Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay

Wanderley, J.L.M.; Benjamin, A.; Real, F.; Bonomo, A.; Moreira, M.E.C.; Barcinski, M.A.

doi:10.1590/S0100-879X2005000600001

Abstract

Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS) exposure and its recognition by a specific receptor (PSR) were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane - an early hallmark of apoptosis - and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.

Apoptosis; Phosphatidylserine; Leishmania; Phagocyte

Braz J Med Biol Res, June 2005, Volume 38(06) 807-812 (Review)

Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay

J.L.M. Wanderley^1,2, A. Benjamin^1,2, F. Real^1,3, A. Bonomo^1,4, M.E.C. Moreira¹ and M.A. Barcinski^1,5

¹Instituto Nacional de Câncer, Rio de Janeiro, RJ, Brasil

2Programa de Pós-Graduação em Ciências Morfológicas, ICB, UFRJ, Rio de Janeiro, RJ, Brasil

3Instituto de Ciências Biomédicas, and ⁴Instituto de Microbiologia Professor Paulo de Góes, UFRJ, Rio de Janeiro, RJ, Brasil

5Departamento de Parasitologia, ICB, USP, São Paulo, SP, Brasil

^Text

References

Correspondence and Footnotes ^{Correspondence and Footnotes} Correspondence and Footnotes

Abstract

Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS) exposure and its recognition by a specific receptor (PSR) were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane - an early hallmark of apoptosis - and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.

Key words: Apoptosis, Phosphatidylserine, Leishmania, Phagocyte

Leishmania parasites

Parasites of the genus Leishmania are the causative agents of human leishmaniasis. The disease ranges from a healing skin lesion to a potentially fatal disseminating disease. Leishmania affects over 80 countries and a half million people, being a global health problem. The parasites are transmitted by sandflies of the subgenera Phlebotomus (Old World) and Lutzomyia (New World) by inoculating infective metacyclic promastigote forms. These forms are internalized by host macrophages and differentiate into non-motile amastigote forms which are able to proliferate inside phagolysosomes, thus being responsible for the maintenance and propagation of the infection (1).

Leishmania parasites have developed different strategies to invade host macrophages. Promastigotes display two major surface molecules - lipophosphoglycan (LPG) and glycoprotein 63 (gp63) - that play a fundamental role in Leishmania virulence. LPG is the major component of the promastigote's glycocalix, which blocks complement-mediated lysis in metacyclic forms due to a specific molecular alteration (2). gp63 is a metalloprotease that cleaves the C3b form of complement into the inactive iC3b form and also blocks complement-mediated lysis (3). Therefore, iC3b can opsonize the parasite for phagocytosis through complement receptors 3 and 1 (CR3 and CR1), targeting the parasite for uptake by macrophages (4). In amastigote forms the antibody-mediated phagocytosis by Fcg receptors plays an important role in parasite internalization (5). However, there is little evidence to date of intrinsic virulence factors in amastigote forms. Recently, we observed that amastigote forms expose phosphatidylserine (PS) on their surface - an early hallmark of apoptosis. This phospholipid plays an important role in amastigote internalization and survival inside host macrophages (6). The possible mechanisms underlying PS participation in the pathogenesis of leishmaniasis and the implication of apoptotic features exhibited by unicellular parasites will be discussed here.

Programmed cell death: unicellularity x multicellularity

Programmed cell death (PCD) is a mechanism involving differential expression of specific target genes. Apoptosis, described by Kerr et al. (7), is the most common phenotype of PCD. Apoptotic cells are characterized by morphological markers such as cell shrinkage, PS exposure, membrane blebbing, DNA fragmentation, and packaging of cell contents into apoptotic bodies (8-11). This kind of cell death is non-inflammatory, especially because it prevents the release of cytoplasmic contents since apoptotic bodies are quickly cleared by phagocytes.

