Microbial Pathogenesis 1992 ; 12 : 9-17

Induction of tumor necrosis factor a by Leishmania infantum in murine macrophages from different inbred mice strains Maria Stefania Chiofalo,' Demetrio Delfino,' Giuseppe Mancuso,' Esmeralda La Tassa, 2 Pietro Mastroeni' and Daniela lannello' 'lstituto di Microbiologia, University di Messina, Piazza XX Settembre, 4, 98100 Messina, Italy, and 2 lstituto di Parassitologia, University di Messina, Via Cesare Battisti, 49, 98100 Messina, Italy (Received April 22, 1991 ; accepted in revised form June 19, 1991)

Chiofalo, M . S . (Istituto di Microbiologia, University di Messina, Piazza XX Settembre, 4 Messina, Italy), D . Delfino, G . Mancuso, E . La Tassa, Pi . Mastroeni and D . lannello . Induction of tumor necrosis factor a by Leishmania infantum in murine macrophages from different inbred mice strains . Microbial Pathogenesis 1992 ; 12 : 9-17 . The present study was undertaken to determine whether the viscerotropic species, Leishmania infantum, endemic in Italy, could induce tumor necrosis factor a (TN Fa) in murine macrophages . Genetically susceptible (Lshs) and resistant (Lshr) mice were used in the attempt to correlate TNFa production with the ability to control parasite growth and replication . Resident peritoneal macrophages of C3H/HeN, DBA/2, CBA (Lsh`), C57BL/10 and BALB/c (Lshs) mice were infected in vitro with promastigotes at a parasite to cell ratio of 8 :1 . No significant differences in the percentages of infected peritoneal cells of Lshs versus Lsh' mice were observed until 72 h of in vitro culture . On the contrary, Kupffer cells from Lsh` mice inhibited Leishmania replication . Peritoneal macrophages of resistant mice produced significantly higher amounts of TNFa as compared to susceptible mice . TNFa production of both resistant and susceptible mice peaked at about 5 h after the challenge with the parasite . No TNFoc was found in supernatants of infected Kupffer cells from all the strains tested . The ability of macrophages from susceptible or resistant mice strains to produce TNFa after challenge with Leishmania infantum does not seem related to their capacity to control parasite replication in vitro . Key words : TNFi ; macrophages; Leishmania .

Introduction It is now generally agreed that the resistance of the host to a variety of infectious agents is often the result of complex interactions between immunocompetent cells such as lymphocytes and macrophages . Among the most important mediators of these interactions are soluble factors released from these cells, comprehensively termed as lymphokines and cytokines . In particular, a large amount of experimental data indicates that these mediators appear to be involved in the response of the host to infection with different microorganisms . A cytokine produced by macrophages, tumor necrosis factor a (TN Foe), was originally described on the basis of its tumor necrotizing activity in sera of BCG primed, endotoxin treated mice .' However, it is now well established that TNFa exerts many other biological activities . 23 An increasing amount of evidence is accumulating on the possible role played by TNFa in the host response to many

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© 1992 Academic Press Limited



