Rapid secretion of prestored interleukin 8 from Weibel-Palade bodies of microvascular endothelial cells

Abstract

Interleukin (IL)-8, a C-X-C chemokine, activates integrin-mediated adhesion of neutrophils. Presentation of IL-8 on the endothelial cell surface may promote leukocyte extravasation. We found that cultured human microvascular endothelial cells from the intestine (HIMEC) and from nasal polyps (PMEC), but not human umbilical vein endothelial cells (HUVEC), contained IL-8 in intracellular granules that coexpressed von Willebrand factor (vWf ). This observation was corroborated by the immunohistochemical observation of double-positive granules (IL-8(+)vWf+) in vessels of small and large intestine, nasal mucosa, and skin, whereas umbilical cords revealed no endothelial IL-8. After treatment of HIMEC or PMEC with histamine or thrombin, a dramatic increase in supernatant IL-8 concentration was observed within 3 min, whereas no increase in IL-8 was detected in supernatants of identically treated HUVEC cultures. Histamine or thrombin treatment also caused IL-8-containing granules to rapidly disappear from HIMEC. In HUVEC, IL-8-containing granules were inducible by treatment with recombinant human IL-1beta for 24 h; additional histamine treatment doubled IL-8 secretion from HUVEC in the same rapid manner observed for mucosal EC. These data suggested that IL-8 prestored in microvascular endothelial cells may provide a rapid pathway for specific activation of neutrophil adhesion at sites of acute inflammation.

Figure 1

Expression of IL-8 and vWf in resting HIMEC. Two-color immunofluorescence staining for IL-8 (a, c, and e) and vWf (b, d, and e) on monolayer culture. Red, IL-8 staining; green, vWf staining; yellow, colocalized IL-8 and vWf staining. Scale bars, 10 μm.

Figure 2

Effect of histamine on release of IL-8. Time course of IL-8 release from confluent EC cultures in the presence (open circles) or absence (filled circles) of histamine (0.1 mM). ELISA measurements in supernatant fluid, representative results from one of four similar experiments (mean ± SD of triplicate wells).

Figure 3

Effect of cycloheximide on histamine-induced IL-8 secretion. Confluent EC were incubated in the absence (white bars) or presence (black bars) of cycloheximide (Cx; 1 mg/ml, 24 h) and washed twice in fresh medium before histamine stimulation (0.1 mM, 15 min). ELISA measurements of IL-8 in EC culture supernatants. Representative results from one of three similar experiments (mean ± SD of triplicate wells).

Figure 4

IL-8⁺ vesicles disappear rapidly after activation with histamine. IL-8 expression in the absence (a) or presence (b) of histamine (0.1 mM, 15 min). Immunofluorescence staining (clone NAP-1) of fixed and permeabilized HIMEC culture (scale bars, 10 μm).

Figure 4

IL-8⁺ vesicles disappear rapidly after activation with histamine. IL-8 expression in the absence (a) or presence (b) of histamine (0.1 mM, 15 min). Immunofluorescence staining (clone NAP-1) of fixed and permeabilized HIMEC culture (scale bars, 10 μm).

Figure 5

In situ detection of IL-8 in human small intestine. EC in submucosal vein of human jejunum stained for IL-8 (a and c) and vWf (b and c). Red, IL-8 staining; green, vWf staining; yellow, colocalized IL-8 and vWf staining. Arrows, Double-positive granules; note variability of staining intensity for the two proteins. Two-color immunofluorescence staining of fixed and permeabilized tissue sections (scale bars, 20 μm).

Figure 6

Effect of histamine on release of IL-8 from IL-1β– activated EC. Confluent EC monolayers were activated with rhIL-1β (100 U/ml, 24 h) and washed twice in fresh medium before histamine stimulation (0.1 mM, 15 min). ELISA measurements of IL-8 in EC culture supernatants, representative results from one of four similar experiments (mean ± SD of triplicate wells).