Serum cytokine TNF-alpha and hemoglobin levels in Plasmodium falciparum malaria Original Article 293major factors for the high disability-adjusted life years affected by P. falciparum along with sex and age matchedattributed to malaria.3 The mechanisms of severe malarial healthy controls.anemia are the subject of intense study.4,5 Many factorshave been reported to inﬂuence its pathogenesis, but themechanisms themselves remain controversial.6 MATERIALS AND METHODS The increased destruction and phagocytosis of infectedand uninfected erythrocytes, the suppression of erythropoi-esis by relatively impaired erythropoietin production, the Study area and patientsautoimmune lysis of both parasitized and normal erythro- The study was carried out in S.C.B. Medical, College,cytes, and reticuloendothelial hyperfunction seem to be Hospital, Cuttack, Odisha, Department of Medicine andimportant causative factors, but they do not adequately Department of Biochemistry. This hospital is the largestexplain the severity and extent of anemia. Furthermore, government institution catering to the entire coastal beltanemia can persist for weeks after effective antimalarial treat- of Odisha. Therefore patients from interior rural areas visitment.7 Although the pathological basis for the development the hospital for treatment.of malarial anemia is not yet well understood, the participa- Subjects were chosen from amongst malaria patientstion of cytokines8 and of autoantibodies has been considered. admitted to medicine indoor ward of S.C.B. Medical Some works have suggested that severe anemia is associ- College Hospital. P. falciparum malaria was deﬁnedated with predominant T-helper 1 (Th1) responses, character- according to modiﬁed World Health Organization criteria.ized by high levels of tumor necrosis factor alpha (TNF-a) in Graph showing correlation between TNF-alpha andrelation to interleukin-10 (IL-10) levels, and conversely, hemoglobin in pf malaria patients.protection from this complication was associated with aninverse relationship, i.e., with a balance toward a highIL-10/TNF-a ratio. Other cytokines and chemokines, partic-ularly those involved in macrophage migration and activity,such as migration inhibitory factor (MIF) and monocytechemotactic protein-1 (MCP-1), also can be involved. Sincehigh TNF-a production by macrophages can be inﬂuencedby phospholipids such as the Plasmodium-derived gly-cosylphosphatidylinositol,9,10 anti-phospholipid antibodies,which are common in malaria infections could play a rolein this system. Antibodies directed to erythrocyte membraneantigens, which also are present in malaria infections,could act by destroying parasitized and non-parasitizederythrocytes. Optimal immune response to malaria infection is charac-terized by early intense pro-inﬂammatory cytokine-mediatedeffectors mechanisms that kill or clear parasite-infected cellsand which are then equally rapidly suppressed by anti-inﬂam-matory effectors once parasite replication has been broughtunder control. The outcome of infection depends on a dedi-cated balance between appropriate and inappropriate induc-tion of these mediators. Early pro-inﬂammatory cytokine As a control group for cytokine determination 30 healthyresponses seem to mediate protective, whereas late response volunteers of same range of age and sex were also includedcontributes to pathology. This suggests that a crucial balance in the study. All volunteers enrolled as control group weremight exist during the inﬂammatory response to malaria negative at the thick smear examination for P. falciparuminfection. without febrile episodes during the last 6 months and The ﬁrst characterized parasite-induced cytokine was without sign of anemia (Hb > 10 g/dl).TNF alpha, induced in macrophages by erythrocytes Inclusion and classiﬁcation of each case were basedinfected by Plasmodium malarial pigment and certain on the symptoms, physical signs and laboratory ﬁndingglycolipids such as GPI moiety. of malaria at the onset of the disease. On the basis of This study was conducted to assess the correlation hematological parameters and evidence of neurologicalbetween TNF alpha levels and Hemoglobin in patients involvement severity of malaria was established. The
