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KATHERINE N. WARD,It* MARY J. WARRELL,1 JOHN RHODES,2t SORNCHAI LOOAREESUWAN,1 AND ..... Taliaferro, W. H., and C. Kluver. 1940.
INFECTION

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IMMUNITY, June 1984, p. 623-626

Vol. 44, No. 3

0019-9567/84/060623-04$02.00/0 Copyright C 1984, American Society for Microbiology

Altered Expression of Human Monocyte Fc Receptors in Plasmodium falciparum Malaria KATHERINE N. WARD,It* MARY J. WARRELL,1 JOHN RHODES,2t SORNCHAI LOOAREESUWAN,1 AND NICHOLAS J. WHITE1 Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand2 and Tropical Medicine Unit, Nuffield

Department of Clinical Medicine, University of Oxford, Oxford OX3 9DU, United Kingdom' Received 31 October 1983/Accepted 10 March 1984

The state of activation of human peripheral blood monocytes was examined by using a rosette assay that detects changes in Fc receptor expression. Monocytes from patients with uncomplicated Plasmodium falciparum malaria showed a significant increase in the number of rosettes relative to healthy controls. In addition, the monocytes from these patients were tested for their ability to phagocytose Candida albicans, but this ability did not differ from that of normal individuals. Finally, the monocytes from patients with cerebral malaria were also tested for Fc receptor expression. In contrast to the results from uncomplicated cases, the activity of the monocytes from these patients was no different from that of controls. We concluded that uncomplicated P. falciparum malaria caused an increase in monocyte Fc receptor expression which did not occur in cerebral malaria and that this difference in activation may be important in the pathogenesis of cerebral malaria. It has long been established that the host response to plasmodia includes the production of enlarged populations of both peripheral blood monocytes and mature macrophages (28, 29). Splenomegaly and hepatomegaly are frequently observed in human malaria cases, and histological studies have demonstrated hyperplasia of the reticuloendothelial system (3). In rodent malaria, experiments have shown that both the spleen (20) and the liver (9) remove parasitized erythrocytes more rapidly from the circulation than they remove uninfected cells. Furthermore, macrophages from infected animals are more phagocytic for parasitized cells than are normal macrophages (8, 27), and this enhanced phagocytic activity depends on the presence of either opsonins (11, 30) or cytophilic antibodies (10) and also on the degree of macrophage activation (27). Although little is known about the nature of this macrophage activation, it seems reasonable to suppose that it includes increased expression of specific surface receptors for the Fc portion of immunoglobulin G (Fc receptors), since this would clearly facilitate the phagocytosis of antibodycoated targets. Moreover, increased Fc receptor expression has been observed in activated macrophages from adjuvantstimulated animals and from experimentally induced inflammatory exudates (4, 21, 31). This finding suggests that changes in the expression of this receptor provide a useful indicator of macrophage activation. Plasmodium falciparum infection is the most serious of the human malarias. Severe forms of the disease cause multiple organ dysfunction, and involvement of the central nervous system leading to coma (cerebral malaria) carries a mortality of 20% despite treatment (32). Cerebral malaria is characterized by a high parasite load but not necessarily a high parasitemia, because mature forms are sequestered in the microcirculation (17). In the present study, we decided to compare macrophage activation in uncomplicated P. falciparum malaria, in which the mononuclear phagocyte system

