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In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection

In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection Background: The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum. Results: Promastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden. Conclusions: Taken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs. many developing countries (OMS). The parasite is inoc- Background The protozoan Leishmania (L.) infantum (or chagasi) ulated as a flagellated extracellular promastigote by a and L. donovani are responsible for visceral leishmania- sandfly vector through the skin of a susceptible host, sis (VL) which remains a significant cause of mortality in mainly dog (L. infantum) and human (L. infantum and BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 L. donovani). Once in the host tissue, the parasite dis- Results seminates throughout the reticulo-endothelial system In vivo ingestion of promastigotes by human PMN reaching its target organs – bone marrow, spleen and liv- When examining bone marrow for diagnosis, observa- er. The main cell target is the macrophage where para- tion of L. infantum amastigotes localised in human pol- sites reside and multiply in parasitophorous vacuoles as ymorphonuclear cells is an infrequent finding. Figure 1 obligate intracellular amastigotes. Progressive intracel- shows the presence of such intracellular amastigotes in a lular infection can be interrupted by generation of T cell patient's bone marrow smear, clearly indicating that the dependent specific immunity that induces, mainly ingestion of Leishmania by PMN occurs in human VL. through IFN gamma secretion or regulation, macro- phage activation, followed by Leishmania killing or by control of its growth. In the liver of experimentally L. infantum or L. donovani infected BALB/c mice, intracellular amastigotes multi- ply rapidly during first weeks of infection and the granu- lomatous tissue response which then occurs, results in a clearing of the parasite [1–9]. By contrast, the spleen be- comes a site of chronic infection, where parasites survive for the life of the host [8,10], with progressive destruc- tion of the architecture and loss of follicular dendritic cells and germinal centres [11–13]. The role of cell-medi- ated immunity and particularly lymphocytes in the spe- cific defence against L. infantum has been considerably studied, but the part played by polymorphonuclear cells (PMN) and more particularly the neutrophils (PN) in Figure 1 Presence of L. infantum in human PMN (arrow) detected at controlling the disease is much less well established. Pre- diagnosis in bone marrow of a VL patient. vious in vitro or in vivo studies on other parasite species have demonstrated the presence of PMN in areas of L. amazonensis destruction during acute and chronic phas- es of the infection [14,15] and showed that PMN could in- gest and kill L. donovani promastigotes in vitro [16–19]. Contrasted results have been obtained in studies on der- motropic L. major species. One study showed that sus- ceptible and resistant mice depleted for neutrophils exhibited accelerated parasite spreading, increased foot- pad swelling and augmented parasite counts in the lesion draining lymph nodes as compared to non-depleted mice [19]. Another study reported that the transient depletion of PMN in BALB/c mice inhibited Th2 cell development and responses and led to a partial resolution of the foot- pad lesions [20]. In L. donovani infected C57BL/6 mice the depletion for neutrophils induced an important en- hancement of parasite growth in both liver and spleen [21]. Only one report has been devoted to ex vivo study, showing that PMN from infected dogs can phagocytize Figure 2 and kill L. infantum promastigotes [22]. Electron microscopy analysis of early parasite phagocytosis in spleen of infected mice. Micrograph of spleen, 1 h after L. In the present report, we investigated PN as L. infantum infantum inoculation (magnification × 6000). phagocytic cell and its role in controlling the early and late stage of experimental disease using neutrophil-de- pleted L. infantum infected BALB/c mice. In mouse, early in vivo involvement of neutrophils occurs in spleen but not in liver In order to study the early in vivo participation of PN in host defence, spleen and liver of mice were examined by electron microscopy 1 h, 2 h, 5 h and 24 h hours after L. BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 infantum inoculation. In liver, the parasites were readily observed already at 1 h and 2 h after the infection. All parasites were localised close to or inside macrophages and no Leishmania were detected inside neutrophil cells, at any time of observation. In contrast, in spleen tissue, parasites were much less abundant and several fields had to be screened in order to find Leishmania. A part of spleen neutrophils was found to contain at least one par- asite in a more or less degraded state. Figure 2 shows that within one hour post infection, the intraneutrophil pro- tozoan, although recognised by parietal tubules, flagellar pocket and longitudinal-cut flagella, generally lost its ul- trastructural integrity. The electron microscopy images do not at all instances allow determining the precise in- tracellular parasite localisation, so a quantitative evalua- tion in spleen of the percentage of parasitised Figure 4 macrophages