Multicellular organisms depend on extensive renewal of senescent, damaged or harmful cells and on an efficient mechanism for death and removal of these cells. Cell death is also required for the maintenance of homeostasis and participates in organogenesis during development. Undoubtedly, apoptosis is advantageous for multicellular organisms but not necessarily for unicellular organisms. However, nowadays much evidence of apoptosis is available also for unicellular organisms. The slime mold Dictyostelium discoideum exposes PS on the surface when submitted to nutritional deprivation (12). Chloroquine-treated strains of Plasmodium falciparum present DNA fragmentation in an oligonucleosomal pattern (13). Some evolutionary forms of Trypanosoma brucei rhodesiense die inside the vector, displaying DNA fragmentation and membrane blebbing (14). A subpopulation of Saccharomyces cerevisiae exposes PS and presents DNA fragmentation in aged cultures (15).

What are the benefits of apoptosis for unicellular organisms? Two main hypotheses have been proposed to answer this question. First, cell death can be important for population size control when there are insufficient nutrients (free-living organisms) or to avoid host death (parasitic organisms). In this case unicellular apoptotic cells show an altruistic behavior, dying for the benefit of others. The second explanation is that apoptotic cells, which will not necessarily die, could provide signals that enhance the survival of the entire population.

Phosphatidylserine and infectivity

PS is a structural phospholipid normally localized on the inner surface of the plasma membrane. Apoptotic cells lose membrane asymmetry, and PS is exposed on the outer leaflet of the plasma membrane. Recognition of PS on the surface of apoptotic cells drives phagocytes to internalize these cells (16,17). In mammals there are redundant receptors for apoptotic cell clearance. Most of them use PS as their ligand but the only one able to recognize PS specifically and directly is the recently described PS receptor (PSR) (18). PS/PSR interaction leads to a macropinocytic activity of macrophages generating the endocytic pathway for apoptotic cell clearance. PS recognition by this receptor also leads to transforming growth factor ß₁ (TGF-ß₁) production by phagocytic cells (19). This cytokine inhibits the inflammatory response in an autocrine and paracrine manner. It has been recently reported that a_vß₃ integrin can also mediate TGF-ß₁ production through PS recognition (20). We have previously shown that amastigote forms of Leishmania (L) amazonensis expose PS on their surface and use this ligand to penetrate host macrophages. PS recognition leads to enhanced TGF-ß₁ secretion and interleukin-10 (IL-10) mRNA production. Pretreatment of amastigotes with annexin V - which binds to PS at high calcium concentrations - inhibited amastigote infectivity by at least 50% and significantly reduced TGF-ß₁ production by infected macrophages (6). Thus, PS signaling in amastigote forms functions like apoptotic cells according to a mechanism denoted by us as "apoptotic mimicry".

Some anti-leishmanial pharmacological agents induce apoptotic death of the parasite. For example, 12 h after antimony addition to macrophages infected with L. donovani, intracellular parasites expose PS and display DNA fragmentation (21). The high levels of drug-induced parasite death very probably preclude any eventual advantage of PS signaling. PS exposure is a natural phenotype displayed by a subpopulation of purified amastigote forms and this characteristic enhances the survival of the entire population. The exact mechanism by which PS exposure increases amastigote infectivity is not fully understood.

Effects of phosphatidylserine signaling: a working hypothesis Arginase activity

Macrophages are the effector cells in the control of Leishmania infection although they are the preferential host cells for these parasites. Macrophage biology defines leishmanial survival. Activated murine macrophages metabolize L-arginine via two main pathways that are catalyzed by the inducible enzymes nitric oxide synthase (iNOS) and arginase. Nitric oxide (NO) is generated from the oxidation of L-arginine by iNOS. L-arginine can be converted by arginase to L-ornithine, the substrate for ornithine decarboxylase (ODC), which is the main enzyme in polyamine synthesis (22). Arginase and iNOS can be differentially induced in murine macrophages by two antagonistic cytokines. The Th1-type pro-inflammatory cytokine IFN-g up-regulates iNOS, whereas Th2-type IL-4 and IL-10, which are anti-inflammatory cytokines, induce arginase activity (23). TGF-ß₁ also enhances arginase activity in macrophages and hence increases polyamine synthesis (24). This dual capacity can be part of distinct metabolic programs since M-1 macrophages - derived from Th1-prone strains (C57Bl/6) - are easily activated to produce NO, and M-2 macrophages - derived from Th2-prone strains (BALB/c) - are easily activated to produce polyamines. Since NO is an inhibitor of cell replication while polyamines can stimulate replication, it seems that macrophages from Th1 and Th2 mice can influence immune reactions in opposite ways. In fact, it has already been shown that N-hydroxyl-L-arginine (a physiological inhibitor of arginase) controls cellular infection of L. infantum and L. major amastigotes, since it was able to inhibit arginase and parasite growth (25). Recently, it was shown that treatment of L. major-infected BALB/c macrophages with IL-4, IL-10 or TGF-ß₁ led to a substantial increase in arginase activity and in the number of intracellular parasites. Furthermore, infected macrophages from C57Bl/6 mice displayed significantly less arginase activity when compared to infected BALB/c macrophages in response to these cytokines (26). These results can help to explain the course of infection observed in susceptible (BALB/c) and resistant (C57Bl/6) mice.