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infectious agents . In particular, recent reports have suggested that TNFa production may be alternatively beneficial or detrimental to the host during infection with different protozoa . 4 6 Intracellular protozoa belonging to the genus Leishmania are the etiologic agents of a spectrum of diseases involving cutaneous and visceral leishmaniasis . It has been recently shown that TNFa is able to influence the evolution of cutaneous leishmaniasis, due to L . major, in mice .` However, less information is at present available on the possible role of TNFa in the host response to the viscerotropic Leishmaniae, L . donovani and L . infantum . It is well established that innate susceptibility or resistance of mice to Leishmania infections is under genetic control .' In particular, the resistance of different strains to L . donovani has been related to the expression of the Lsh gene in macrophages, which in turn play a crucial role in the inhibition of Leishmania replication and survival in the infected host . 1011 The experiments described in this paper were carried out in the attempt to assess whether the species Leishmania infantum, endemic in Italy, could induce TNFa in murine macrophages . Genetically susceptible (Lshs) and resistant (Lsh') mice were used in order to correlate the ability to produce TNFa in vitro upon challenge with the parasite with the leishmanicidal activity of these cells . Results Infection of macrophages from susceptible and resistant mice The course of in vitro infection of peritoneal macrophages from BALB/c (Lshs) and C3H/HeN (Lshr) mice is shown in Fig . 1 . As was previously observed for L . donovani by Crocker, 10 we also found no significant difference between the two macrophage populations, in the ability to inhibit or support replication of L . infantum . However, a significant difference was found only at 48 h from infection (P < 0 .01) . Similar results were obtained with macrophages from other mice strains such as C57BL/10, DBA/2 and CBA (data not shown) . Moreover, liver macrophages from BALB/c mice were more heavily infected, starting from 48 h from infection, with respect to liver macrophages from C3H/HeN mice as shown in Fig . 1 (P < 0 .005, P < 0 .001 after 48 and 72 h, respectively) or other resistant strains (data not shown) . Liver macrophages, clearly, expressed the resistance/ susceptibility phenotype when infected in vitro . Induction of TNFa in macrophages from susceptible and resistant mice I n order to assess whether promastigotes of L . infantum could induce TN Fa production, monolayers of peritoneal or liver macrophages were infected, and the supernatants harvested after 1, 2, 5 and 24 h . The kinetics of TNFa release from peritoneal macrophages during the first 5 h of infection with L . infantum are shown in Fig . 2 . The parasite appears to induce TNFoc production in macrophage monolayers . However, peritoneal cells from different mice strains produced significantly different amounts of TNFa . In particular, BALB/c and C57BL/10 mice peritoneal macrophages produced almost undetectable quantities of TNFa which were significantly lower, during all the hours post-infection (p .i .), compared to all the tested strains (P < 0 .005, P < 0 .001, P < 0 .001 vs DBA/2, CBA and C3H/HeN respectively) . No TNFa was found in supernatants of macrophage monolayers at 24 h p .i ., regardless of the strain of mice . In addition no cytotoxic activity for L929 cells was found in supernatants of Kupffer cells infected with L . infantum . On the contrary, these cells produced consistent amounts of TNFa when stimulated with LPS (Fig . 3) .



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Induction of TNFa by Leishmania infantum 100 (a) 80

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Fig . 1 . In vitro infection by L . infantum promastigotes of (a) peritoneal macrophages and (b) Kupffer cells from BALB/c ( •) and C3H/HeN (o) mice . Results are expressed as the mean of three experiments±SD . Statistically significant : the P values are reported in the text .

Effect of polymyxin B on the induction of TNFa To establish whether the induction of TNFa could be attributed to the parasites and not to contamination of the preparations with LPS, promastigotes were suspended in RPMI and pretreated for 1 h at 37°C in 5% CO 2 with polymyxin B (20 µg/ml), a reagent that neutralizes the TNF inducing Lipid A component of LPS . 12 Polymyxin B treatment did not abrogate the production of TNFa in macrophages in response to promastigotes of L . infantum. However, the same dose of polymyxin B was capable of inhibiting the TNFa release induced by exogenous LPS (1 ug/ml) . The pretreatment of the supernatants from infected monolayers with rabbit antiserum to murine rTNFa (200 U/ml) neutralized the cytotoxic activity, as shown in Table 1 . Pre-incubation of macrophages with m-rTNFa To determine whether exogenous recombinant TNFa could activate macrophages for anti-leishmanial activity, rTNFa (50 and 500 U/ml) was added to BALB/c peritoneal macrophage monolayers for 48 h before infection . Moreover, in order to assess



M . S . Chiofalo et al

12 140

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Fig . 2 . Production of TNFa by peritoneal macrophages from BALB/c (0), C57BL/10 (0), DBA/2 (U), CBA ( •) and C3H/HeN (A) mice infected with L . infantum promastigotes . Results are expressed as the mean of three experiments. The P values are reported in the text .

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Fig . 3 . Production of TNFa by liver macrophages from BALB/c (open symbols) and C3H/HeN mice (closed symbols) respectively infected with L . infantum promastigotes (0, A) or stimulated with 10 pg/ml of LPS (o, 0) .



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Induction of TNFa by Leishmania infantum

Table 1 Identification of cytotoxin as TNFa in supernatants from infected macrophages from two mice strains Cytotoxicity of L929 (Mean+SD)a

L929 Normal rabbit serum Anti-mouse TNFa serum

BALB/c

C3H/HeN

65+0 .4 68+1 .0