| PrefaceMalaria is a kind of infectious diseases with high concern all over the world.Nature's latest reports indicate that the amount of the world's malaria patients is 515million and the population threated is 2.2 billion in 2002. Malaria, AIDS andtuberculosis as infectious diseases have been taken as the focal points of prevention andtherapy by WHO. An improved understanding of the mechanisms of host protectionimmunity against malaria may facilitate the development of new therapeuticinterventions.Monocyte-macrophages obviously impact the Plasmodium infectious process indifferent aspects.As nonspecific immune system cells ,macrophages play thenonspecific phagocytosis and killing effect, also may take part in specific immuneresponse, antigen presentation, and play an important role in immune regulation. Anumber of recent studies indicated that, the distinctive characteristics of blood-stageprotective immunity is orderly activation of Th1 and Th2-type immune response,withCD4+Thl cells and antibodies's participation. Macrophage function is up-regulated bythe potent macrophage activating factor IFN-γ(Th1-type cytokines), which is producedearly during acute blood-stage malaria. Subsequently host protection immuntiytransition to Th2 -type response,finally through antibody-mediated immune responseeliminate malaria parasites. Ours previous research demonstrated that, activatemacrophages suppressed acute parasitemia and improved the survival of mice duringearly stage Plasmodium yoelii infection. However, during whole process of malarial infection,the activation and effect of macrophage still need to be further explored andexplained.In the experiments reported here, we investigated the function of splenicmacrophages from Plasmodium yoelii-infected resistant DBA/2 mice with respect totheir ability to express surface membrane molecular,secret effector molecules NO,andtheir phagocytic function. In order to further clarify activation and effect of splenicmacrophages during malarial infection, we aimed at providing a new theoretical andexperimental basis to an effective malarial vaccine and anti-malarial drugs'sdevelopment.Materials and methodsUtilizing Giemsa-stained blood smears from tail blood, count of erythrocyticinfected rate through optical microscope; applying Flow Cytometry to quantitativelyanalyze splenic macrophages and the expression of CD36, CD64 and MHCâ…¡onsplenic macrophages; concentrations of NO in cell supernatants were measured by theGriess reaction; phagocytosis of mononuclear macrophages was also observed. Datawere presented as means±standard errors of the means (SEM). Statistical significanceof differences were analyzed by Student's t test. A value of P＜0.05 was consideredsignificant.ResultsPrimary parasitemia in vein blood was developed on day 2 postinfection, whichpeaked with a level of 33.87% on day 7. Most mice cleared the infection and survivedby day 15 postinfection; splenocytes from infected mice produced significantly higherlevel of NO on day3and5 postinfection and rate of macrophage phagocytosis increasedon day 5, which peaked on day 10, then continued to remain high level; the level of MHCâ…¡and CD36 increased at day 3 which peaked on day 10,CD64 increased at day5 then continued to remain high in DBA/2 mice, simultaneously with the amount ofmacrophage significantly increased on day 3 which peaked on day 10.DiscussionIncreasing evidence indicates that innate immune responses contribute to thecontrol of blood-stage malaria infection, lighten the parasite burden, and decreaseprogression to severe disease. Macrophage has the remarkable non-specificphagocytosis and killing capability, its merit could be further strengthen by the potentmacrophage activating factor IFN-γ. Previous reports indicate that circulatingmonocytes and tissue resident macrophages in the liver,spleen,and elsewhere facilitatethe control and resolution of infection by cleating parasitized erythrocytes. Our earlierstudies already confirmed that, CD4+ Thl cell from resistant DBA/2 mice may producehigh level of IFN-γin the early stage of infection, and activated macrophages play animportant role in controlling early blood parasitemia levels during acute infection.Here,this research used lethal Plasmodium yoelii (P. y 17XL)and resistant DBA/2 micemodel, systematically observed activation and effect of splenic macrophage in thewhole course of P. y 17XL infection.Result showed that, splenic macrophages wasrapidly activated in the early stage, and killed malaria parasites by secreting effectormolecule NO and nonspecific phagocytosis of pRBCs.The results suggested thatsplenic macrophages is important effector cells in protection immune against murinemalaria.Past research have suggested, as a professional antigen-presenting cells,macrophages presents antigen to T cells by expressing MHC molecules andcostimulatory molecules, provides the essential activated signal of T celldifferentiation,and then controlls the subsequent outcome of the malarial infection.MHCâ…¡is an important indicator of macrophage activation. MHCâ…¡knockout micestudies has shown that protection against blood-stage infection of non-lethalPlasmodium yoelii and P. chabaudi chabaudi completely dependented on MHCâ…¡molecules.After infected non-lethal Plasmodium yoelii, macrophages from BALB/c mice obviously increased the expression of CD80 and MHCâ…¡, could promotedactivation of T-cell and production of IFN-γ. This research further discovered that, inearly stage of P. y 17XL infection ,macrophages markedly enhanced the expression ofMHCâ…¡,and simultaneously accompanied by a notable increasing in macrophagesquantity, it suggested splenic macrophages had been effectively activated under IFN-γ,stimulating.Evidences already confirmed that macrophages is the most important source ofnonspecific immune effector molecule NO. Nitrogen peroxide is formed by oxidationof nitric oxide ,and it is the major effector molecule of macrophages displaying killingtoxic effect. In vitro experiment demonstrated, NO display cytotoxicity and inhibitoryof parasite growth in blood-stage infection. Although it is still disputing that NO play aprotective role in controlling of blood-stage malaria, but our earlier research alreadydiscovered that, in the early stage