On the other hand, high concentrations of TNF- and IL-6 were detected in the co-cultures and both peaked at 72 h. the leishmanicidal activity, and 5) destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-, as reported for otherLeishmaniaspecies. == Conclusions/Significance == The CZC-8004 present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect onL. (L.) amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by thisLeishmaniaspecies. == Introduction == Neutrophils, key players of the innate immune system, provide a first line of defense against invading pathogens. Neutrophils may be also implicated in immunoregulation as a source of cytokines, such as interleukin-2 (IL-12), interleukin-10 (IL-10), gamma interferon (IFN-) and TNF-[1],[2], thus establishing a link between innate and adaptative immunity during parasitic infection[3],[4]. Studies have shown that neutrophils could protect or enhance infection withLeishmania (Leishmania) major[5][8]. Neutrophils were also shown to behave like Trojan horses, sponsoring the invasion of macrophages byL. (L.) major in vitro[9]andin vivo[10]. A role for neutrophil in protective immune responses toL. (Viannia) braziliensis[11]and to visceralizingLeishmaniaspecies was also reported[12][15]. In previous studies neutrophils were detected inL. (L.) amazonensislesions soon after infection[16]. Neutrophils were also implicated in chemotactic responses toL. (L.) amazonensispromastigotes, in the destruction of these parasites and in the release of leishmanicidal effectors[17][19]. More recently, human apoptotic and necrotic neutrophils were shown to increase and to reduce, respectively,L. (L.) amazonensisparasite burden in infected macrophages[20]. We have previously observed that neutrophils predominate at the sites of infection withL. (L.)amazonensis in resistant C3H/HePas mice which displayed a low parasite burden. In contrast, few neutrophils were found in the parasite-rich lesions of susceptible BALB/c mice (unpublished results). These observations suggest that neutrophils could play a role in the resistance of C3H/HePas mice to the parasite. In the present study we investigated thein vitrointeraction of neutrophils withL. (L.) amazonensis-infected macrophages. We report thatL. (L.) amazonensisamastigotes were destroyed when infected peritoneal macrophages from either susceptible BALB/c or resistant C3H/HePas mice were co-cultured with syngeneic inflammatory neutrophils. The leishmanicidal activity did not require cell to cell contact and was mediated by TNF-, neutrophil elastase and platelet activating factor. These Rabbit Polyclonal to RAB6C findings indicate that inflammatory neutrophils may play a role in innate host defense againstL. (L.) amazonensis. == CZC-8004 Results == == L. (L.) amazonensisamastigotes are killed after addition of neutrophils to infected macrophages == Inflammatory neutrophils isolated from peritoneal cavities of BALB/c mice 7 h after they had received an intraperitoneal injection of starch were co-cultured for four days with mouse peritoneal macrophages previously infected withL. (L.) amazonensis. The macrophage infection was followed by light microscopy and immunofluorescence. Destruction of amastigotes could be observed by the presence of parasite debris CZC-8004 insideL. (L.) amazonensis-infected macrophages co-cultured with neutrophils, as indicated by the arrows inFigure 1, E to H. In contrast, healthy amastigotes could be visualized in control cultures (arrows inFigure 1, A to D). == Figure 1. Light microscopy and immunofluorescence of neutrophils co-cultured withL. (L.) amazonensis-infected macrophages. == L. (L.) amazonensis-infected macrophages were either cultured in the absence (upper sequence) or in the presence of inflammatory neutrophils (lower sequence). After 4 days the cultures were stained with HE (A, E) or DAPI (C, G) or incubated with a rabbit serum anti-L. (L.) amazonensisamastigotes and stained with DAPI (D, H). B, F, Nomarski interference contrast. Red arrows indicate intact amastigotes; white arrows show parasite debris. A, E Magnification, x1,000. CZC-8004 B, C, D, F, G, H – Magnification, x400. == Effect of neutrophils.