Freire-de-Lima et al. (20) reported convincing data correlating apoptotic cell recognition with polyamine synthesis and survival of intracellular Trypanosoma cruzi. Peritoneal macrophages treated with apoptotic cells showed a 5-fold enhancement in putrescine production and a 20-fold increase in ODC activity. Moreover, putrescine addition increased parasite growth in infected macrophages. ODC activity induced by apoptotic cell recognition was inhibited by an anti-TGF-ß₁ monoclonal antibody.

Based on these published data, we can postulate a model for the mechanisms by which PS enhances macrophage permissiveness to Leishmania. Recognition of PS on the surface of amastigotes leads to TGF-ß₁ release by infected macrophages. This cytokine then stimulates the arginase activity of infected and surrounding cells, consequently reducing NO production and increasing polyamine synthesis (Figure 1). Furthermore, we cannot exclude the fact that direct activation of PSR can lead to increased arginase activity, since PSR activation by an agonist antibody induces arginase protein expression (27).

Figure 1.
Macrophage modulation by amastigotes exposing phosphatidylserine. ARG = arginase activity; NO = nitric oxide; PS = phosphatidylserine; TGF-ß₁ = transforming growth factor ß₁.

Immune system modulation

Apoptotic cells seem to influence immune system physiology through interactions with dendritic cells (DC). Sauter et al. (28) showed that DC exposed to tumoral apoptotic cells do not mature. These cells, in contact with T lymphocytes, are unable to induce T cell proliferation because they lack proper expression of CD86 and CD80 molecules, contrasting with those that have engulfed fresh or necrotic tumor cells (28). These findings suggest that apoptotic cells can induce toleration of T lymphocytes, acting as an antigen carrier to maintain peripheral tolerance. This hypothesis was corroborated by Albert et al. (29) who demonstrated the necessity of DC maturation for cross-toleration of CD8⁺ T cells. They used a system to test the role of CD4⁺T cell/DC interplay in the activation of cross-presentation by DC. When antigen delivery to DC was through an apoptotic cell infected with influenza virus, these cells were unable to activate CD8⁺ T cells. The same occurred when DC did not interact with CD4⁺ T cells or lacked a CD40 stimulus (29). Many questions are still open concerning the mechanism by which apoptotic cells modulate antigen processing and presentation. In a recent review, Albert (30) postulated that many immunosuppressor signals given by apoptotic cells can participate in this mechanism such as TGF-ß₁, IL-10 and PS recognition by CD36 and PSR.

The role of DC in immune responses against Leishmania is well characterized, but only recently these cells were characterized as host cells for amastigote and promastigote proliferation, at least in vitro. Interestingly, only antibody-opsonized amastigotes or metacyclic promastigotes can induce DC maturation while amastigotes derived from nude mice - which produce only IgM antibodies - internalize and proliferate inside DC without inducing their maturation (31).

We propose that PS on the surface of amastigote forms of Leishmania may play a role in DC maturation and antigen presentation. In fact, DC matured in the presence of a modified PS molecule of Schistosoma mansoni induce a subpopulation of T cells that produce high levels of IL-10 (32). This suggests a potential role of PS signaling in the regulation of immune responses against pathogens.

Address for correspondence: M.A. Barcinski, Coordenação de Pesquisa, Instituto Nacional de Câncer, Rua André Cavalcanti, 3720231-050, Rio de Janeiro, RJ, Brasil. E-mail: barcinsk@inca.gov.br

Presented at the XI Congresso Brasileiro de Biologia Celular, Campinas, SP, Brazil, July 15-18, 2004. Publication supported by FAPESP. Received July 16, 2004. Accepted Janeiro 20, 2005.