versus parasitised neutrophils would be Electron microscopy analysis of early parasite phagocytosis in liver of infected mice. Micrograph of liver, 1 h after L. infan- non-reliable. Twenty-four hours after the infection, neu- tum inoculation (magnification × 4000). trophil influx was still observed in the spleen (Figure 3) but no intraneutrophil parasites were detected. In the parasitised monocytes (Figure 4) Leishmania appeared Table 1: White cell subpopulations in blood of RB6-8C5 mAb- relatively intact as compared to that phagocytised by treated and control mice. PMN. NeutroΦ Lympho⊂ Mono⊂ EosinoΦ 48 h after mAb injection Control IgG2b 36 ± 11 51 ± 88 ± 25 ± 2 RB6-8C5 4 ± 189 ± 75 ± 53 ± 1 6 days after mAb injection Control IgG2b 32 ± 15 56 ± 13 7 ± 25 ± 1 RB6-8C5 55 ± 535 ± 78 ± 32 ± 1 Mice (4 animals per group) received intraperitoneally RB6-BC5 mAb or irrelevant IgG2b rat mAb (200 µg per mice). Cells were counted on blood smears and results are expressed as percentage of total number of cells (mean ± SD). NeutroΦ = Neutrophils, Lympho⊂ = Lym- phocyes, Mono⊂ = Monocytes, EosinoΦ = Eosinophils. Figure 3 Neutrophil influx to the infected spleen, shown by electron microscopy. Micrograph of spleen, 24 h after L. infantum cytometric analyses of cell suspensions were performed inoculation (magnification × 1200) 22 days after the RB6-8C5 mAb injection. Although the total number of cells was notably increased [(105 ± 10) × 6 6 6 10 , (362 ± 60) × 10 , (545 ± 65) × 10 cells per spleen of control, infected RB6-8C5-untreated, infected RB6-8C5- Effect of mAb RB6-8C5 on cell counts in blood and spleen treated mice, respectively], the mAb treatment showed To carry out depletion experiments, we used mAb RB6- no effect on the percentage of B cells, macrophages, 8C5, which has been shown to bind and lyse neutrophils Mac1+ cells and CD4+ cells. The only sub-population af- and eosinophils in vivo and in vitro [19,23,24]. Table 1 fected was that of CD8+ cells, percentage of which was shows that in mouse blood, 48 h after a single intra peri- decreased two-fold (Figure 5). toneal injection of the mAb, the neutrophil percentage was reduced 9-fold and lymphocyte percentage was in- Effect of neutrophil depletion on parasite burden creased two-fold while 6 days after the injection, the In the first series of experiments, we studied the role of counts from depleted and control mice (which received neutrophils in the early stage of infection. RB6-8C5 mAb isotype-matched mAb) were comparable. In spleen, flow was administered as a single injection to four groups of BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 Figure 5 Figure 6 Influence of RB6-8C5 mAb treatment on CD8+ cell sub-pop- Liver and spleen amastigote load in neutrophil-depleted and ulation in spleen, assessed by flow cytometry 22 days after control mice. Animals received intraperitoneally 200 µg of Leishmania and antibody injection, as described in Methods. RB6-BC5 or of irrelevant IgG2b rat mAb 5 h prior to L. infan- Results are expressed as percentage of total number of cells tum inoculation (10 stationary-phase promastigote per in the spleen for each group [mean ± sem (4 mice per mouse). The parasite counts were determined 22 days after group)]. The total number of cells is indicated in the text. the infection from Giemsa-stained spleen and liver touch prints, and are expressed (mean ± SEM) in Leishman Dono- van units (LDU = number of amastigotes per 1000 nucleated cells × organ weight (in grams) × 2 × 10 ). The experiment mice (5 mice per group), 48 h and 5 h before and 72 h and was repeated twice. 168 h after L. infantum inoculation. Amastigote burden in spleen and liver was examined 22 days post infection. Figure 6 shows that whereas hepatic amastigote loads in host resistance is thought to depend on the development depleted and control groups were similar, in the spleen of specific cell-mediated immunity acting through acti- the parasite load increased 12-fold in neutrophil deplet- vation and expansion of CD4+ T lymphocytes of the Th1 ed mice as compared to controls. The data shown in Fig. subset [26,27]. These activated T cells secrete cytokines 6 relate to mice that were RB6-8C5-treated 5 h before such as gamma interferon that activate the mononuclear Leishmania infection; the same enhancing effect of neu- phagocytes turning them into cells with potent leishm- trophil depletion on parasite multiplication was ob- anicidal activity (reviewed in [2]). This activity of macro- served in all four groups of mice treated with anti- phages is non-specific in nature since it is also effective neutrophil mAb at different times. These results indicate against heterotologous microorganisms with unrelated that early in infection, in the spleen but not in liver, the antigenic specificities [28]. Among other immune de- neutrophil cells play a role in controlling L. infantum fence mechanisms, polymorphonuclear neutrophils, burden, and this effect is well detectable even after the which have been shown to play a crucial role in the early state of depletion wears off. stage of innate resistance to listeriosis [24,29], have been reported to participate to the control of Leishmania in- Next, to examine the effect of neutrophil depletion in the fection. Indeed, studies showing that PN are present in late stage of infection, mice infected 3 months earlier vivo in areas of Leishmania destruction [14,15,30] or can were treated with