294 Apollo Medicine 2012 December; Vol. 9, No. 4 Sarangi et al.malaria patients admitted to Medicine ward from correlation between hemoglobin and TNF-alphaNovember 2009 to October 2010 were included in the (r ¼ À0.96) was found indicating an important associationstudy. If as per the inclusion criteria the patient was correlating TNF-alpha levels with anemia.found ﬁt to be included in the study then the consentform was obtained duly signed and the patients wereincluded in the study. Blood hemoglobin was measured DISCUSSIONby cyanmethemoglobin method. Patient informed consentform and Volunteer’s informed consent form were obtained In human malaria altered immune reactivity appears late infrom the participants in the study and the ethical clearance the acute phase of the diseases and can last a long time aftercertiﬁcate from the Institutional Ethics Committee of the clearance of parasites from the circulation. An explana-S.C.B Medical College, Cuttack is attached. tion for the poor acquisition of malaria unit in naturally exposed population is that the parasite actively modulates of speciﬁc immune response.11 The inﬂammatory responseSample collection that is needed to remove parasites lead to considerableBlood samples were drawn from malaria patients admitted tissue damage and activation of phagocytes to kill intracel-to medicine ward and transferred to Department for Hb esti- lular or extracellular parasites requires the production ofmation and ELISA for TNF alpha. Blood samples were inﬂammatory cytokines which can cause systemic effectscollected for immunological assessment in sterile tubes. such as severe anemia and cerebral malaria.12 The immuneAll the samples were centrifuged and serum was refriger- response to malaria likely is regulated by the balance ofated at À70 C in the Department of Biochemistry for pro- and anti-inﬂammatory cytokines that culminate indetermination of TNF-alpha. either immunoprotection or pathogenesis. In our study the Blood samples were drawn from healthy volunteers not TNF alpha was elevated in all patients of malaria as expres-infected with malaria and transferred to Department of sion of immune activation in response to the presence ofBiochemistry for Hb estimation and ELISA for TNF-alpha. parasites are determinants of malaria severity and outcome13 and can represent potential target for therapeutic interventions if their effect will be highlighted.Cytokine determination Cytokines TNF-alpha induced in macrophages by eryth- rocytes infected by Plasmodium malaria pigment andCytokine analysis was performed by Ray Biotech ELISA certain glycolipids such as GPI moiety. It has been shownassay kits (Ray Biotech Inc 3607 Parkway Lane, Suite that GPI moiety induces NOS in macrophages and activates200, Norcross GA 30092) for TNF-alpha as speciﬁed by endothelial cells by tyrosineekinase-mediated signal trans-the manufacturer. Each plate included a standard curve of duction. The amount of TNF-alpha produced by malariarecombinant human cytokine on logelog graph paper. All parasites seems to vary between people in the samespecimens were measured in duplicate and the mean of endemic area exposed to similar parasites and inoculationthe two values was taken. rates .TNF alpha has a role in the regulation of macrophage, interleukin 12 induced production and it has been shownStatistical analysis that TNF alpha is an important co-factor for interleukin production of interferon gamma by NK cells. Luty et al14Serum cytokine concentration was determined in dupli- showed a close association between the presence of severecate and expressed as mean Æ SE of the mean. Compari- anemia, high TNF alpha concentrations and large numberssons between groups were made using Z test with of circulating haemozoin-containing monocytes, suggestingstatistical signiﬁcance set at SE value of 1% level of that haemozoin-induced TNF alpha reduction plays a partsigniﬁcance. in either initiation or exacerbation of anemia as a clinical outcome of chronic, uncontrolled parasitemia. TNF alpha levels in falciparum malaria in the presentRESULTS study showed signiﬁcant elevation (272.48 Æ 335.05 pg/ ml) compared to healthy controls (42.90 Æ 13.5 pg/ml).In our study TNF alpha in falciparum malaria was signiﬁ- TNF alpha levels were higher in falciparum malaria signi-cantly elevated (631.25 Æ 382.26 pg/ml) compared to fying that TNF alpha may be an important component inhealthy controls (42.90 Æ 13.5 pg/ml) whereas Hb levels the pathogenesis of severe falciparum malaria and in partic-were signiﬁcantly reduced (8.2 Æ 0.54 g/dl) compared to ular in the cerebral syndrome and the hypoglycemia whichhealthy controls (13.21 Æ 0.85). A strong negative can complicate this disease.
