Effect of antimalarial drugs and malaria pigment ( *-haematin) on monocyte phagocytosis and GTP-cyclohydrolase 1 gene expression.

Abstract

During the erythrocytic stage, the malaria parasite digests host cell haemoglobin into amino acids. Toxic haeme is released and is incorporated into an insoluble non-toxic crystal called haemozoin. Haemozoin is released into the blood stream along with the merozoites when the erythrocyte bursts and is phagocytosed by circulating monocytes and macrophages resident in tissues. Phagocytosed haemozoin impairs many functions of the monocytes, including antigen presentation and adhesion to T cells, differentiation and maturation to dendritic cells, erythropoiesis and thrombopoiesis, but stimulates the release of proinflammatory cytokines and activation of metalloproteinase 9 expression. In response to interferon-g secretion by T-helper cells subtype 1, monocytes secrete neopterin, which is used as a marker of a cell mediated immune response. Neopterin is an oxidation product of 7,8-dihydroneopterin, produced by the dephosphorylation of 7,8- dihydroneopterin triphosphate which results from the conversion of guanosine triphosphate that is catalysed by GTP-cyclohydrolase 1. Elevated plasma and urine neopterin levels have been detected in malaria infections and are associated with severe anaemia, respiratory distress, peak temperatures as well as fever- and parasite-clearance times. It has also been reported that monocytic U937 cells treated with P. falciparum-infected red blood cell lysate secrete elevated levels of neopterin. Antimalarial drugs are known to modulate the functions of monocytes, including inhibition of cytokine release, changes in phospholipid metabolism, decrease in expression of cytoadherance receptors as well as TNF receptors and MHC Class I and II molecules, changes in the production of reactive oxygen and nitrogen intermediates, and decreased phagocytosis. However, the effects of antimalarial drugs on haemozoin phagocytosis and GTP-cyclohydrolase 1 mRNA expression by monocytes are unknown. This study aimed to determine the effects of seven antimalarial drugs, amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of latex beads and b-haematin, a synthetic equivalent of haemozoin. Phagocytosis of b-haematin and latex beads by two monocytic cell lines, J774A.1 and U937, as well as peripheral blood mononuclear cells were monitored by enumeration and a novel spectrophotometric method. Patterns of inhibition and activation differed with each cell type investigated, due to the differing stages of cell differentiation. In general, artemisinin, primaquine, pyrimethamine and quinine activated the phagocytosis of b-haematin, whereas amodiaquine and chloroquine inhibited b-haematin phagocytosis. Doxycycline had different effects on each cell type investigated. Artemisinin, chloroquine, primaquine and quinine inhibited latex bead phagocytosis. The remaining drugs had minimal effects on latex bead phagocytosis. Thus, the effects of antimalarial drugs on monocyte phagocytosis appear to be dependent on the substance being phagocytosed. The effects of antimalarial drugs, b-haematin, latex beads, non-infected- and P. falciparuminfected cell lysates on interferon-g-induced neopterin secretion by U937 cells was monitored by GTP-cyclohydrolase 1 mRNA expression using quantitative PCR. Artemisinin, primaquine and quinine down-regulated the interferon-g-induced expression of GTPcyclohydrolase 1 mRNA, but by no greater than 1.7-fold. b-haematin up-regulated mRNA expression by 1.2-fold whereas P. falciparum-infected red blood cell lysate down-regulated the mRNA expression of GTP-cyclohydrolase 1 by 1.6-fold. Quinine and artemisinin, currently used to treat malaria, increased b-haematin phagocytosis suggesting that quinine and artemisinin might promote increased phagocytosis of infected red blood cells and enhance clearance of the parasite from circulation. Increased b- haematin phagocytosis also reduces ICAM-1 expression on the monocyte surface, thereby leading to reduced cytoadherance and sequestration, thus increasing the number of circulating monocytes to phagocytose infected red blood cells. Down regulation of GTPcyclohydrolase 1 mRNA expression by quinine and artemisinin suggested that the drugs reduce the responsiveness of the monocyte to interferon-g. Thus, quinine and artemisinin might also decrease the production of interferon-g-induced proinflammatory cytokines by monocytes, and potentially play a role in maintaining the balance between the pro- and antiinflammatory cytokines that determines the progression from acute to severe malaria. Therefore, in addition to the drug’s ability to kill the malaria parasite, the immunomodulatory effects of the antimalarial drugs may play a role in controlling the pathophysiology associated with the malaria infection.