RB6-8C5 mAb twice a week, for 4 kill the parasites in vitro [16–18] have suggested that weeks (4 RB6-8C5-treated mice and 4 controls). No sig- these cells might be involved in the inhibition of the par- nificant increase of amastigote load was found either in asite multiplication. However, only very few studies us- the spleen, or in the liver (not shown), indicating that ing neutrophil-depleted mice infected with L. major[19] neutrophils are not significantly involved in the host pro- or L. donovani[21] have provided direct evidence for the tection against L. infantum in chronically infected mice. participation of PN in non-specific resistance to these in- fections and only one report [22], studying the ex vivo Discussion phagocytosis by PN from infected dogs, has suggested a Obligate intracellular micro-organisms such as Leish- possible role for these cells in the infection with L. infan- mania, have adopted many diverse strategies for their tum. replication inside the host (reviewed in [25]) and the BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 In this report we show in vivo that neutrophil granulo- In chronically infected mice, long term administration of cytes are effectively capable to ingest and kill L. infantum anti-neutrophil antibody, did not significantly enhance promastigotes. The in vivo implication of PN, occasion- parasite counts in either liver or spleen. Therefore, neu- ally observed in human bone marrow as illustrated in Fig trophil cells do not appear to be involved in the late 1, was examined in mouse spleen and liver. The very ear- phase of L. infantum infection, in contrast to the early ly engagement of neutrophils and their ability to effi- phase where they crucially contribute to parasite killing. ciently eliminate L. infantum were confirmed in the The precise mechanisms of Leishmania destruction re- spleen using electron microscopy analyses. Indeed, as main to be established. Some reports [34–36] suggest early as one hour after the infection, many PN were that PN could lyse infected host cells. A second hypothe- found to harbour 1 or more parasites presenting a sis is that PN kill extracellular parasite before they enter marked loss of ultrastructural integrity. By contrast, in macrophage or even after they have been released by the liver more numerous parasites and in a less degraded necrotic cells. In this case, as already hypothesised [21], state were observable. In both organs, parasites ap- reactive oxygen intermediates, which play an important peared undegraded in monocytic cells. role in the early regulation of parasite growth and which are among others produced by PN under the control of To further characterise the role of PN, we next performed TNFα, might be responsible for the parasite destruction. depletion experiments in early and late phases of L. in- Finally, PN could function as immunoregulatory cells by fantum infection, using the mAb RB6-8C5 directed releasing soluble mediators such as TNFα [37] that reg- against mature mouse granulocytes. The apparent health ulate the entry and intracellular multiplication of para- status of mice was unaffected neither by a single mAb ad- site in host cells. ministration (which efficiently reduced the number of circulating PN for at least 48 h) nor by repeated injec- We hypothesise, that the neutrophil-generated early de- tions. We showed that in the early phase of L. infantum struction of Leishmania, although beneficient for the infection, the neutrophil cells play a protective role in host in terms of the early control of parasite growth, spleen, since early PN depletion resulted in marked in- might be also responsible for induction of inflammatory crease of the parasite growth. At the time of the parasite pathology in the spleen which subsequently contributes load assessment, the subpopulation of CD8+ cells was to the lack of parasite clearance and chronicity [33]. significantly decreased in the spleen of PN depleted ani- mals. This reduction of CD8+ cells could be due to their Materials and methods neutrophil-dependent recruitement as observed in some Parasites studies [31] but also may be related to a possible recogni- L. infantum MON1 (MHOM/FR/94/LPN101), isolated tion of this cell subset by RB6-8C5 [32]. No significant from a patient with visceral leishmaniasis, was main- effect of neutrophils depletion on the parasite load was tained by serial passages in Syrian hamsters. The pro- evidenced in liver. mastigote form was cultured and prepared for infection experiments as described previously [8–10]. Very interesting results published recently [21], reported that the resistance of B cell-deficient C57/BL6 mice to L. Infections of mice donovani infection was accompanied by neutrophil de- Six-week-old female BALB/c mice (Iffa Credo, L'Ar- pendent destructive pathology in liver. In C57/BL6 mice bresle, France) were inoculated in the caudal vein with neutrophil depletion led to enhancement of parasite 10 stationary-phase promastigotes/mouse or left unin- growth both in liver and in spleen of B-cell deficient and fected, split randomly into groups of 2 to 5 animals, and wild type animals. It is noteworthy that in this study [21], maintained in positive pressure chamber (Esi-Flufrance, neutrophil depletion of BALB/c mouse caused in spleen Wissous, France). For electron microscopy (EM) analy- the enhancement of parasite growth close to that report- ses, mice were examined 1 h, 2 h, 