Endothelial cell "memory" of inflammatory stimulation: human venular endothelial cells store interleukin 8 in Weibel-Palade bodies

Abstract

The expression and secretion of interleukin (IL)-8, the prototype member of the C-X-C subfamily of chemokines, can be induced by diverse inflammatory stimuli in many cells, including endothelial cells (EC). Upon de novo synthesis, IL-8 localizes intracellularly in the Golgi apparatus, from where it is secreted. In addition to this constitutive secretory pathway, we describe a depot storage and separate regulated secretory pathway of IL-8 in EC. The prolonged stimulation of primary human EC with inflammatory mediators resulted in the accumulation of IL-8 in Weibel-Palade bodies, where it colocalized with von Willebrand factor. IL-8 was retained in these storage organelles for several days after the removal of the stimulus and could be released by EC secretagogues such as phorbol myristate acetate, the calcium ionophore A23187, and histamine. These findings suggest that storage of IL-8 in Weibel-Palade bodies may serve as the EC "memory" of a preceding inflammatory insult, which then enables the cells to secrete IL-8 immediately without de novo protein synthesis.

Figure 1

Immunolocalization of IL-8 in HUVEC: Golgi apparatus and Weibel-Palade bodies. HUVEC were stimulated with 1,000 U/ml IL-1β for 4 h (a–c) or overnight (d–l). Cells were fixed and permeabilized (a–i) or fixed only (j–l), and colocalization experiments were performed as described in Materials and Methods. Images (a–f    ) were recorded with the confocal laser scanning microscope (scale bar, 10 μm). Localization of IL-8 (a) and the 58-kD Golgi protein (b) is shown in two separate cells and of IL-8 (d) and vWf (e) in one individual cell. Images a and b were merged to yield c, and the merged image of d and e is shown in f; yellow, areas of colocalization. Pictures g–l were photographed from an Olympus IX 70 fluorescence microscope (scale bar, 20 μm): IL-8 (g and j), vWf (h and k), and DAPI (i and l).

Figure 2

Immunoelectron microscopic colocalization of IL-8 and vWf in Weibel-Palade bodies. HUVEC were stimulated with 1,000 U/ml IL-1β overnight and processed for electron microscopy as indicated in Materials and Methods. Large granules (18-nm gold) represent vWf; small granules (6-nm gold) correspond to IL-8. Short arrows, IL-8 in the Golgi apparatus; long arrows, Weibel-Palade bodies that contain both IL-8 and vWf. Top left inset, The absence of granules from Weibel-Palade bodies stained with the irrelevant antibodies rabbit antioccludin and mouse anti–E-selectin. Bottom right inset, Weibel-Palade bodies in unstimulated HUVEC contained only vWf, but no IL-8. Scale bar, 100 nm.

Figure 3

Removal of IL-1β from stimulated HUVEC: effect on intracellular and secreted IL-8. HUVEC were stimulated with 1,000 U/ml IL-1β overnight and then further incubated in IL-1–containing medium (a) or IL-1–free medium (b and c) for 4 h (b) or the times indicated (c). Secreted (open circles) and intracellular (filled circles) IL-8 were determined as described in Materials and Methods, and are expressed as nanograms of IL-8 per 10⁶ cells and as arbitrary fluorescence units (FU) × 10⁻⁶ per 10⁶ cells, respectively. Results are means ± SD of triplicate samples.

Figure 3

Removal of IL-1β from stimulated HUVEC: effect on intracellular and secreted IL-8. HUVEC were stimulated with 1,000 U/ml IL-1β overnight and then further incubated in IL-1–containing medium (a) or IL-1–free medium (b and c) for 4 h (b) or the times indicated (c). Secreted (open circles) and intracellular (filled circles) IL-8 were determined as described in Materials and Methods, and are expressed as nanograms of IL-8 per 10⁶ cells and as arbitrary fluorescence units (FU) × 10⁻⁶ per 10⁶ cells, respectively. Results are means ± SD of triplicate samples.

Figure 4

Release of IL-8 from Weibel-Palade bodies by secretagogues: intracellular and secreted IL-8. HUVEC were stimulated with 1,000 U/ml IL-1β overnight and then released into IL-1–free medium for 4 h. Control cells (a) and cells that were induced to release IL-8 from Weibel-Palade bodies by the addition of 10 nM PMA (b and c), 10 μM A23187 (c), and 5 mM histamine (c) were examined after an additional 1 h. IL-8 was determined as described in Materials and Methods; values are means ± SD of triplicate samples.

Figure 4

Release of IL-8 from Weibel-Palade bodies by secretagogues: intracellular and secreted IL-8. HUVEC were stimulated with 1,000 U/ml IL-1β overnight and then released into IL-1–free medium for 4 h. Control cells (a) and cells that were induced to release IL-8 from Weibel-Palade bodies by the addition of 10 nM PMA (b and c), 10 μM A23187 (c), and 5 mM histamine (c) were examined after an additional 1 h. IL-8 was determined as described in Materials and Methods; values are means ± SD of triplicate samples.