. Karnovsky. 1980. Active oxygen species and the functions of phagocytic leukocytes. Annu. Rev. Biochem. 49:695-726. 4. Carell, R. W., C. C. Winterbourn, and E. A. Rachmilewitz. 1975. Activated oxygen and hemolysis. Br. J. Haematol. 30:259-264. 5. Celada, A., A. Cruchaud, and L. H. Perrin. 1983. Phagocytosis of Plasmodium falciparum-parasitized erythrocytes by human polymorphonuclear leukocytes. J. Parasitol. 69:49-53. 6. Chaudry, A. N., J. T. Santinga, and T. G. Gabig. 1982. The subcellular particulate NADPH-dependent 0O- -generating oxidase from human blood monocytes: comparison to the neutrophil system. Blood 60:979-983. 7. Clark, I. A., A. C. Allison, and F. E. G. Cox. 1976. Protection of mice against Babesia and Plasmodium with BCG. Nature (London) 259:309-311. 8. Clark, I. A., W. B. Cowden, and G. A. Butcher. 1983. Free oxygen radical generators as antimalarial drugs. Lancet i:234. 9. Clark, I. A., and N. H. Hunt. 1983. Evidence for reactive oxygen intermediates causing hemolysis and parasite death in malaria. Infect. Immun. 39:1-6. 10. Clark, I. A., J.-L. Virelizier, E. A. Carswell, and P. R. Wood. 1981. Possible importance of macrophage-derived mediators in acute malaria. Infect. Immun. 32:1058-1066. 11. De Chatelet, L. R., and P. S. Shirley. 1981. Evaluation of chronic granulomatous disease by a chemiluminescence assay of microliter quantities of whole blood. Clin. Chem. 27:1739-1741. 12. Dockrell, H. M., and J. H. L. Playfair. 1983. Killing of bloodstage murine malaria parasites by hydrogen peroxide. Infect. Immun. 39:456-459 13. Easmon, C. S. F., P. J. Cole, A. J. Williams, and M. Hastings. 1980. The measurement of opsonic and phagocytic function by luminol-dependent chemiluminescence. Immunology 41:67-74. 14. Jensen, J. B., M. T. Boland, J. S. Allan, J. M. Carlin, J. A. Vande waa, A. A. Divo, and M. A. S. Akood. 1983. Association between human serum-induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan. Infect. Immun. 41:1302-1311. 14a.Jensen, J. B., S. L. Hoffman, M. T. Boland, M. A. S. Akood, L. W. Laughlin, L. Kurniawan, and H. A. Marwoto. 1984. Comparison of immunity to malaria in Sudan and Indonesia: crisis-form versus merozoite-invasion inhibition. Proc. Natl. Acad. Sci. U.S.A. 81:922-925. 15. Khusmith, S., P. Druilhe, and M. Gentilini. 1982. Enhanced Plasmodiumfalciparum merozoite phagocytosis by monocytes from immune individuals. Infect. Immun. 35:874-879. 16. Lynch, R. E., and I. Fridovich. 1978. Effects of superoxide on the erythrocyte membrane. J. Biol. Chem. 253:1838-1845. 17. Lynch, R. E., and I. Fridovich. 1978. Permeation of the erythrocyte stroma by superoxide radical. J. Biol. Chem. 253:46974699. 18. Macgraith, B. 1948. Pathological processes in malaria, p. 345379. In B. Macgraith (ed.), Pathological processes in malaria

712

19.

20.

21.

22.

23.

INFECT. IMMUN.

STOCKER ET AL. and blackwater fever. Blackwell Scientific Publications. Oxford. Makimura, S., V. Brinkmann, H. Mossmann, and H. Fischer. 1982. Chemiluminescence response of peritoneal macrophages to parasitized erythrocytes and lysed erythrocytes from Plasmodium berghei-infected mice. Infect. Immun. 37:800-804. Makimura, S., and N. Suzuki. 1977. Studies on the phagocytosis of parasitized erythrocytes from mice experimentally infected with Plasmodiuim1 berghei by mouse peritoneal macrophages. Res. Bull. Obihiro Univ. 10:401-406. Ohno, Y.-I., K.-I. Hirai, T. Kanoh, H. Uchino, and K. Ogawa. 1982. Subcellular localization of H.O, production in human neutrophils stimulated with particles and effect of cytochalasinB on the cells. Blood 60:253-260. Ohno, Y.-I., K.-I. Hirai, T. Kanoh, H. Uchino, and K. Ogawa. 1982. Subcellular localization of hydrogen peroxide production in human polymorphonuclear leukocytes stimulated with lectins, phorbol myristate acetate, and digitonin: an electron microscopic study using CeCl3. Blood 60:1195-1202. Picard-Maureau, A., E. Hempelmann, G. Krammer, R. Jakisch, and A. Jung. 1975. Glutathionstatus in Plasmodiuin i'inckei

24. 25. 26. 27.

28. 29. 30.

parasitierten Erythrozyten in Abhangigkeit vom intraerythrozytaren Entwicklungsstadium des Parasiten. Tropenmed. Parasitol. 26:405-416. Shear, H. L., R. S. Nussenzweig, and C. Bianco. 1979. Immune phagocytosis in murine malaria. J. Exp. Med. 149:1288-1298. Taliaferro, W. H., and P. R. Cannon. 1936. The cellular reactions and superinfections of Plasinodiini b1rasilianirn in Panamanian monkeys. J. Infect. Dis. 59:72-125. Taliaferro, W. H., and H. W. Mulligan. 1937. The histopathology of malaria with special reference to the function and origin of the macrophages in defense. Indian Med. Res. Mem. 29:1-138. Tono-Oka, T., N. Ueno, T. Matsumoto, M. Ohkawa, and S. Matsumoto. 1983. Chemiluminescence of whole blood. Clin. Immunol. Immunopathol. 26:66-75. Trubowitz, S., and B. Masek. 1968. Plasinodium falciparum: phagocytosis by polymorphonuclear leukocytes. Science 162:273-274. Vernes, A. 1980. Phagocytosis of P. falciparum parasitised erythrocytes by peripheral monocytes. Lancet ii:1297-1298. Weiss, S. J. 1982. Neutrophil-mediated methemoglobin formation in the erythrocyte. J. Biol. Chem. 257:2947-2953.