5 h, and 24 h after in- ed here, and a much lower, but significant, increase of fection. For neutrophil depletion experiments, rat anti- liver burden. Therefore, the effect of neutrophil deple- mouse PMN cells mAb RB6-8C5 was used [23]. The hy- tion on liver parasite multiplication might depend both bridoma producing RB6-8C5 mAb was kindly provided on the mouse strain as well as the infecting parasite spe- by Dr Appelberg (Centro di Citologia Experimental, Por- cies. The lack of effect on liver parasite load observed in to, Portugal); the antibody was purified from ascitic flu- our study might be related to the relatively weak neu- ids produced in nude mice, using protein G Sepharose trophil influx elicited by L. infantum infection to this or- affinity chromatography (Pharmacia). Purity of RB6- gan, as compared to spleen. It is noteworthy that in 8C5 mAb was controlled by SDS-PAGE and specificity mouse liver amastigote load is initially larger than that in was verified by cell sorting on whole mouse blood. Mice spleen but it resolves spontaneously, while the spleen were injected intraperitoneally with 200 µg mAb RB6- supports a sustained parasite load [33]. 8C5 or isotype-matched rat IgG (IgG2b, CliniSciences, BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 animal models, and in humans. Diagn Microbiol Infect Dis 1990, France) 48 h and 5 h before or 72 h and 168 h after L. in- 13:411-421 fantum inoculation and sacrificed 22 days post infection. 3. Murray HW, Squires KE, Miralles CD, Stoeckle MY, Granger AM, Alternatively, animals previously infected for three Granelli-Piperno A, Bogdan C: Acquired resistance and granulo- ma formation in experimental visceral leishmaniasis. Differ- months were injected as above twice a week during four ential T cell and lymphokine roles in initial versus established weeks and analysed 2 days after the last injection. The immunity. J Immunol 1992, 148:1858-1863 4. Buffet PA, Sulahian A, Gari YJF, Nassar N, Derouin F: Culture mi- assessment of the amastigote burden in livers and crotitration: a sensitive method for quantifying Leishmania spleens was carried as described previously [8–10]. The infantum in tissues if infected mice. Antimicrob Agents Chemother neutrophil depletion was assessed by microscopic exam- 1995, 39:2167-2168 5. Leclercq V, Lebastard M, Belkaid Y, Louis J, Milon G: The outcome ination of Giemsa-stained blood smears, or cell sorting of the parasitic process initiated by Leishmania infantum in analyses of mouse blood. Neutrophil depletion did not laboratory mice. J Immunol 1996, 157:4537-4543 lead to any deterioration in apparent health status of 6. Wilson ME, Sandor M, Blum AM, Young BM, Metwali A, Elliott D, Lynch RG, Weinstock JV: Local suppression of IFNγ in hepatic mice. granulomas correlates with tissue-specific replication of Leishmania chagasi. J Immunol 1996, 156:2231-2238 7. Cotterell SEJ, Engwerda CR, Kaye PM: Leishmania donovani in- Tissue processing for electron microscopy fection initiates T-cell independent chemokine responses, Samples of livers and spleens were fixed for 1 h with 2.5 which are subsequently amplified in a T-cell dependent man- % glutaraldehyde (Merck) in PBS buffer and cut into ner. Eur J Immunol 1999, 29:203-214 8. Rousseau D, Le Fichoux Y, Stien X, Suffia I, Ferrua B, Kubar J: Pro- pieces of approximately 1 mm3. The material was fixed gression of visceral leishmaniasis due to Leishmania infan- for another 24 h washed in the buffer, postfixed in 1% os- tum in BALB/c mice is markedly slowed by prior infection with Trichinella spiralis. Infect Immun 1997, 65:4978-4983 mium tetroxide in buffer for 2 h, washed in buffer, dehy- 9. Le Fichoux Y, Rousseau D, Ferrua B, Ruette S, Lelièvre A, Grousson drated with graded ethanol, and embedded in Epon D, Kubar J: Short- and long-term efficacy of hexadecylphos- (TAAB). Ultrathin sections were poststained with uranyl phocholine against established Leishmania infantum infec- tion in BALB/c mice. Antimicrob Agents Chemother 1998, 42:654- acetate and lead citrate, examined, and photographed using a Zeiss 1200EXII transmission electron micro- 10. Rousseau D, Suffia I, Ferrua B, Philip P, Le Fichoux Y, Kubar J: Pro- scope. longed administration of dexamethasone induces limited re- activation of visceral leishmaniosis in chronically infected BALB/c mice. Eur Cytokine Netw 1998, 9:655-661 Flow cytometry 11. Smelt SC, Engwerda CR, McCrossen M, Kaye PM: Destruction of follicular dendritic cells during chronic visceral leishmania- Cell suspensions from spleen (20 mg tissu per ml) were sis. J Immunol 1997, 158:3813-3821 counted in a Malassez haemacytometer after erythro- 12. Engwerda CR, Murphy ML, Cotterell SEJ, Smelt SC, Kaye PM: Neu- cytes lysis and two washes. Phenotypic analyses of spleen tralization of IL-12 demonstrates the existence of discrete organ-specific phases in the control of Leishmania donovani cells subpopulations were performed after triple staining Eur J Immunol 1998, 28:669-680 with FITC-conjugated anti-B220 (clone RA3-6B2, Calt- 13. Gorak PMA, Engwerda CR, Kaye PM: Dendritic cells, but not ag), phycoerythrin-conjugated anti-CD8 (clone 53-6.72, macrophages, produce IL-12 immediately following Leish- mania donovani infection. Eur J Immunol 1998, 28:687-695 Pharmingen) and anti-CD4 (clone CT-CD4, Caltag), and 14. Pompeu ML, Freitas LA, Santos MLV, Khouri M, Barral-Netto M: biotin-conjugated anti CD11b (clone Mac1, Caltag) or F4/ Granulocytes in the inflammatory process of BALB/c mice infected by Leishmania amazonensis. A quantitative ap- 80 (clone C1-A31, Caltag), followed by streptavidin-tri- proach. Acta Tropica 1991, 48:185-193 color (TC, Caltag). All the staining steps were carried out 15. Beil WJ, Meinardus-Hager G, Neugebauer D, Sorg C: Differences in at 4°C in PBS containing 5 mM EDTA and 3% (v/v) FCS. the onset of the inflammatory response to cutaneous Leish- maniasis in resistant and susceptible mice. J Leukocyte Biol 1992, Analysis was performed on a FACScan flow cytometer 52:135-142 (Becton Dickinson, Mountain View, CA), using Cellquest 16. Chang KP: Leishmanicidal mechanisms of human polymor- software (Becton Dickinson). phonuclear phagocytes. Am J Trop Med Hyg 1981, 30:322-333 17. Chang KP: Antibody-mediated inhibition of phagocytosis in Leishmania donovani-human phagocyte interactions in vit- Acknowledgements ro. Am J Trop Med Hyg 1981, 30:334-339 This work was supported by grants from the Ministry of Research (EA 18. Pearson RD, Steigbigel RT: Phagocytosis and killing of the pro- tozoan Leishmania donovani by human polymorphonuclear 2675) and from the University of Nice, and by gifts from le Groupe d'Action Contre la Leishmaniose (GACL). We thank Dr R. Appelberg (Centro di Ci- leukocytes. J Immunol 1981, 127:1438-1443 19. Lima GMAC, Vallochi AL, Silva UR, Bevilacqua EMAF, Kiffer MMF, Ab- tologia Experimental, Porto, Portugal) for providing us with the RB6-8C5 hybridoma, Dr. J. Bayle (Laboratoire d'Hématologie, Hôpital de l'Archet, rahamsohn IA: The role of polymorphonuclear leukocytes in the resistance to cutaneous Leishmaniasis. Immunol Letters Nice) for providing Figure 1, Mireille Mari and Dominique Sadoulet for 1998, 64:145-151 technical assistance with EM experiments, Gilbert Dabbene for taking care 20. Tacchini-Cottier F, Zweifel C, Belkaid Y, Mukankundiye C, Vasei M, of the animal facility, Aurore Grima for illustration work and Georgette Pagliardini for administrative assistance. Launois P, Milon G, Louis JA: An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major. J Inmmunol References 2000, 165:2628-2636 1. 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In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection

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Springer Journals
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Copyright © 2001 by Rousseau et al; licensee BioMed Central Ltd.
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Life Sciences; Microbiology; Biological Microscopy; Mycology; Parasitology; Virology; Life Sciences, general
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Abstract

Background: The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum. Results: Promastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden. Conclusions: Taken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs. many developing countries (OMS). The parasite is inoc- Background The protozoan Leishmania (L.) infantum (or chagasi) ulated as a flagellated extracellular promastigote by a and L. donovani are responsible for visceral leishmania- sandfly vector through the skin of a susceptible host, sis (VL) which remains a significant cause of mortality in mainly dog (L. infantum) and human (L. infantum and BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 L. donovani). Once in the host tissue, the parasite dis- Results seminates throughout the reticulo-endothelial system In vivo ingestion of promastigotes by human PMN reaching its target organs – bone marrow, spleen and liv- When examining bone marrow for diagnosis, observa- er. The main cell target is the macrophage where para- tion of L. infantum amastigotes localised in human pol- sites reside and multiply in parasitophorous vacuoles as ymorphonuclear cells is an infrequent finding. Figure 1 obligate intracellular amastigotes. Progressive intracel- shows the presence of such intracellular amastigotes in a lular infection can be interrupted by generation of T cell patient's bone marrow smear, clearly indicating that the dependent specific immunity that induces, mainly ingestion of Leishmania by PMN occurs in human VL. through IFN gamma secretion or regulation, macro- phage activation, followed by Leishmania killing or by control of its growth. In the liver of experimentally L. infantum or L. donovani infected BALB/c mice, intracellular amastigotes multi- ply rapidly during first weeks of infection and the granu- lomatous tissue response which then occurs, results in a clearing of the parasite [1–9]. By contrast, the spleen be- comes a site of chronic infection, where parasites survive for the life of the host [8,10], with progressive destruc- tion of the architecture and loss of follicular dendritic cells and germinal centres [11–13]. The role of cell-medi- ated immunity and particularly lymphocytes in the spe- cific defence against L. infantum has been considerably studied, but the part played by polymorphonuclear cells (PMN) and more particularly the neutrophils (PN) in Figure 1 Presence of L. infantum in human PMN (arrow) detected at controlling the disease is much less well established. Pre- diagnosis in bone marrow of a VL patient. vious in vitro or in vivo studies on other parasite species have demonstrated the presence of PMN in areas of L. amazonensis destruction during acute and chronic phas- es of the infection [14,15] and showed that PMN could in- gest and kill