Serum cytokine TNF-alpha and hemoglobin levels in Plasmodium falciparum malaria Original Article 295 The anemia of malaria is due to a combination of prema- fall was soon realized to be out of proportion to the numberture red cell destruction and inadequate red cell production. of red cells parasitized, so other factors were realized toThe two mechanisms often overlap and each may be the contribute. Phagocytosis of unparasitized red cells wasresult of different pathways. Red cell destruction, for also recorded decades ago in monkey and human malaria,example, involves both infected and uninfected cells whose and for many years was regarded as sufﬁcient explanationlifespan may be shortened by nonimmune and immune for this discrepancy.mechanisms. Inadequate production may be due to the Others had been investigating dyserythropoiesis in thesuppression of erythropoiesis, or dysfunctional erythropoi- bone marrow of patients with falciparum malaria andesis (dyserythropoiesis), where the bone marrow erythroid stressed its contribution to malarial anemia. A group incells are increased but mature red cell output is inadequate. Oxford seeking an explanation for this dyserythropoiesis As recently reviewed,15 critical illness associated with an through an electron microscopy study of bone marrow,inﬂammatory response invariably causes multifactorial observed sequestration of parasitized red cells and arguedanemia. It has often been noted that anemia could that this caused the bone marrow dysfunction in falciparumcontribute to poor oxygenation of tissues in malaria and malaria by restricting blood ﬂow and thus inducing hypoxicthere is general acceptance that it can be severe enough changes.to reduce the supply of oxygen to mitochondria to danger- Twenty-ﬁve years ago our group proposed that TNFously low levels. Thus it can be a major component of might cause the bone marrow depression seen in malaria.malarial pathology. Subsequently an undeﬁned product in macrophage superna- Erythrocytes have a limited life, determined by how long tants, later identiﬁed as TNF, was found to inhibit thethey can remain ﬂexible enough to squeeze through fenes- growth and differentiation of erythroid progenitor cells.trations in specialized vessels in the red pulp of the spleen. When rTNF became available (but before it had becomeA red cell that cannot pass this test is phagocytosed by adja- technically possible to assay for this cytokine in humancent macrophages, and lost. In health this loss is balanced serum) the dyserythropoiesis and erythrophagocytosisby erythropoiesis, and hematocrit remains normal. Should seen in terminal Plasmodium vincke infected mice werered cells develop a premature poor deformability they are reproduced when a single injection of rTNF was given earlyremoved from the circulation correspondingly earlier. in the course of the infection. Like other cells, erythrocytes stay intact by constantly Phagocytosis of erythroblasts in bone marrow,extruding Naþ in exchange for Kþ through an energy a phenomenon also reported by Wickramasinghe in humandependant “pump” in their cell membrane that was deﬁned malaria, was commonly observed. Decreased erythropoiesisby the ability of certain digitalis gylcosides to block it. This was subsequently reported in mice receiving continuousNaþ/Kþ pump fails, and intracellular Naþ accumulates TNF infusions via implanted osmotic pumps, and increasedin (non-parasitized as well as parasitized) red cells erythropoiesis in malarial mice after injecting neutralizingduring human or monkey malaria. Since inhibition of the antibody directed against murine TNF. TNF-induced dyser-Naþ/Kþ pump in vitro correlates with a reduced red cell ythropoiesis has since been conﬁrmed in rats, and micedeformability plus a parallel decrease in red cell ﬁlter- expressing high levels of human TNF become markedlyability, any inﬂuence, such as NO, that inhibits this pump anemic during malaria infections, even though parasitecould potentially cause poor red cell deformability. Cyto- numbers, and therefore red cell loss post-schizogony, arekine-induced iNOS provides a demonstrable16 way for considerably reduced.these changes to occur in severe malaria. There is good During the intraerythrocytic phase of its life cycle, theevidence that, when measured on admission, a severe malaria parasite matures within a cell in which hemo-reduction in red cell deformability is a strong predictor of globin is the single major cytosolic protein. While in themalarial mortality, but whether this is cause and effect, or trophozoite stage, the parasite avidly ingests and degradesthe two phenomena are simply inevitable co-travelers in host erythrocyte hemoglobin by means of a specializeda strong pro-inﬂammatory milieu, is unclear. It seems clear structure called a cytostome, which spans the doublethat poor red cell deformability (which affects parasitized membrane between erythrocyte and parasite cytoplasm.and unparasitized red cells equally) and dyserythropoiesis Hemoglobin-containing vesicles are pinched off from thecan lead to severe anemia in various diseases, particularly cytostome and travel to the digestive vacuole where thein chronic infections such as malaria. Because the parasite hemoglobin is broken down. The process of hemoglobininhabits erythrocytes, which must burst if the parasite is degradation releases heme, which accumulates in crystal-to propagate, the obvious initial conclusion was that this line particles within the digestive vacuoles .The formationsource of red cell loss was central to the fall in hematocrit of these pigmented crystals (called hemozoin) is poorlyseen in this disease. As reviewed nearly 60 years ago this understood.