1. Melby PC (2002). Recent developments in leishmaniasis. Current Opinion in Infectious Diseases, 15: 485-490.
2. Cunningham AC (2002). Parasitic adaptive mechanisms in infection by Leishmania Experimental and Molecular Pathology, 72: 132-141.
3. Brittingham A, Morrison CJ, McMaster WR, McGwire BS, Chang KP & Mosser DM (1995). Role of the Leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis. Journal of Immunology, 155: 3102-3111.
4. Wilson ME, Hardin KK & Donelson JE (1989). Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes. Journal of Immunology, 143: 678-684.
5. Dominguez M & Torano A (1999). Immune adherence-mediated opsonophagocytosis: the mechanism of Leishmania infection. Journal of Experimental Medicine, 189: 25-35.
6. de Freitas Balanco JM, Moreira ME, Bonomo A, Bozza PT, Amarante-Mendes G, Pirmez C & Barcinski MA (2001). Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity. Current Biology, 11: 1870-1873.
7. Kerr JF, Wyllie AH & Currie AR (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer, 26: 239-257.
8. Ohyama H, Yamada T, Ohkawa A & Watanabe I (1985). Radiation-induced formation of apoptotic bodies in rat thymus. Radiation Research, 101: 123-130.
9. Oberhammer FA, Hochegger K, Froschl G, Tiefenbacher R & Pavelka M (1994). Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase. Journal of Cell Biology, 126: 827-837.
10. Tounekti O, Belehradek Jr J & Mir LM (1995). Relationships between DNA fragmentation, chromatin condensation, and changes in flow cytometry profiles detected during apoptosis. Experimental Cell Research, 217: 506-516.
11. Daniel PT, Sturm I, Ritschel S, Friedrich K, Dorken B, Bendzko P & Hillebrand T (1999). Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers. Analytical Biochemistry, 266: 110-115.
12. Tatischeff I, Petit PX, Grodet A, Tissier JP, Duband-Goulet I & Ameisen JC (2001). Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis. European Journal of Cell Biology, 80: 428-441.
13. Al-Olayan EM, Williams GT & Hurd H (2003). Apoptosis in the malaria protozoan, Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito. International Journal of Parasitology, 32: 1133-1143. Erratum in: International Journal of Parasitology, 33: 105.
14. Welburn S, Barcinski M & Williams G (1997). Programmed cell death in trypanosomatids. Parasitology Today, 13: 22-26.
15. Herker E, Jungwirth H, Lehmann KA, Maldener C, Frohlich KU, Wissing S, Buttner S, Fehr M, Sigrist S & Madeo F (2004). Chronological aging leads to apoptosis in yeast. Journal of Cell Biology, 164: 501-507.
16. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL & Henson PM (1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. Journal of Immunology, 148: 2207-2216.
17. van den Eijnde SM, Boshart L, Baehrecke EH, De Zeeuw CI, Reutelingsperger CP & Vermeij-Keers C (1998). Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved. Apoptosis, 3: 9-16.
18. Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RA & Henson PM (2000). A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature, 405: 85-90.
19. McDonald PP, Fadok VA, Bratton D & Henson PM (1999). Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-beta in macrophages that have ingested apoptotic cells. Journal of Immunology, 163: 6164-6172.
20. Freire-de-Lima CG, Nascimento DO, Soares MB, Bozza PT, Castro-Faria-Neto HC, de-Mello FG, DosReis GA & Lopes MF (2000). Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature, 403: 199-203.
21. Sudhandiran G & Shaha C (2003). Antimonial-induced increase in intracellular Ca²⁺ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes. Journal of Biological Chemistry, 278: 25120-25132.
22. Mori M & Gotoh T (2000). Regulation of nitric oxide production by arginine metabolic enzymes. Biochemical and Biophysical Research Communications, 275: 715-719.
23. Modolell M, Corraliza IM, Link F, Soler G & Eichmann K (1995). Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines. European Journal of Immunology, 25: 1101-1104.
24. Boutard V, Havouis R, Fouqueray B, Philippe C, Moulinoux JP & Baud L (1995). Transforming growth factor-beta stimulates arginase activity in macrophages. Implications for the regulation of macrophage cytotoxicity. Journal of Immunology, 155: 2077-2084.
25. Iniesta V, Gomez-Nieto LC & Corraliza I (2001). The inhibition of arginase by N(omega)-hydroxy-arginine controls the growth of Leishmania inside macrophages. Journal of Experimental Medicine, 193: 777-784.
26. Iniesta V, Gomez-Nieto LC, Molano I, Mohedano A, Carcelen J, Miron C, Alonso C & Corraliza I (2002). Arginase I induction in macrophages, triggered by Th2-type cytokines, supports the growth of intracellular Leishmania parasites. Parasite Immunology, 24: 113-118.
27. Freire-de-Lima CG, Xiao YQ, Gardai SJ, Bratton DL, Fadok VA & Henson PM (2003). The interaction of apoptotic cells and phagocytes is non-inflammatory, actively anti-inflammatory and pleiotropic. XXVIII Meeting of the Brazilian Society of Immunology, ClubMed Village Rio das Pedras, Mangaratiba, RJ, Brazil, October 5-8.
28. Sauter B, Albert ML, Francisco L, Larsson M, Somersan S & Bhardwaj N (2000). Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. Journal of Experimental Medicine, 191: 423-434.
29. Albert ML, Jegathesan M & Darnell RB (2001). Dendritic cell maturation is required for the cross-toleration of CD8+ T cells. Nature Immunology, 2: 1010-1017.
30. Albert ML (2004). Death-defying immunity: do apoptotic cells influence antigen processing and presentation? Nature Reviews. Immunology, 4: 223-231.
31. Prina E, Abdi SZ, Lebastard M, Perret E, Winter N & Antoine JC (2004). Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation. Journal of Cell Science, 117 (Part 2): 315-325.
32. van der Kleij D, Latz E, Brouwers JF et al. (2002). A novel host-parasite lipid cross-talk. Schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and affects immune polarization. Journal of Biological Chemistry, 277: 48122-48129.