infection, the tendency which the percentage ofinfected erythrocyte unceasingly rising was inhibited along with NO synthesisincreasing, and a lot of merozoite attaching reticulocyte surface could be seen. It isspeculated that the increasing level of NO in the early stage could block invasion ofPlasm od ium merozoites into red blood cells. The results of ours study showed that, onday 4 after infection, although splenic macrophage from infected DBA/2 mice has yetnot been shown phagocytosis function, however the level of NO in spleen cell culturesupernatants were found significantly increased on day 3 after infection, meanwhile,parasitemia presented slowly rising trendency. These data indicated that in early stagemalarial infection stage, nitric oxide which was secreted by macrophages may play ainhibitory action of plasmodium.Two types of macrophage non-specific phagocytosis have been described:opsonin-dependent and opsonin-independent phagocytosis. For opsonin-independentphagocytosis, macrophages recognize and phagocytose the microorganism viapattern-recognition receptors, such as scavenger and mannose receptors,it has beenshown to contribute to early or innate resistance to a number of bacterial pathogens.CD36 is a member of a novel family of scavenger receptors, at present we consider that CD36 play an important role to mediate macrophage nonopsonic phagocytosis. Studiesfrom areas where malaria is endemic have reported that higher levels of binding ofplasmodium parasitized erythrocytes to CD36 are associated with nonsevere malaria.In addition, a population-based study by Aitman et al reported that CD36-deficientindividuals were at greater risk of developing severe and cerebral malaria. Someevidence indicates that pretreatment macrophages with PPARγ, agonists, which increasethe expression of CD36, macrophage from human and C57BL/6 mice and BALB/cmice significantly increases the phagocytosis of P. y 17XL-parasitized erythrocytes.Using MAb blockade of CD36 and CD36-null (KO) murine macrophages, Kodjo Ayiet al have found that CD36 was the major receptor involved in the phagocytic processin vitro, human monocyte-derived macrophages and murine macrophages were able tophagocytose Ring-stage Plasmodium falciparum-parasitized Erythrocytes(RPEs) invitro even in the absence of opsonization and in the presence of an Fc receptorblockade, that nonopsonic uptake in vitro appears to be largely dependent on CD36 andnot on other macrophage receptors such as ICAM-1,αvβ3, or PECAM-1 or patternrecognition receptors or CD14. Todd G. Smith's research found that phagocytosis ofstageâ… and IIA gametocytes by monocytes and macrophages appears to be mediated toa large extent by the interaction of PfEMP-1 and CD36, suggesting that CD36 may playa role in innate clearance of these early sexual stages. In the present study, we havedemonstrated that the expression of CD36 in splenic macrophages from DBA/2 miceinfected P.y 17XL began to increase on day 3 post-infection, with the infected processgradually increased and reached the peak on day 10, correspondingly, macrophagephagocytosis function was obviously enhancing. These data indicated that CD36 havebeen implicated in innate clearance of P. y 17XL-parasitized erythrocytes bymacrophages in early infection.After acute infection stage, against blood-stage malarial infection protectionfinally depended on the specificity antibody-mediated immune responses to eliminateplasmodium. Ab-dependent opsonic phagocytosis is one of the important effector mechanism to the acquired immunity of blood-stage malaria infection. FcR-mediatedAb-dependent phagocytosis is important as an effector mechanism to eliminate PRBCin blood-stage P. berghei XAT infection ,phagocytic activity of splenic macrophageswas demonstrated to be important for the resistance by using its inhibitor, carrageenan.FcRγ-/- mice which lack the ability to mediate Ab-dependent phagocytosis and ADCCmarkedly enhanced the susceptibility of P. berghei XAT and all of these mice diedeventually. Mota and colleagues have demonstrated that peritoneal macrophages fromCBA mice show increased phagocytosis of antibody-opsonized P. chabaudi-infectedred blood cells (PRBCs) and that this antibody-mediated phagocytosis is parasitespecies specific. We previously demonstrated that the level of IgG antibody in DBA/2mice increased on dayl0 after P. y 17XL infection. This research found that theexpression of Fcγ,R I (CD64) started to elevate on day 5 after the infection, andgradually rised and continued to maintain the high level. At the same time, macrophagephagocytosis rate was in the higher level during 8d-15d after infection. It wassuggested that,in the late stage of infection, with specific antibodyproducing ,macrophage may play opsonic phagocytosis effect through CD64.In sum, this study dynamically observated activation and effect of splenicmacrophage from resistant DBA/2 mice infected P. y 17XL, the result revealsmacrophages may play an important role in both innate immunity and acquiredimmunity in malarial infection. Realizing the immune effect of macrophagessufficiently especially clarifying the role and mechanism may possess an importantscientific significance for the development of effective malaria vaccine andanti-malaria drugs.ConclusionThis study dynamically observated activation and effect of splenic macrophagefrom resistant DBA/2 mice infected P. y 17XL, the result reveals macrophages mayplay an important role in both innate immunity and acquired immunity in malarial infection.1. The quantity of splenic macrophages from DBA/2 mice has gradually increasedduring P. yl 7XL infection.2. Through the expression of MHCâ…¡, it suggested that splenic macrophages hadbeen effectively activated under IFN-γstimulating.3. The obviously increased expression of the scavenger receptor CD36 in the earlystage indicates that CD36 is involved in mediating non-opsonic phagocytosis ofmacrophages.4. The significantly increased expression of FcγRI(CD64) in the late stageindicates that CD64 maybe involved in antibody-mediated opsonic phagocytosis ofmacrophages.5. In early stage of malarial infection, nitric oxide, secreted by macrophages, mayexert an inhibitory effect on plasmodium Infection. |
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