Limits...
Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol

View Article: PubMed Central - PubMed

ABSTRACT

Neutrophils harbor a number of preformed effector proteins that allow for immediate antimicrobial functions without the need for time-consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, including interleukin (IL)-1ra, CXCL8 and CXCL2. In the search for additional preformed cytokines, a cytokine array analysis identified IL-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in lysates from human primary neutrophils. Both IL-16 and MIF are unconventional cytokines because they lack a signal sequence. Using confocal immunofluorescence microscopy as well as western blot analysis of subcellular fractions, IL-16 and MIF were found to be stored in the cytosol rather than in the granules of human neutrophils, which implies an unconventional secretion mechanism for both cytokines. IL-16 is synthesized and stored as a precursor (pre-IL-16). We present evidence that the processing of pre-IL-16 to the biologically active IL-16C is mediated by caspase-3 and occurs during both spontaneous and UV-induced apoptosis of human neutrophils. Although IL-16 processing occurs during apoptosis, IL-16C and MIF release was observed only during secondary necrosis of neutrophils. Screening a panel of microbial substances and proinflammatory cytokines did not identify a stimulus that induced the release of IL-16C and MIF independent of secondary necrosis. The data presented here suggest that IL-16 and MIF are neutrophil-derived inflammatory mediators released under conditions of insufficient clearance of apoptotic neutrophils, as typically occurs at sites of infection and autoimmunity.

No MeSH data available.


IL-16C release upon treatments inducing secondary necrosis of neutrophils. (a) Neutrophils were irradiated with 0, 200, 800 and 1600 mJ/cm2 UV light (256 nm). After 6 h of incubation at 37 °C, the supernatants were analyzed with ELISA for released IL-16 and MIF. Neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the means±S.E.M. of three independent experiments (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 compared with the 0-h sample by one-way ANOVA followed by Holm Sidak multiple comparison correction). Individual data points are shown as black dots. (b) Neutrophils were infected with A. phagocytophilum (A. phago) for 6 at 37 °C. The supernatants were analyzed with ELISA for released IL-16 and MIF, and neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the means±S.E.M. of four independent experiments (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 compared with the uninfected control by paired t-test). Individual data points are shown as black dots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4979515&req=5

fig5: IL-16C release upon treatments inducing secondary necrosis of neutrophils. (a) Neutrophils were irradiated with 0, 200, 800 and 1600 mJ/cm2 UV light (256 nm). After 6 h of incubation at 37 °C, the supernatants were analyzed with ELISA for released IL-16 and MIF. Neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the means±S.E.M. of three independent experiments (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 compared with the 0-h sample by one-way ANOVA followed by Holm Sidak multiple comparison correction). Individual data points are shown as black dots. (b) Neutrophils were infected with A. phagocytophilum (A. phago) for 6 at 37 °C. The supernatants were analyzed with ELISA for released IL-16 and MIF, and neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the means±S.E.M. of four independent experiments (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 compared with the uninfected control by paired t-test). Individual data points are shown as black dots.

Mentions: Our results revealed that IL-16C and MIF are released from secondary necrotic rather than apoptotic neutrophils. In an attempt to clarify the role of apoptosis in the release of IL-16C and MIF, we modulated neutrophil apoptosis. For this purpose, spontaneous neutrophil apoptosis was facilitated using UV-light irradiation (256 nm). UV-light irradiation enhanced neutrophil apoptosis in a dose-dependent manner (Figure 5a). Importantly, although irradiation with 200 mJ/cm2 UV-light-induced apoptosis in ~80% of neutrophils, no significant release of IL-16 and MIF was observed. However, irradiation with higher UV doses led to the release of high IL-16 and MIF levels (Figure 5a). Notably, increasing the UV dose enhanced the degree of apoptosis only marginally but led to the induction of secondary necrosis in ~25% of neutrophils (Figure 5a). These results demonstrate once more that IL-16C and MIF are released not from apoptotic neutrophils but from secondary necrotic neutrophils, as has been shown during spontaneous cell death.


Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol
IL-16C release upon treatments inducing secondary necrosis of neutrophils. (a) Neutrophils were irradiated with 0, 200, 800 and 1600 mJ/cm2 UV light (256 nm). After 6 h of incubation at 37 °C, the supernatants were analyzed with ELISA for released IL-16 and MIF. Neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the means±S.E.M. of three independent experiments (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 compared with the 0-h sample by one-way ANOVA followed by Holm Sidak multiple comparison correction). Individual data points are shown as black dots. (b) Neutrophils were infected with A. phagocytophilum (A. phago) for 6 at 37 °C. The supernatants were analyzed with ELISA for released IL-16 and MIF, and neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the means±S.E.M. of four independent experiments (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 compared with the uninfected control by paired t-test). Individual data points are shown as black dots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979515&req=5

fig5: IL-16C release upon treatments inducing secondary necrosis of neutrophils. (a) Neutrophils were irradiated with 0, 200, 800 and 1600 mJ/cm2 UV light (256 nm). After 6 h of incubation at 37 °C, the supernatants were analyzed with ELISA for released IL-16 and MIF. Neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the means±S.E.M. of three independent experiments (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 compared with the 0-h sample by one-way ANOVA followed by Holm Sidak multiple comparison correction). Individual data points are shown as black dots. (b) Neutrophils were infected with A. phagocytophilum (A. phago) for 6 at 37 °C. The supernatants were analyzed with ELISA for released IL-16 and MIF, and neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the means±S.E.M. of four independent experiments (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 compared with the uninfected control by paired t-test). Individual data points are shown as black dots.
Mentions: Our results revealed that IL-16C and MIF are released from secondary necrotic rather than apoptotic neutrophils. In an attempt to clarify the role of apoptosis in the release of IL-16C and MIF, we modulated neutrophil apoptosis. For this purpose, spontaneous neutrophil apoptosis was facilitated using UV-light irradiation (256 nm). UV-light irradiation enhanced neutrophil apoptosis in a dose-dependent manner (Figure 5a). Importantly, although irradiation with 200 mJ/cm2 UV-light-induced apoptosis in ~80% of neutrophils, no significant release of IL-16 and MIF was observed. However, irradiation with higher UV doses led to the release of high IL-16 and MIF levels (Figure 5a). Notably, increasing the UV dose enhanced the degree of apoptosis only marginally but led to the induction of secondary necrosis in ~25% of neutrophils (Figure 5a). These results demonstrate once more that IL-16C and MIF are released not from apoptotic neutrophils but from secondary necrotic neutrophils, as has been shown during spontaneous cell death.

View Article: PubMed Central - PubMed

ABSTRACT

Neutrophils harbor a number of preformed effector proteins that allow for immediate antimicrobial functions without the need for time-consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, including interleukin (IL)-1ra, CXCL8 and CXCL2. In the search for additional preformed cytokines, a cytokine array analysis identified IL-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in lysates from human primary neutrophils. Both IL-16 and MIF are unconventional cytokines because they lack a signal sequence. Using confocal immunofluorescence microscopy as well as western blot analysis of subcellular fractions, IL-16 and MIF were found to be stored in the cytosol rather than in the granules of human neutrophils, which implies an unconventional secretion mechanism for both cytokines. IL-16 is synthesized and stored as a precursor (pre-IL-16). We present evidence that the processing of pre-IL-16 to the biologically active IL-16C is mediated by caspase-3 and occurs during both spontaneous and UV-induced apoptosis of human neutrophils. Although IL-16 processing occurs during apoptosis, IL-16C and MIF release was observed only during secondary necrosis of neutrophils. Screening a panel of microbial substances and proinflammatory cytokines did not identify a stimulus that induced the release of IL-16C and MIF independent of secondary necrosis. The data presented here suggest that IL-16 and MIF are neutrophil-derived inflammatory mediators released under conditions of insufficient clearance of apoptotic neutrophils, as typically occurs at sites of infection and autoimmunity.

No MeSH data available.