296 Apollo Medicine 2012 December; Vol. 9, No. 4 Sarangi et al. Since the parasite has a limited capacity to synthesize 4. McDevitt MA, Xie J, Shanmugasundaram G, et al. A criticalamino acids de novo or to take them up exogenously the role for the host mediator macrophage migration inhibitoryhemoglobin is thought to be broken down to provide amino factor in the pathogenesis of malarial anemia. J Exp Med.acids for its growth and maturation. It has been estimated 2006;203:1185e1196.that between 25% and 75% of the hemoglobin in an 5. Mishra SK, Mohanty S, Mohanty A, et al. Management ofinfected erythrocyte is degraded. Therefore, in an average severe and complicated malaria. J Postgrad Med. 2006;52:patient with about 750 g of circulating hemoglobin and 281e287.a heavy malaria infection at 20% parasitemia, up to 100 g 6. Nussenblatt V, Mukasa G, Metzger A, et al. Anemia and inter-of hemoglobin is utilized during a single cycle. leukin-10, tumor necrosis factor alpha, and erythropoietin levels In our study the Hb values were signiﬁcantly reduced among children with acute, uncomplicated Plasmodium falcipa-(8.2 Æ 0.54 g/dl) in Pf patients indicating signiﬁcant degra- rum malaria. Clin Diagn Lab Immunol. 2001;8:1164e1170.dation of hemoglobin consequently leading to anemia, poor 7. Angulo I, Fresno M. Cytokines in the pathogenesis of andred cell deformability, dyserythropoiesis contributing protection against malaria. Clin Diagn Lab Immunol. 2002;9:a major component in malarial pathology. TNF causes 1145e1152.bone marrow depression seen in malaria, further was found 8. Chang KH, Stevenson MM. Effect of anemia and renal cyto-to inhibit the growth and differentiation of erythroid kine production on erythropoietin production during blood-progenitor cells leading to TNF-induced dyserythropoiesis stage malaria. Kidney Int. 2004;65:1640e1646.and anemia. We found strong negative correlation between 9. Debierre-Grockiego F, Schoﬁeld L, Azzouz N, et al. Fattyhemoglobin and TNF-alpha (r ¼ À0.96) indicating acids from Plasmodium falciparum down-regulate the toxica predominant role of TNF alpha in inducing anemia, activity of malaria glycosylphosphatidylinositols. Infecta major component of malarial pathology. Immun. 2006;74:5487e5496. 10. Weatherall DJ, Miller LH, Baruch DI, et al. Malaria and the red cell. Hematology; 2002:35e57.CONFLICTS OF INTEREST 11. Plebanski M, Hill AV. The immunology of malaria. Curr Opin Immunol. 2000;12:437e441.All authors have none to declare. 12. McGuire W, Hill AV, Allsopp CE, et al. Variation in the TNF-alpha promoter region associated with susceptibility toREFERENCES cerebral malaria. Nature. 1994;371:508e510. 13. Malaguarnera L, Musumeci S. The immune response to Plasmo-1. World Health Organization. Malaria. WHO Fact Sheet. dium falciparum malaria. Lancet Infect Dis. 2002;2:472e478. 1995;94:1e3. 14. Luty AJ, Perkins DJ, Lell B, et al. Low interleukin 12 activity2. Murphy SC, Breman JG. Gaps in the childhood malaria burden in severe Plasmodium falciparum malaria. Infect Immun. in Africa: cerebral malaria, neurological sequelae, anemia, 2000;68:3909e3915. respiratory distress, hypoglycaemia, and complications of preg- 15. Scharte M, Fink MP. Red blood cell physiology in critical nancy. Am J Trop Med Hyg. 2001;64(suppl 1e2):57e67. illness. Crit Care Med. 2003;31:S651eS657.3. Owusu-Agyei S, Fryauff DJ, Chandramohan D, et al. Charac- 16. Clark IA, Awburn MM, Whitten RO, et al. Tissue distribution teristics of severe anemia and its association with malaria in of migration inhibitory factor and inducible nitric oxide syn- young children of the KassenaeNankana district of northern thase in falciparum malaria and sepsis in African children. Ghana. Am J Trop Med Hyg. 2002;67:371e377. Malar J. 2003;2:6.