might be activated and effectively aiding in defense against the parasite, with that in cerebral malaria, in which such defenses might have failed. Fc receptor expression by peripheral blood monocytes was chosen as an index of macrophage activation, and this paper reports the results. MATERIALS AND METHODS This study was conducted at Pra Pokklao Hospital, Chantaburi, Eastern Thailand, and ethical committee approval was granted by Mahidol University, Bangkok, Thailand. Subjects. The groups of Thais in the study consisted of the following. (i) Twelve normal, healthy individuals aged between 16 and 47 years (mean age, 29 years). (ii) Thirteen inpatients with acute uncomplicated P. falciparum malaria who were receiving treatment with quinine. They were aged between 9 and 63 years (mean age, 24 years) with a median parasite count of 24,000 (range, 3,360 to 125,000) P. falciparum asexual forms per ,u of blood. (iii) Five patients with cerebral malaria who were being treated with quinine. They were aged between 19 and 25 years (mean age, 22 years) and had a median parasite count of 3,400 (range, 1,320 to 62,500) P. falciparum asexual forms per ,u of blood. Two of the five patients also had acute renal failure. Cerebral malaria was defined as the presence of asexual forms of P. falciparum in the blood of patients in a coma that could not be attributed to other causes (32). All samples were taken within 24 h of admission to the hospital. Peripheral blood monocytes. From each subject, 10 ml of blood was obtained. It was defibrinated in nonwettable plastic containers with a U-shaped glass rod. This process also removed platelets. Defibrinated blood was diluted 1 in 3 with phosphate-buffered saline, layered onto Ficoll-Paque (density, 1.078 g/ml) (Pharmacia Fine Chemicals, Uppsala, Sweden) and centrifuged for 40 min at 400 x g. The mononuclear cells at the interface were aspirated, washed, suspended in Hanks balanced salt solution (HBSS) containing 20% heat-inactivated fetal calf serum, then placed in tissue-culture chamber slides (Lab-Tek 4 chamber slides; Miles Laboratories, Slough, U.K.), and incubated for 1 h at 37°C. In these circumstances, the resultant adherent cell monolayers consist of more than 95% monocytes after being

* Corresponding author. t Present address: Lucy Cavendish College, Cambridge CB3 OBU, U.K. t Present address: Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K.

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washed with HBSS to remove nonadherent cells (23). The monocytes of patients and normal donors were compared simultaneously in the following assays. Rosette assay for Fc receptor expression (23). Antibodysensitized sheep erythrocytes were prepared by incubating 2% washed erythrocytes with a range of concentrations of rabbit anti-sheep erythrocyte serum (Wellcome Research Laboratories, Beckenham, England) for 30 min at room temperature. The cells were then washed twice and suspended in HBSS at a concentration of 1%. Sensitized erythrocytes in 1-ml volumes were added to each monocyte monolayer and allowed to settle at room temperature for 1 h. The monolayers were then washed three times with HBSS and fixed with 0.5% glutaraldehyde for at least 1 h. The cells were stained with citrate-buffered Giemsa, the chambers were removed, and the slides were mounted with cover slips. Two hundred cells were counted per slide, and those cells bearing three or more erythrocytes were scored as rosettes and expressed as a percentage of the total monocyte population. A range of suboptimal concentrations of antibody was used so that the binding of the reagent, visualized as rosette formation, could be expressed as a function of the dose of antibody. The resultant dose-response curves provided a sensitive reflection of differences between activated and nonactivated monocytes. At higher doses, however, all curves reached 100% rosette formation, indicating that all peripheral blood monocytes expressed detectable Fc receptors, whether activated or not. The rabbit reagent employed in the present study binds to the same receptor as human immunoglobulin G and is merely a convenient tool for assaying human monocyte Fc receptor expression (24). Phagocytosis assay. Samples of washed Candida albicans (1 ml) suspended at suitable concentrations in HBSS with 5% fetal calf serum (Fig. 2) were incubated with the monocyte layers for 45 min at 37°C. The slides were then prepared, fixed, and stained as in the Fc receptor assay. Two hundred cells were counted on each slide, and the results were expressed as the average number of C. albicans ingested per monocyte. Statistical analysis. The parasite counts were not normally distributed, and the median value is therefore quoted rather than the mean. A normal distribution was assumed for the results obtained in the rosette assay for Fc receptor expression. In every experiment, monocytes from one or more patients with malaria were simultaneously compared with monocytes from a normal individual so that there would be a control for day-to-day variation in the assay. The data were therefore analyzed with a paired two-tailed t test, and the results are expressed as the mean + the standard error of the mean. RESULTS Fc receptor expression in monocytes from patients with uncomplicated malaria was compared with that in normal individuals (Fig. 1). It is evident that there is a statistically significant increase in antibody-binding capacity of the monocytes from the infected individuals relative to the controls at all concentrations of sensitizing antiserum except the highest where each curve approached its maximum and, presumably, the cell sites were almost saturated. This change was clearly a function of increased Fc receptor activity, although no distinction could be made between greater receptor affinity and an increase in the number of available receptors. The increased monocyte Fc receptor activity in uncompli-

