in Nonhealing BALB/c Mice Activation Delays Cutaneous Leishmaniasis Impairment of Alternative Macrophage

Expressed on various cell types, the IL-4R (cid:1) is a component of both receptors for IL-4 and IL-13. Susceptibility of BALB/c mice to Leishmania major is believed to be dependent on the development of IL-4- and IL-13-producing Th2 cells, while IFN- (cid:2) secretion by Th1 cells is related to resistance. Despite a sustained development of Th2 cells, IL-4R (cid:1) -deﬁcient BALB/c mice are able to control acute cutaneous leishmaniasis, suggesting that IL-4R (cid:1) -bearing cells other than Th2 cells contribute to susceptibility. To analyze the contribution of the IL-4R (cid:1) on macrophages, recently generated macrophage/neutrophil-speciﬁc IL-4R (cid:1) -deﬁcient mice on a susceptible BALB/c genetic background were infected with L. major . Strikingly, macrophage/neutrophil-speciﬁc IL-4R (cid:1) deﬁcient mice showed a signiﬁcantly delayed disease progression with normal Th2 and type 2 Ab responses but improved macrophage leishmanicidal effector functions and reduced arginase activity. Together, these results suggest that alternative macrophage activation contributes to susceptibility in cutaneous leishmaniasis. The Journal of Immunology, 2006, 176: 1115–1121. the organism that has most in-tensely studied and from which the original Th1/Th2 paradigm of immunological development during infectious was determined (1). Protective an IL-12-driven cell-mediated immune response mainly characterized the of IFN- (cid:1) in CD4 (cid:1) Th1 cells and metal sieve. any additional peritoneal exudate cells ﬂushing the peritoneal cavity of infected mice with After depletion of erythrocytes, cells resuspended in complete IMDM (Invitrogen Life Technologies) supplemented with 10% FCS (Invitrogen Life Technologies), 0.05 mM 2-ME (Sigma-Aldrich), and pen-icillin and streptomycin (100 U/ml and 100 (cid:4) g/ml; Invitrogen Life Tech- nologies), cultured in triplicates at 2 (cid:3) 10 6 /ml in 48-well plates. Restimulation performed with L. major Ag (10 6 /ml inactivated or LPS 15 ng/ml). After 48 h, the content of IFN- (cid:1) , IL-4, and NO was quantiﬁed in culture supernatants Arginase activity was measured in cell lysates as Brieﬂy, cells were lysed 50 (cid:4) l of 0.1% Triton X-100 (Sigma-Aldrich). After 30 min on a shaker, 50 (cid:4) l of 10 mM MnCl 2 (Merck), 50 mM Tris-HCl (Merck) added, and the was activated by heating for 10 min at 55°C. Ar- ginine hydrolysis was conducted by incubating 25 (cid:4) l of the activated lysate with 25 (cid:4) l of 0.5 M L -arginine (Merck; pH 9.7) at 37°C for 60 min. The reaction was stopped with 400 (cid:4) l of H 2 SO 4 (96%)/H 3 PO 4 (85%)/H 2 O (1/ 3/7, v/v/v). As a degree of arginase activity, the urea concentration was measured at 540 nm after addition of 25 (cid:4) l of (cid:2) -isonitrosopropiophenone that IL-4R (cid:2) responsiveness is necessary for optimal IFN- (cid:1) production. This suggests that caM (cid:3) may promote Th1 responses as previously found in Schistosoma mansoni -infected LysM cre IL-4R (cid:2) ﬂox/ (cid:2) BALB/c mice (16). Because IL-4R (cid:2) en-gagement suppresses IL-12 production in macrophages (8), mac-rophage-speciﬁc IL-4R (cid:2) deﬁciency may uncover IL-4R (cid:2) -depen-dent proinﬂammatory mechanisms such as the IL-4-mediated instruction of dendritic cells to produce IL-12 which has been shown to promote Th1 cell development and resistance in L. major -infected BALB/c mice (28). However, we did not consistently ﬁnd increased IL-12 in infected LysM cre IL-4R (cid:2) BALB/c mice (data not shown). Factors produced by alternatively activated macrophages may also be directly involved in the regulation of Th1 immune responses. Accordingly, we recently identiﬁed reactive oxygen intermediates and 12/15 lipoxygenase to be suppressive molecules produced by alternatively activated myeloid cells capable of inhibiting T cell proliferation (29).