L. donovani promastigotes in vitro [16–19]. Contrasted results have been obtained in studies on der- motropic L. major species. One study showed that sus- ceptible and resistant mice depleted for neutrophils exhibited accelerated parasite spreading, increased foot- pad swelling and augmented parasite counts in the lesion draining lymph nodes as compared to non-depleted mice [19]. Another study reported that the transient depletion of PMN in BALB/c mice inhibited Th2 cell development and responses and led to a partial resolution of the foot- pad lesions [20]. In L. donovani infected C57BL/6 mice the depletion for neutrophils induced an important en- hancement of parasite growth in both liver and spleen [21]. Only one report has been devoted to ex vivo study, showing that PMN from infected dogs can phagocytize Figure 2 and kill L. infantum promastigotes [22]. Electron microscopy analysis of early parasite phagocytosis in spleen of infected mice. Micrograph of spleen, 1 h after L. In the present report, we investigated PN as L. infantum infantum inoculation (magnification × 6000). phagocytic cell and its role in controlling the early and late stage of experimental disease using neutrophil-de- pleted L. infantum infected BALB/c mice. In mouse, early in vivo involvement of neutrophils occurs in spleen but not in liver In order to study the early in vivo participation of PN in host defence, spleen and liver of mice were examined by electron microscopy 1 h, 2 h, 5 h and 24 h hours after L. BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 infantum inoculation. In liver, the parasites were readily observed already at 1 h and 2 h after the infection. All parasites were localised close to or inside macrophages and no Leishmania were detected inside neutrophil cells, at any time of observation. In contrast, in spleen tissue, parasites were much less abundant and several fields had to be screened in order to find Leishmania. A part of spleen neutrophils was found to contain at least one par- asite in a more or less degraded state. Figure 2 shows that within one hour post infection, the intraneutrophil pro- tozoan, although recognised by parietal tubules, flagellar pocket and longitudinal-cut flagella, generally lost its ul- trastructural integrity. The electron microscopy images do not at all instances allow determining the precise in- tracellular parasite localisation, so a quantitative evalua- tion in spleen of the percentage of parasitised Figure 4 macrophages versus parasitised neutrophils would be Electron microscopy analysis of early parasite phagocytosis in liver of infected mice. Micrograph of liver, 1 h after L. infan- non-reliable. Twenty-four hours after the infection, neu- tum inoculation (magnification × 4000). trophil influx was still observed in the spleen (Figure 3) but no intraneutrophil parasites were detected. In the parasitised monocytes (Figure 4) Leishmania appeared Table 1: White cell subpopulations in blood of RB6-8C5 mAb- relatively intact as compared to that phagocytised by treated and control mice. PMN. NeutroΦ Lympho⊂ Mono⊂ EosinoΦ 48 h after mAb injection Control IgG2b 36 ± 11 51 ± 88 ± 25 ± 2 RB6-8C5 4 ± 189 ± 75 ± 53 ± 1 6 days after mAb injection Control IgG2b 32 ± 15 56 ± 13 7 ± 25 ± 1 RB6-8C5 55 ± 535 ± 78 ± 32 ± 1 Mice (4 animals per group) received intraperitoneally RB6-BC5 mAb or irrelevant IgG2b rat mAb (200 µg per mice). Cells were counted on blood smears and results are expressed as percentage of total number of cells (mean ± SD). NeutroΦ = Neutrophils, Lympho⊂ = Lym- phocyes, Mono⊂ = Monocytes, EosinoΦ = Eosinophils. Figure 3 Neutrophil influx to the infected spleen, shown by electron microscopy. Micrograph of spleen, 24 h after L. infantum cytometric analyses of cell suspensions were performed inoculation (magnification × 1200) 22 days after the RB6-8C5 mAb injection. Although the total number of cells was notably increased [(105 ± 10) × 6 6 6 10 , (362 ± 60) × 10 , (545 ± 65) × 10 cells per spleen of control, infected RB6-8C5-untreated, infected RB6-8C5- Effect of mAb RB6-8C5 on cell counts in blood and spleen treated mice, respectively], the mAb treatment showed To carry out depletion experiments, we used mAb RB6- no effect on the percentage of B cells, macrophages, 8C5, which has been shown to bind and lyse neutrophils Mac1+ cells and CD4+ cells. The only sub-population af- and eosinophils in vivo and in vitro [19,23,24]. Table 1 fected was that of CD8+ cells, percentage of which was shows that in mouse blood, 48 h after a single intra peri- decreased two-fold (Figure 5). toneal injection of the mAb, the neutrophil percentage was reduced 9-fold and lymphocyte percentage was in- Effect of neutrophil depletion on parasite burden creased two-fold while 6 days after the injection, the In the first series of experiments, we studied the role of counts from depleted and control mice (which received neutrophils in the early stage of infection. RB6-8C5 mAb isotype-matched mAb) were comparable. In spleen, flow was administered as a single injection to four groups of BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 Figure 5 Figure 6 Influence of RB6-8C5 mAb treatment on CD8+ cell sub-pop- Liver and spleen amastigote load in neutrophil-depleted and ulation in spleen, assessed by flow cytometry 22 days after control mice. Animals received intraperitoneally 200 µg of Leishmania and antibody injection, as described in Methods. RB6-BC5 or of irrelevant IgG2b rat mAb 5 h prior to L. infan- Results are expressed as percentage of total number of cells tum inoculation (10 stationary-phase promastigote per in the spleen for each group [mean ± sem (4 mice per mouse). The parasite counts were determined 22 days after group)]. The total number of cells is indicated in the text. the infection from Giemsa-stained spleen and liver touch prints, and are expressed (mean ± SEM) in Leishman Dono- van units (LDU = number of amastigotes per 1000 nucleated cells × organ weight (in grams) × 2 × 10 ). The experiment mice (5 mice per group), 48 h and 5 h before and 72 h and was repeated twice. 