Correspondence and Footnotes

Publication Dates

Publication in this collection
28 Sept 2005
Date of issue
June 2005

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] 1. Melby PC (2002). Recent developments in leishmaniasis. Current Opinion in Infectious Diseases, 15: 485-490.

[2] 2. Cunningham AC (2002). Parasitic adaptive mechanisms in infection by Leishmania Experimental and Molecular Pathology, 72: 132-141.

[3] 3. Brittingham A, Morrison CJ, McMaster WR, McGwire BS, Chang KP & Mosser DM (1995). Role of the Leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis. Journal of Immunology, 155: 3102-3111.

[4] 4. Wilson ME, Hardin KK & Donelson JE (1989). Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes. Journal of Immunology, 143: 678-684.

[5] 5. Dominguez M & Torano A (1999). Immune adherence-mediated opsonophagocytosis: the mechanism of Leishmania infection. Journal of Experimental Medicine, 189: 25-35.

[6] 6. de Freitas Balanco JM, Moreira ME, Bonomo A, Bozza PT, Amarante-Mendes G, Pirmez C & Barcinski MA (2001). Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity. Current Biology, 11: 1870-1873.

[7] 7. Kerr JF, Wyllie AH & Currie AR (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer, 26: 239-257.

[8] 8. Ohyama H, Yamada T, Ohkawa A & Watanabe I (1985). Radiation-induced formation of apoptotic bodies in rat thymus. Radiation Research, 101: 123-130.

[9] 9. Oberhammer FA, Hochegger K, Froschl G, Tiefenbacher R & Pavelka M (1994). Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase. Journal of Cell Biology, 126: 827-837.

[10] 10. Tounekti O, Belehradek Jr J & Mir LM (1995). Relationships between DNA fragmentation, chromatin condensation, and changes in flow cytometry profiles detected during apoptosis. Experimental Cell Research, 217: 506-516.

[11] 11. Daniel PT, Sturm I, Ritschel S, Friedrich K, Dorken B, Bendzko P & Hillebrand T (1999). Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers. Analytical Biochemistry, 266: 110-115.

[12] 12. Tatischeff I, Petit PX, Grodet A, Tissier JP, Duband-Goulet I & Ameisen JC (2001). Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis. European Journal of Cell Biology, 80: 428-441.

