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.


Cytosolic storage of IL-16 and MIF in neutrophils. (a) Neutrophils were fixed and stained for IL-16 (green, I and III), MIF (green, V and VII), MMP9 (red, I and V), S100A8 (red, III and VII) and the nuclei (DAPI, blue; I, III, V and VII). Staining was analyzed using confocal microscopy. Colocalization studies of the fluorescent signals for IL-16 and MMP9 (II), IL-16 and S100A8 (IV), MIF and MMP9 (VI), MIF and S100A8 (VIII) were performed. White signals indicate colocalization of the green and red fluorescent signals. Bars, 3 μm. The presented data are representative of three independent experiments. (b) Freshly isolated neutrophils were fractionated in a two-step Percoll gradient and analyzed for its content of IL-16, MIF, pro-MMP9 and S100A8 using western blot analysis. The presented data are representative of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Cytosolic storage of IL-16 and MIF in neutrophils. (a) Neutrophils were fixed and stained for IL-16 (green, I and III), MIF (green, V and VII), MMP9 (red, I and V), S100A8 (red, III and VII) and the nuclei (DAPI, blue; I, III, V and VII). Staining was analyzed using confocal microscopy. Colocalization studies of the fluorescent signals for IL-16 and MMP9 (II), IL-16 and S100A8 (IV), MIF and MMP9 (VI), MIF and S100A8 (VIII) were performed. White signals indicate colocalization of the green and red fluorescent signals. Bars, 3 μm. The presented data are representative of three independent experiments. (b) Freshly isolated neutrophils were fractionated in a two-step Percoll gradient and analyzed for its content of IL-16, MIF, pro-MMP9 and S100A8 using western blot analysis. The presented data are representative of two experiments.

Mentions: IL-16 and MIF share a feature that is rather uncommon for cytokines: they both lack a signal sequence.22,23 Having observed that these two cytokines with this unusual feature are present in neutrophils, we addressed the issue of the subcellular localization and release of IL-16 and MIF by primary human neutrophils in vitro by applying immunofluorescence staining and confocal fluorescence microscopy. Imaging confirmed the intracellular presence of preformed IL-16 and MIF in neutrophils (Figure 2a). The white spots/areas shown represent the results of the computer-assisted analysis of colocalization of the green IL-16 signals with the red signals of S100A8 (Figure 2a, III and IV). The analysis showed a strong colocalization of IL-16 with S100A8, an abundant cytosolic protein in neutrophils (Figure 2a, IV). In contrast, colocalization analysis of IL-16 with gelatinase (MMP9; Figure 2a, I and II), a protein contained in both gelatinase and specific granules, showed a weak colocalization with IL-16. Similarly, a very strong colocalization between MIF and S100A8 signals was also observed (Figure 2a, VII and VIII), whereas only very weak colocalization was observed between the MIF and MMP9 signals (Figure 2a, V and VI).


Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol
Cytosolic storage of IL-16 and MIF in neutrophils. (a) Neutrophils were fixed and stained for IL-16 (green, I and III), MIF (green, V and VII), MMP9 (red, I and V), S100A8 (red, III and VII) and the nuclei (DAPI, blue; I, III, V and VII). Staining was analyzed using confocal microscopy. Colocalization studies of the fluorescent signals for IL-16 and MMP9 (II), IL-16 and S100A8 (IV), MIF and MMP9 (VI), MIF and S100A8 (VIII) were performed. White signals indicate colocalization of the green and red fluorescent signals. Bars, 3 μm. The presented data are representative of three independent experiments. (b) Freshly isolated neutrophils were fractionated in a two-step Percoll gradient and analyzed for its content of IL-16, MIF, pro-MMP9 and S100A8 using western blot analysis. The presented data are representative of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Cytosolic storage of IL-16 and MIF in neutrophils. (a) Neutrophils were fixed and stained for IL-16 (green, I and III), MIF (green, V and VII), MMP9 (red, I and V), S100A8 (red, III and VII) and the nuclei (DAPI, blue; I, III, V and VII). Staining was analyzed using confocal microscopy. Colocalization studies of the fluorescent signals for IL-16 and MMP9 (II), IL-16 and S100A8 (IV), MIF and MMP9 (VI), MIF and S100A8 (VIII) were performed. White signals indicate colocalization of the green and red fluorescent signals. Bars, 3 μm. The presented data are representative of three independent experiments. (b) Freshly isolated neutrophils were fractionated in a two-step Percoll gradient and analyzed for its content of IL-16, MIF, pro-MMP9 and S100A8 using western blot analysis. The presented data are representative of two experiments.
Mentions: IL-16 and MIF share a feature that is rather uncommon for cytokines: they both lack a signal sequence.22,23 Having observed that these two cytokines with this unusual feature are present in neutrophils, we addressed the issue of the subcellular localization and release of IL-16 and MIF by primary human neutrophils in vitro by applying immunofluorescence staining and confocal fluorescence microscopy. Imaging confirmed the intracellular presence of preformed IL-16 and MIF in neutrophils (Figure 2a). The white spots/areas shown represent the results of the computer-assisted analysis of colocalization of the green IL-16 signals with the red signals of S100A8 (Figure 2a, III and IV). The analysis showed a strong colocalization of IL-16 with S100A8, an abundant cytosolic protein in neutrophils (Figure 2a, IV). In contrast, colocalization analysis of IL-16 with gelatinase (MMP9; Figure 2a, I and II), a protein contained in both gelatinase and specific granules, showed a weak colocalization with IL-16. Similarly, a very strong colocalization between MIF and S100A8 signals was also observed (Figure 2a, VII and VIII), whereas only very weak colocalization was observed between the MIF and MMP9 signals (Figure 2a, V and VI).

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.