168 h after L. infantum inoculation. Amastigote burden in spleen and liver was examined 22 days post infection. Figure 6 shows that whereas hepatic amastigote loads in host resistance is thought to depend on the development depleted and control groups were similar, in the spleen of specific cell-mediated immunity acting through acti- the parasite load increased 12-fold in neutrophil deplet- vation and expansion of CD4+ T lymphocytes of the Th1 ed mice as compared to controls. The data shown in Fig. subset [26,27]. These activated T cells secrete cytokines 6 relate to mice that were RB6-8C5-treated 5 h before such as gamma interferon that activate the mononuclear Leishmania infection; the same enhancing effect of neu- phagocytes turning them into cells with potent leishm- trophil depletion on parasite multiplication was ob- anicidal activity (reviewed in [2]). This activity of macro- served in all four groups of mice treated with anti- phages is non-specific in nature since it is also effective neutrophil mAb at different times. These results indicate against heterotologous microorganisms with unrelated that early in infection, in the spleen but not in liver, the antigenic specificities [28]. Among other immune de- neutrophil cells play a role in controlling L. infantum fence mechanisms, polymorphonuclear neutrophils, burden, and this effect is well detectable even after the which have been shown to play a crucial role in the early state of depletion wears off. stage of innate resistance to listeriosis [24,29], have been reported to participate to the control of Leishmania in- Next, to examine the effect of neutrophil depletion in the fection. Indeed, studies showing that PN are present in late stage of infection, mice infected 3 months earlier vivo in areas of Leishmania destruction [14,15,30] or can were treated with RB6-8C5 mAb twice a week, for 4 kill the parasites in vitro [16–18] have suggested that weeks (4 RB6-8C5-treated mice and 4 controls). No sig- these cells might be involved in the inhibition of the par- nificant increase of amastigote load was found either in asite multiplication. However, only very few studies us- the spleen, or in the liver (not shown), indicating that ing neutrophil-depleted mice infected with L. major[19] neutrophils are not significantly involved in the host pro- or L. donovani[21] have provided direct evidence for the tection against L. infantum in chronically infected mice. participation of PN in non-specific resistance to these in- fections and only one report [22], studying the ex vivo Discussion phagocytosis by PN from infected dogs, has suggested a Obligate intracellular micro-organisms such as Leish- possible role for these cells in the infection with L. infan- mania, have adopted many diverse strategies for their tum. replication inside the host (reviewed in [25]) and the BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 In this report we show in vivo that neutrophil granulo- In chronically infected mice, long term administration of cytes are effectively capable to ingest and kill L. infantum anti-neutrophil antibody, did not significantly enhance promastigotes. The in vivo implication of PN, occasion- parasite counts in either liver or spleen. Therefore, neu- ally observed in human bone marrow as illustrated in Fig trophil cells do not appear to be involved in the late 1, was examined in mouse spleen and liver. The very ear- phase of L. infantum infection, in contrast to the early ly engagement of neutrophils and their ability to effi- phase where they crucially contribute to parasite killing. ciently eliminate L. infantum were confirmed in the The precise mechanisms of Leishmania destruction re- spleen using electron microscopy analyses. Indeed, as main to be established. Some reports [34–36] suggest early as one hour after the infection, many PN were that PN could lyse infected host cells. A second hypothe- found to harbour 1 or more parasites presenting a sis is that PN kill extracellular parasite before they enter marked loss of ultrastructural integrity. By contrast, in macrophage or even after they have been released by the liver more numerous parasites and in a less degraded necrotic cells. In this case, as already hypothesised [21], state were observable. In both organs, parasites ap- reactive oxygen intermediates, which play an important peared undegraded in monocytic cells. role in the early regulation of parasite growth and which are among others produced by PN under the control of To further characterise the role of PN, we next performed TNFα, might be responsible for the parasite destruction. depletion experiments in early and late phases of L. in- Finally, PN could function as immunoregulatory cells by fantum infection, using the mAb RB6-8C5 directed releasing