Low concentration of CpG-ODN type A, in GM-CSF-pretreated cells, suppressed the production of TGF-β by neutrophils. However, at higher concentrations of CpG-ODN type A or B in the presence of GM-CSF, the TGF-β levels were maintained (Figure 1b). As shown, neither GM-CSF (Figure 1c) nor CpG-ODN class A (Table 2) elicited TNF-α production by neutrophils on their

own. On the other hand, CpG-ODN type A, at concentrations of 15 and 40 μg/mL, significantly stimulated GM-CSF-pretreated cells to secrete selleck compound TNF-α whereas control ODN did not (P < 0·05). Consequently, the production of TNF-α by neutrophils depends on the co-stimulation with GM-CSF and CpG-ODN type A. The secretion of IL-8 and TNF-α was not observed in the presence BAY 80-6946 of different concentrations of CpG-ODN class B regardless

of GM-CSF treatment (Figure 1a,c). Dose–response assessment showed that the optimum concentration of CpG-ODN to stimulate neutrophils was 40 μg/mL. In addition, with regard to TNF-α production, GM-CSF at concentration of 50 ng/mL possesses a co-stimulatory action on neutrophils in the presence of CpG-ODN. Ten healthy individuals were, therefore, tested simultaneously at these concentrations. As shown in Table 3, the obtained results from these experiments confirmed previous data. That is, the level of TNF-α in cells co-stimulated with the combination of these agents increased threefold and fivefold as compared to that in cells stimulated only with GM-CSF and CpG-ODN class A, respectively. The results obtained in healthy donors were followed up by assessment of the same parameters in asymptomatic and nonhealing CL individuals. IL-8, TNF-α and TGF-β were measured in cell supernatants PRKACG after 18 h (Figure 2a). Neutrophils

from all three groups produced similar levels of IL-8 upon stimulation with L. major. Moreover, in all groups, in comparison with infected neutrophils, IL-8 secretion by infected neutrophils, pretreated with GM-CSF and stimulated with CpG-ODN class A, decreased approximately 1·3-folds. TGF-β was detected, but not induced by either stimulation or infection in both healthy donors and nonhealing individuals. However, neutrophils from asymptomatic subjects did not produce measurable levels of TGF-β regardless of stimulation (Figure 2b). TNF-α production by neutrophils was induced by L. major infection (P < 0·05) in asymptomatic and nonhealing individuals (Figure 2c). Co-stimulation with GM-CSF and CpG-ODN class A did not increase TNF-α levels induced by L. major in nonhealing donors, but did so in asymptomatic individuals (P < 0·05). There was no difference in the level of TNF-α between unstimulated and stimulated infected neutrophils in normal and nonhealing individuals (Figure 2c). High-quality RNAs were isolated from all individuals (healthy, nonhealing and asymptomatic) for further analysis using real-time PCR.