Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression

Abstract

Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.

Figure 1.

Anti-migratory effect of ANX-A1 and detection of endogenous ANX-A1 in inflamed mesenteries. Rats (n = 4) were treated intravenously with vehicle, PBS (1 ml/kg), or ANX-A1 (20 to 100 μg/kg) 5 minutes before intraperitoneal injection of carrageenin (1.5 mg/kg). a: Dose-dependent reduction by ANX-A1 of neutrophil extravasation into the peritoneal cavity and in the mesenteric tissue. b: Detection of endogenous ANX-A1 in the inflamed mesenteries by Western blot analysis and reduction by the pharmacological treatment. Human recombinant ANX-A1, 500 ng, was used as positive control. c and d: Densitometric analysis for the ANX-A1 standard curve and the mesenteric tissue homogenates (n = 4). Data are mean ±SEM. #, P < 0.05 versus control; *, P < 0.05 versus vehicle group.

Figure 2.

Light micrographs of rat mesenteries showing ANX-A1 immunoreactivity in intravascular and extravascular neutrophils. a: Inflamed mesentery as seen 4 hours after carrageenin with neutrophils adherent to the postcapillary venule (arrowheads) and in the extravascular tissue (open arrow). Endothelium (arrow). Section (0.5 μm) was stained with toluidine blue. b: Immunofluorescence labeling of ANX-A1 in sections of rat mesentery by LCS3. Neutrophils adherent (arrowheads) to the postcapillary endothelium (arrow) were positive for ANX-A1. c, e, and f: Examples of LCS3 (c and d) and LCPS1 (e and f) immunoreactivity in extravasated neutrophils (arrowhead). Staining was obtained with both antibodies (c and e) and greatly diminished after pre-adsorption with the respective antigen (ANX-A1 or peptide Ac2–26) (d and f). Scale bar, 10 μm.

Figure 3.

Immunocytochemistry for ANX-A1 using LCPS1 and LCS3 in intravascular neutrophils. a: LCPS1 staining for ANX-A1 shows a significant proportion in the plasma membrane (arrowheads). Immunoreactivity throughout the cytosol and in granules (arrows) can also be seen. En, endothelial cells. b: Similar but higher immunostaining with LCS3. Arrowheads indicate gold particles in the plasma membrane. c: Example of ANX-A1 labeling at points of contact (arrows) between an adherent neutrophil (Nø) and the endothelial cell (En). d: Absence of gold labeling in sections incubated with control nonimmune sheep serum. Scale bars: 0.5 μm (a); 0.2 μm (b–d).

Figure 4.

Immunocytochemistry for ANX-A1 using LCPS1 and LCS3 in extravascular neutrophils. Extravasated neutrophils were greatly activated as indicated by the presence of large vacuoles in the cytosol (asterisks). a: LCS3 produced intense immunoreaction in the cytosol and nucleus. Foci of ANX-A1 expression are found in the vacuoles. b: A lower degree of immunoreactivity was obtained with LCPS1. Scale bars, 0.5 μm.

Figure 5.

Analysis of ANX-A1 and gelatinase localization in intravascular and extravasated neutrophils. Co-localization of ANX-A1 with gelatinase, after immunogold labeling with 5-nm particles (anti-ANX-A1 serum, LCPS1) and 15-nm particles (anti-gelatinase serum). a: Intravascular neutrophil with examples of gelatinase and ANX-A1 immunoreactivity in close vicinity (arrowheads); En, endothelium. b: Extravascular neutrophil with almost absent gelatinase immunoreactivity, and a high degree of ANX-A1 protein expression. As in Figure 4 ▶ , the presence of large vacuoles in the cytosol is indicated by asterisks. C, collagen fibers. Scale bars: 0.2 μm (a), 0.5 μm (b).

Figure 6.

ANX-A1 mRNA expression in extravasated neutrophils as assessed by in situ hybridization in flat mount rat mesenteries. a: ANX-A1 mRNA is detected in neutrophils after tissue incubation with specific antisense oligonucleotide (arrows). a, b, and c: The identity of the cell type was confirmed by staining of the same sections with propidium iodide (color staining of the nucleus) and analysis in the fluorescence microscope (interference filters at two excitation wavelengths of 334 to 365 and 546 nm). In b the two images were superimposed. d: In situ hybridization product formation after incubation of mesenteric tissue with the antisense oligonucleotides, but not in e after tissue incubation with the sense oligonucleotide. Scale bars, 10 μm.

Figure 7.

Model to summarize neutrophil ANX-A1 mobilization during the extravasation process. a: Circulating (resting) neutrophils have the majority of their ANX-A1 intact (37-kd isoform). b: Neutrophil adhesion to the endothelium causes externalization of the protein on the leukocyte plasma membrane (this study and Harricane et al 22 ). c: A cleavage process may here take place. d: Extravasated neutrophils contain mainly cleaved ANX-A1 (33-kd isoform), with a predominant intracellular localization. Neutrophil contact with extracellular matrix proteins leads to de novo ANX-A1 synthesis as seen by in situ hybridization. Cleaved ANX-A1 (33 kd) is mainly found in the endothelial cells after neutrophil diapedesis.