[13] 13. Al-Olayan EM, Williams GT & Hurd H (2003). Apoptosis in the malaria protozoan, Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito. International Journal of Parasitology, 32: 1133-1143. Erratum in: International Journal of Parasitology, 33: 105.

[14] 14. Welburn S, Barcinski M & Williams G (1997). Programmed cell death in trypanosomatids. Parasitology Today, 13: 22-26.

[15] 15. Herker E, Jungwirth H, Lehmann KA, Maldener C, Frohlich KU, Wissing S, Buttner S, Fehr M, Sigrist S & Madeo F (2004). Chronological aging leads to apoptosis in yeast. Journal of Cell Biology, 164: 501-507.

[16] 16. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL & Henson PM (1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. Journal of Immunology, 148: 2207-2216.

[17] 17. van den Eijnde SM, Boshart L, Baehrecke EH, De Zeeuw CI, Reutelingsperger CP & Vermeij-Keers C (1998). Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved. Apoptosis, 3: 9-16.

[18] 18. Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RA & Henson PM (2000). A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature, 405: 85-90.

[19] 19. McDonald PP, Fadok VA, Bratton D & Henson PM (1999). Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-beta in macrophages that have ingested apoptotic cells. Journal of Immunology, 163: 6164-6172.

[20] 20. Freire-de-Lima CG, Nascimento DO, Soares MB, Bozza PT, Castro-Faria-Neto HC, de-Mello FG, DosReis GA & Lopes MF (2000). Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature, 403: 199-203.

[21] 21. Sudhandiran G & Shaha C (2003). Antimonial-induced increase in intracellular Ca²⁺ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes. Journal of Biological Chemistry, 278: 25120-25132.

[22] 22. Mori M & Gotoh T (2000). Regulation of nitric oxide production by arginine metabolic enzymes. Biochemical and Biophysical Research Communications, 275: 715-719.

[23] 23. Modolell M, Corraliza IM, Link F, Soler G & Eichmann K (1995). Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines. European Journal of Immunology, 25: 1101-1104.

[24] 24. Boutard V, Havouis R, Fouqueray B, Philippe C, Moulinoux JP & Baud L (1995). Transforming growth factor-beta stimulates arginase activity in macrophages. Implications for the regulation of macrophage cytotoxicity. Journal of Immunology, 155: 2077-2084.

[25] 25. Iniesta V, Gomez-Nieto LC & Corraliza I (2001). The inhibition of arginase by N(omega)-hydroxy-arginine controls the growth of Leishmania inside macrophages. Journal of Experimental Medicine, 193: 777-784.

[26] 26. Iniesta V, Gomez-Nieto LC, Molano I, Mohedano A, Carcelen J, Miron C, Alonso C & Corraliza I (2002). Arginase I induction in macrophages, triggered by Th2-type cytokines, supports the growth of intracellular Leishmania parasites. Parasite Immunology, 24: 113-118.

[27] 27. Freire-de-Lima CG, Xiao YQ, Gardai SJ, Bratton DL, Fadok VA & Henson PM (2003). The interaction of apoptotic cells and phagocytes is non-inflammatory, actively anti-inflammatory and pleiotropic. XXVIII Meeting of the Brazilian Society of Immunology, ClubMed Village Rio das Pedras, Mangaratiba, RJ, Brazil, October 5-8.

[28] 28. Sauter B, Albert ML, Francisco L, Larsson M, Somersan S & Bhardwaj N (2000). Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. Journal of Experimental Medicine, 191: 423-434.

[29] 29. Albert ML, Jegathesan M & Darnell RB (2001). Dendritic cell maturation is required for the cross-toleration of CD8+ T cells. Nature Immunology, 2: 1010-1017.

[30] 30. Albert ML (2004). Death-defying immunity: do apoptotic cells influence antigen processing and presentation? Nature Reviews. Immunology, 4: 223-231.

[31] 31. Prina E, Abdi SZ, Lebastard M, Perret E, Winter N & Antoine JC (2004). Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation. Journal of Cell Science, 117 (Part 2): 315-325.

[32] 32. van der Kleij D, Latz E, Brouwers JF et al. (2002). A novel host-parasite lipid cross-talk. Schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and affects immune polarization. Journal of Biological Chemistry, 277: 48122-48129.