cated malaria might reflect a more general cellular activation. We therefore decided to investigate the phagocytic activity of monocytes from these patients. Figure 2 shows the average number of C. albicans phagocytosed per cell at different concentrations of added C. albicans, and it can be seen that a dose-response curve was obtained. Monocytes were also tested from normal individuals, and there was no significant difference in phagocytic ability between the two groups. Finally, the monocytes from the five patients with cerebral malaria were also tested for Fc receptor expression (Fig. 3). In contrast to the findings in uncomplicated malaria cases, the receptor activity in comatose patients did not differ from that of the healthy controls at any concentration of antibody tested. DISCUSSION that peripheral blood demonstrated The present study monocytes from patients with uncomplicated P. falciparum malaria show increased Fc receptor activity (Fig. 1). Enhanced Fc receptor expression has also been reported in monocytes from patients with malignancies (22, 23) and in patients with granulomatous diseases, namely, active pulmonary tuberculosis, sarcoidosis, and Crohn's disease (22, 25). These latter diseases are all chronic inflammatory conditions in which macrophage activation is expected. The observed increase in the activity of monocyte Fc receptors in patients with uncomplicated malaria should facilitate antibody-dependent phagocytosis of parasites. It is already established that immune serum enhances the ability of normal monocytes to phagocytose P. falciparum-infected erythrocytes containing mature trophozoites and schizonts 100

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FIG. 1. The percentage of rosette-forming monocytes as a function of the degree of erythrocyte sensitization in nine patients with uncomplicated malaria (0) and seven normal individuals (0). Each values is expressed as the mean + standard error of the mean. Results of paired two-tailed t tests at different dilutions of sensitizing antiserum: 80 x 10-6, P = 0.04; 160 x 10-6, P = 0.01; 240 x 10-6, p = 0.03; 320 x 10-6, P = 0.0006; 640 x 10-6, no significant difference.

MONOCYTE Fc RECEPTORS IN P. FALCIPARUM MALARIA

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by the reticuloendothelial system in rodent malaria (7, 16) and human malaria (26). However, this en,hancement in vivo probably reflects an increase in the number of macrophages in the spleen (15, 33) and the liver (9), rather than an increase in individual macrophage activity. The pathophysiological mechanisms underlying cerebral malaria are unknown, but some researchers have suggested that it has an immunological basis. It is interesting therefore that in the present study, monocytes from patients with cerebral malaria did not show significantly increased Fc receptor expression (Fig. 3). This result contrasts with the enhanced Fc receptor activity observed in uncomplicated malaria. It is possible that the difference merely reflects the lower parasitemia observed in the peripheral venous blood of patients with cerebral malaria. However, such an explanation is unlikely because in cerebral malaria there is a marked dissociation between peripheral parasitemia and parasite load, owing to pooling of mature trophozoites and schizonts in the deep tissue capillaries, particularly in the brain (17). The parasite concentration in the blood may therefore grossly underestimate the total parasite numbers within the patient. In fact, increased circulating immune complexes have been demonstrated in cerebral malaria patients (2), and it seems most likely that the monocytes from comatose patients do have increased Fc receptor expression, as in uncomplicated malaria, but that the receptors are blocked by immune complexes. Such impairment of monocyte function could be important in the pathogenesis of cerebral malaria, and further studies should be carried out to examine this

FIG. 2. Phagocytosis of C. albicans by monocytes as a function of C. albicans concentration in 10 patients with uncomplicated malaria (0) and 6 normal individuals (0). Each value is expressed as the mean ± the standard error of the mean.

