Simultaneous Determination of Phagocytosis of Plasmodium Falciparum-parasitized and Non-parasitized Red Blood Cells by Flow Cytometry

Overview

Journal Malar J

Publisher Biomed Central

Specialty Tropical Medicine

Date 2012 Dec 25

PMID 23259636

Citations 12

Authors

Valentina Gallo

Oleksii A Skorokhod

Evelin Schwarzer

Paolo Arese

Affiliations

Soon will be listed here.

Abstract

Background: Severe falciparum malaria anaemia (SMA) is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs) in excess of loss of parasitized (p-) RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC.Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample.

Methods And Results: Phagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFNγ to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE) and the DNA label ethidium bromide (EB). EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia.

Conclusions: The assay allowed the analysis of phagocytosis rapidly and with low subjective error, and the differentiation between phagocytosed p- and np-RBCs in the same sample. The presented method may help to analyse the factors or conditions that modulate the share of np-RBC removal in vitro and in vivo and lead to a better understanding of the pathogenesis of SMA.

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References

Fendel R, Mordmuller B, Kreidenweiss A, Rudat A, Steur C, Ambrosch C . New method to quantify erythrophagocytosis by autologous monocytes. Cytometry A. 2007; 71(4):258-64. DOI: 10.1002/cyto.a.20360. View

Murphy S, Breman J . Gaps in the childhood malaria burden in Africa: cerebral malaria, neurological sequelae, anemia, respiratory distress, hypoglycemia, and complications of pregnancy. Am J Trop Med Hyg. 2001; 64(1-2 Suppl):57-67. DOI: 10.4269/ajtmh.2001.64.57. View

Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K . Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer. 1980; 26(2):171-6. DOI: 10.1002/ijc.2910260208. View

Uyoga S, Skorokhod O, Opiyo M, Orori E, Williams T, Arese P . Transfer of 4-hydroxynonenal from parasitized to non-parasitized erythrocytes in rosettes. Proposed role in severe malaria anemia. Br J Haematol. 2012; 157(1):116-24. PMC: 3412292. DOI: 10.1111/j.1365-2141.2011.09015.x. View

Abdalla S . Peripheral blood and bone marrow leucocytes in Gambian children with malaria: numerical changes and evaluation of phagocytosis. Ann Trop Paediatr. 1988; 8(4):250-8. DOI: 10.1080/02724936.1988.11748582. View

Drevets D, Leenen P, Campbell P . Complement receptor type 3 mediates phagocytosis and killing of Listeria monocytogenes by a TNF-alpha- and IFN-gamma-stimulated macrophage precursor hybrid. Cell Immunol. 1996; 169(1):1-6. DOI: 10.1006/cimm.1996.0083. View

Jakeman G, Saul A, Hogarth W, Collins W . Anaemia of acute malaria infections in non-immune patients primarily results from destruction of uninfected erythrocytes. Parasitology. 1999; 119 ( Pt 2):127-33. DOI: 10.1017/s0031182099004564. View

Wang X, Duan X, Liu L, Fang Y, Tan Y . Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling. Acta Biochim Biophys Sin (Shanghai). 2005; 37(6):379-85. DOI: 10.1111/j.1745-7270.2005.00051.x. View

Goka B, Kwarko H, Kurtzhals J, Gyan B, Ohene S, Hviid L . Complement binding to erythrocytes is associated with macrophage activation and reduced haemoglobin in Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg. 2001; 95(5):545-9. DOI: 10.1016/s0035-9203(01)90036-7. View

10.

Collins W, Jeffery G, Roberts J . A retrospective examination of anemia during infection of humans with Plasmodium vivax. Am J Trop Med Hyg. 2003; 68(4):410-2. View

11.

Evans K, Hansen D, van Rooijen N, Buckingham L, Schofield L . Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes. Blood. 2005; 107(3):1192-9. PMC: 1895912. DOI: 10.1182/blood-2005-08-3460. View

12.

Turrini F, Ginsburg H, Bussolino F, Pescarmona G, Serra M, Arese P . Phagocytosis of Plasmodium falciparum-infected human red blood cells by human monocytes: involvement of immune and nonimmune determinants and dependence on parasite developmental stage. Blood. 1992; 80(3):801-8. View

13.

Lutz H . Innate immune and non-immune mediators of erythrocyte clearance. Cell Mol Biol (Noisy-le-grand). 2004; 50(2):107-16. View

14.

Looareesuwan S, Merry A, Phillips R, Pleehachinda R, Wattanagoon Y, Ho M . Reduced erythrocyte survival following clearance of malarial parasitaemia in Thai patients. Br J Haematol. 1987; 67(4):473-8. DOI: 10.1111/j.1365-2141.1987.tb06171.x. View

15.

Schwarzer E, Turrini F, Arese P . A luminescence method for the quantitative determination of phagocytosis of erythrocytes, of malaria-parasitized erythrocytes and of malarial pigment. Br J Haematol. 1994; 88(4):740-5. DOI: 10.1111/j.1365-2141.1994.tb05112.x. View

16.

Facer C, Brown J . Monocyte erythrophagocytosis in falciparum malaria. Lancet. 1981; 1(8225):897-8. DOI: 10.1016/s0140-6736(81)92172-3. View

17.

Skorokhod O, Schwarzer E, Ceretto M, Arese P . Malarial pigment haemozoin, IFN-gamma, TNF-alpha, IL-1beta and LPS do not stimulate expression of inducible nitric oxide synthase and production of nitric oxide in immuno-purified human monocytes. Malar J. 2007; 6:73. PMC: 1904226. DOI: 10.1186/1475-2875-6-73. View

18.

Lyons A . Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J Immunol Methods. 2000; 243(1-2):147-54. DOI: 10.1016/s0022-1759(00)00231-3. View

19.

Casals-Pascual C, Roberts D . Severe malarial anaemia. Curr Mol Med. 2006; 6(2):155-68. DOI: 10.2174/156652406776055159. View

20.

Kitagawa S, Yuo A, Yagisawa M, Azuma E, Yoshida M, Furukawa Y . Activation of human monocyte functions by tumor necrosis factor: rapid priming for enhanced release of superoxide and erythrophagocytosis, but no direct triggering of superoxide release. Exp Hematol. 1996; 24(4):559-67. View