soluble mediators such as TNFα [37] that reg- against mature mouse granulocytes. The apparent health ulate the entry and intracellular multiplication of para- status of mice was unaffected neither by a single mAb ad- site in host cells. ministration (which efficiently reduced the number of circulating PN for at least 48 h) nor by repeated injec- We hypothesise, that the neutrophil-generated early de- tions. We showed that in the early phase of L. infantum struction of Leishmania, although beneficient for the infection, the neutrophil cells play a protective role in host in terms of the early control of parasite growth, spleen, since early PN depletion resulted in marked in- might be also responsible for induction of inflammatory crease of the parasite growth. At the time of the parasite pathology in the spleen which subsequently contributes load assessment, the subpopulation of CD8+ cells was to the lack of parasite clearance and chronicity [33]. significantly decreased in the spleen of PN depleted ani- mals. This reduction of CD8+ cells could be due to their Materials and methods neutrophil-dependent recruitement as observed in some Parasites studies [31] but also may be related to a possible recogni- L. infantum MON1 (MHOM/FR/94/LPN101), isolated tion of this cell subset by RB6-8C5 [32]. No significant from a patient with visceral leishmaniasis, was main- effect of neutrophils depletion on the parasite load was tained by serial passages in Syrian hamsters. The pro- evidenced in liver. mastigote form was cultured and prepared for infection experiments as described previously [8–10]. Very interesting results published recently [21], reported that the resistance of B cell-deficient C57/BL6 mice to L. Infections of mice donovani infection was accompanied by neutrophil de- Six-week-old female BALB/c mice (Iffa Credo, L'Ar- pendent destructive pathology in liver. In C57/BL6 mice bresle, France) were inoculated in the caudal vein with neutrophil depletion led to enhancement of parasite 10 stationary-phase promastigotes/mouse or left unin- growth both in liver and in spleen of B-cell deficient and fected, split randomly into groups of 2 to 5 animals, and wild type animals. It is noteworthy that in this study [21], maintained in positive pressure chamber (Esi-Flufrance, neutrophil depletion of BALB/c mouse caused in spleen Wissous, France). For electron microscopy (EM) analy- the enhancement of parasite growth close to that report- ses, mice were examined 1 h, 2 h, 5 h, and 24 h after in- ed here, and a much lower, but significant, increase of fection. For neutrophil depletion experiments, rat anti- liver burden. Therefore, the effect of neutrophil deple- mouse PMN cells mAb RB6-8C5 was used [23]. The hy- tion on liver parasite multiplication might depend both bridoma producing RB6-8C5 mAb was kindly provided on the mouse strain as well as the infecting parasite spe- by Dr Appelberg (Centro di Citologia Experimental, Por- cies. The lack of effect on liver parasite load observed in to, Portugal); the antibody was purified from ascitic flu- our study might be related to the relatively weak neu- ids produced in nude mice, using protein G Sepharose trophil influx elicited by L. infantum infection to this or- affinity chromatography (Pharmacia). Purity of RB6- gan, as compared to spleen. It is noteworthy that in 8C5 mAb was controlled by SDS-PAGE and specificity mouse liver amastigote load is initially larger than that in was verified by cell sorting on whole mouse blood. Mice spleen but it resolves spontaneously, while the spleen were injected intraperitoneally with 200 µg mAb RB6- supports a sustained parasite load [33]. 8C5 or isotype-matched rat IgG (IgG2b, CliniSciences, BMC Microbiology 2001, 1:17 http://www.biomedcentral.com/1471-2180/1/17 animal models, and in humans. Diagn Microbiol Infect Dis 1990, France) 48 h and 5 h before or 72 h and 168 h after L. in- 13:411-421 fantum inoculation and sacrificed 22 days post infection. 3. Murray HW, Squires KE, Miralles CD, Stoeckle MY, Granger AM, Alternatively, animals previously infected for three Granelli-Piperno A, Bogdan C: Acquired resistance and granulo- ma formation in experimental visceral leishmaniasis. Differ- months were injected as above twice a week during four ential T cell and lymphokine roles in initial versus established weeks and analysed 2 days after the last injection. The immunity. J Immunol 1992, 148:1858-1863 4. Buffet PA, Sulahian A, Gari YJF, Nassar N, Derouin F: Culture mi- assessment of the amastigote burden in livers and crotitration: a sensitive method for quantifying Leishmania spleens was carried as described previously [8–10]. The infantum in tissues if infected mice. Antimicrob Agents Chemother neutrophil depletion was assessed by microscopic exam- 1995, 39:2167-2168 5. Leclercq V, Lebastard M, Belkaid Y, Louis J, Milon G: The outcome ination of Giemsa-stained blood smears, or cell sorting of the parasitic process initiated by Leishmania infantum in analyses of mouse blood. Neutrophil depletion did not laboratory mice. J Immunol 1996, 157:4537-4543 lead to any deterioration in apparent health status of 6. Wilson ME, Sandor M, Blum AM, Young BM, Metwali A, Elliott D, Lynch RG, Weinstock JV: Local suppression of IFNγ in hepatic mice. granulomas correlates with tissue-specific replication of Leishmania chagasi. J Immunol 1996, 156:2231-2238 7. 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