possibility. In conclusion, although the exact role of the mononuclear phagocyte in resistance to malaria is unknown, this study shows that P. falciparum malaria can cause changes in

(1, 5) and merozoites (13), but remarkably little is known about the state of activation of monocytes and macrophages in acute human malaria. Abdalla and Weatherall (1) have shown that monocytes from malarious Gambian children with various degrees of anemia and parasitemia do not show increased activity with respect to phagocytosis of anti-Dimmunoglobulin G-coated human erythrocytes. However, it is difficult to relate this result directly to the present findings, as the natural history of P. falciparum malaria in the hyperendemic regions of West Africa is so different from that in Southeast Asia where transmission is seasonal and immunity is variable. The relationship between monocyte activation and antibody-mediated parasite phagocytosis was therefore considered to deserve further investigation. Since the monocytes from patients with uncomplicated malaria were activated with respect to Fc receptor expression, it might be expected that the inherent phagocytic ability of such monocytes would be similarly increased. However, preliminary experiments showed that the percentage of phagocytic cells was the same in normal individuals and in those with uncomplicated malaria, and there was no significant difference in the number of C. albicans phagocytosed per cell by monocytes from the two groups (Fig. 2). This result has also been observed by others (14), and it is well established that not all macrophage functions change in concert (6, 12, 19). For example, Meltzer and Stevenson (18), who used a syngeneic murine system, showed that macrophage Fc receptor activity increases and chemotactic responsiveness decreases in tumor recipients. The lack of increased nonspecific phagocytic activity in monocytes from patients with uncomplicated malaria is in direct contrast to the observed increase in colloidal particle clearance shown

significance in the cellular immune response to the parasite.

monocyte Fc receptor activity which are likely to be of

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ACKNOWLEDGMENTS We thank David Warrell for enabling us to carry out the study. We are also indebted to the Director of Pra Pokklao Hospital, Chaisit Dharakul, and his staff and to Damrong Bhantkumkosol and his laboratory staff. This study was part of the Wellcome-Mahidol University, Oxford Tropical Medicine Research Programme, funded by the Wellcome Trust of Great Britain. K.N.W. was also assisted by the H. E. Durham Fund, King's College, Cambridge, the British Medical Students' Trust, and the Royal College of Pathologists.

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110:1455-1463. 5. Celada, A., A. Cruchaud, and L. H. Perrin. 1982. Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodiumfalciparum infected red blood cells by monocytes. Clin. Exp. Immunol. 47:635-644. 6. Cohn, Z. A. 1978. The activation of mononuclear phagocytes: fact, fancy and future. J. Immunol. 121:813-816. 7. Cox, F. E. G., and D. L. J. Bilbey, and T. Nicol. 1964. Reticuloendothelial activity in mice infected with Plasmodium vinckei. J. Protozool. 11:229-230. 8. Criswell, B. S., W. T. Butler, R. D. Rossen, and V. Knight. 1971. Murine malaria: the role of humoral factors and macrophages in destruction of parasitized erythrocytes. J. Immunol. 107:212221. 9. Dockrell, H. M., J. B. de Souza, and J. H. L. Playfair. 1980. The role of the liver in immunity to blood stage murine malaria. Immunology 41:421-430. 10. Green, T. J., and J. P. Kreier. 1978. Demonstration of the role of cytophilic antibody in resistance to malaria parasites (Plasmodium berghei) in rats. Infect. Immun. 19:138-145. 11. Hunter, K. W., J. A. Winkelstein, and T. W. Simpson. 1979. Serum opsonic activity in rodent malaria: functional and immunochemical characteristics in vitro. J. Immunol. 123:2582-2587. 12. Karnovsky, M. L., and J. K. Lazdins. 1978. Biochemical criteria for activated macrophages. J. Immunol. 121:809-813. 13. Khusmith, S., and P. Druilhe. 1983. Antibody dependent ingestion of P. falciparum merozoites by human blood monocytes. Parasite Immunol. 5:357-368. 14. Khusmith, S., P. Druilhe, and M. Gentilini. 1982. Enhanced Plasmodium falciparum merozoite phagocytosis by monocytes from immune individuals. Infect. Immun. 35:874-879. 15. Lelchuk, R., J. Taverne, P. U. Agomo and J. H. L. Playfair.

INFECT. IMMUN. 1979. Development and suppression of a population of lateadhering macrophages in mouse malaria. Parasite Immunol. 1:61-78. 16. Lucia, H. L., and R. S. Nussenweig. 1969. Plasmodium chabaudi and Plasmodium vinckei: phagocytic activity of mouse reticuloendothelial system. Exp. Parasitol. 25:319-323.

17. Maegraith, B. G., and A. Fletcher. 1972. The pathogenesis of mammalian malaria. Adv. Parasitol. 10:49-75. 18. Meltzer, M. S., and M. M. Stevenson. 1978. Macrophage function in tumor bearing mice: dissociation of phagocytic and chemotactic responsiveness. Cell. Immunol. 35:99-111. 19. North, R. J. 1978. The concept of the activated macrophage. J. Immunol. 121:806-809. 20. Quinn, T. C., and D. J. Wyler. 1979. Intravascular clearance of parasitised erythrocytes in rodent malaria. J. Clin. Invest. 63:1187-1194. 21. Rhodes, J. 1975. Macrophage heterogeneity in receptor activity: the activation of macrophage Fc receptor function in vivo and in vitro. J. Immunol. 114:976-981. 22. Rhodes, J. 1977. Altered expression of human monocyte Fc receptors in malignant disease. Nature (London) 265:253-255. 23. Rhodes, J., P. Plowman, M. Bishop, and D. Lipscomb. 1981. Human macrophage function in cancer: systemic and local changes detected by an assay for Fc receptor expression. J. Natl. Cancer Inst. 66:423-429. 24. Rhodes, J., and P. Stokes. 1982. Interferon-induced changes in the monocyte membrane: inhibition by retinol and retinoic acid.

Immunology 45:531-536. 25. Schmidt, M. E., and S. D. Douglas. 1977. Monocyte lgG receptor activity, dynamics and modulation-normal individuals and patients with granulomatous diseases. J. Lab. Clin. Med. 89:332-340. 26. Sheagren, J. N., J. E. Tobie, L. M. Fox, and S. M. Wolff. 1970. Reticulo-endothelial system phagocytic function in naturally acquired human malaria. J. Lab. Clin. Med. 75:481-487. 27. Shear, H. L., R. S. Nussenweig, and C. Bianco. 1979. Immune phagocytosis in murine malaria. J. Exp. Med. 149:1288-1298. 28. Singer, I. 1954. The cellular reactions to infections with Plasmodium berghei in the white mouse. J. Infect. Dis. 94:241-261. 29. Taliaferro, W. H., and C. Kluver. 1940. The haematology of malaria (Plasmodium brasilianum) in Panamanian monkeys. I. Numerical changes in leukocytes. J. Infect. Dis. 67:121-161. 30. Tosta, C. E., and N. Wedderburn. 1980. Immune phagocytosis of Plasmodium yoellii-infected erythrocytes by macrophages and eosinophils. Clin. Exp. Immunol. 42:114-120. 31. Unkeless, J. C., and H. M. Eisen. 1975. Binding of monomeric immunoglobulins to Fc receptors of mouse macrophages. J. Exp. Med. 142:1520-1533. 32. Warrell, D. A., S. Looareesuwan, M. J. Warrell, P. Kasemsarn, R. Intaraprasert, D. Bunnag, and T. Harinasuta. 1982. Dexamethasone proves deleterious in cerebral malaria. A doubleblind trial in 100 comatose patients. N. Engl. J. Med. 306:313319. 33. Wyler, D. J., and J. I. Gallin. 1977. Spleen-derived mononuclear cell chemotactic factor in malaria infections: a possible mechanism of splenic macrophage accumulation. J. Immunol. 118:478-486.