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. 2003 Sep;140(1):133-45.
doi: 10.1038/sj.bjp.0705413. Epub 2003 Aug 11.

Dexamethasone enhances interaction of endogenous annexin 1 with L-selectin and triggers shedding of L-selectin in the monocytic cell line U-937

Catherine de Coupade  1 , Egle Solito, Jon D Levine
Affiliations

Dexamethasone enhances interaction of endogenous annexin 1 with L-selectin and triggers shedding of L-selectin in the monocytic cell line U-937

Catherine de Coupade et al. Br J Pharmacol. 2003 Sep.

Abstract

(1) L-selectin, constitutively expressed by leukocytes, is involved in the initial binding of leukocytes to activated endothelium. Anti-inflammatory drugs like glucocorticoids can induce shedding of L-selectin, but the mechanism is still unknown. Annexin 1, a protein whose synthesis and externalization/secretion are induced during the inflammatory response, has been proposed as a mediator of the anti-inflammatory actions of glucocorticoids. (2) The monocytic cell line U-937 strongly expresses Annexin 1 after 24 h of phorbol 12-myristate 13-acetate (PMA, 1 nm) treatment and externalizes/releases the protein after additional 16 h of dexamethasone (1 microm) treatment. (3) This study investigated the possible regulation of cell surface L-selectin shedding by endogenous Annexin 1, and its role in glucocorticoid-induced L-selectin shedding in the U-937 cell line. (4) PMA- and dexamethasone treatment-induced L-selectin shedding was potentially mediated by Annexin 1, since neutralizing antibodies against Annexin 1 reduced dexamethasone- and Annexin 1-induced shedding. (5) Immunoprecipitation and binding assays provided support for the suggestion that this effect could be mediated by an interaction between externalized Annexin 1 and L-selectin. Such interaction involved the N-terminal domain of Annexin 1 and was calcium-dependent. Confocal microscopy studies demonstrated increased colocalization of Annexin 1 and L-selectin on the cell surface. (6) Overall, our study provides new insights into the potential role of endogenous ANXA1 as a mediator of dexamethasone-induced L-selectin shedding, which may contribute to the anti-inflammatory activity of glucocorticoids.

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Figures

Figure 1
Figure 1
Effect of dexamethasone-induced ANXA1 expression and externalization/release on L-selectin cell-surface expression in differentiated U-937 cells. Nonstimulated U-937 cells (C) were treated with 1 nM PMA for 24 h (P) or with 1 μM dexamethasone for 16 h (D) or with the combination PMA (24 h)+dexamethasone (additional 16 h) (P+D). Control samples were incubated in RPMI+5% serum medium for the same period (24 h+16 h). Flow cytometry histograms show ANXA1 (a), L-selectin (c) and CD11a (d) immunoreactivities on U-937 cell surface after the indicated treatments. Data are shown as relative fluorescence intensity (RFI) and represent mean±s.e.m. of five independent experiments done in duplicate, *P⩽0.05. FITC-conjugated antibody alone was used as an internal control for background fluorescence, (b) Analysis of the amount of cell membrane-associated ANXA1 that was EDTA-extracted (white bars) or released in U-937 supernatants (black bars) from nonstimulated (C), PMA 24 h (P), dexamethasone 16 h (D) or PMA and dexamethasone (P+D)-treated cells. Cell-free supernatants were harvested from U-937 cells and concentrated three times before ELISA; EDTA-wash was used for the assay. Soluble and membrane-associated ANXA1 were captured with a polyclonal antibody anti-ANXA1, and detected with a monoclonal antibody as described in Methods. Soluble ANXA1 levels were quantitated by constructing a standard curve using human recombinant ANXA1 protein. Data represent mean±s.e.m. of three independent experiments done in quadruplicate, *P≤0.05.
Figure 2
Figure 2
Exogenous human recombinant ANXA1 mimicked the endogenous protein by inducing L-selectin shedding. Both effects were reversed by a neutralizing antibody. Nonstimulated U-937 cells (C) were treated with 1 nM PMA for 24 h (P) or with 1 μM dexamethasone for 16 h (D) or with the combination PMA (24 h)+dexamethasone (additional 16 h) (P+D). Control samples were incubated in RPMI+5% serum medium for the same period (24 h+16 h), (a) The contribution of ANXA1 to the downregulation of L-selectin expression was confirmed by PMA treatment of U-937 cells for 24 h, followed by 30 min incubation at 37°C with 5 μg ml−1 of human recombinant ANXA1 (PMA+hrANXA1). Cells were treated with PMA for 24 h, then incubated with a specific anti-ANXA1 mAb at a concentration of 20 μg ml−1 (+mAB1-A) for 10 min, and finally incubated with either hrANXA1 for 30 min or dexamethasone for 16 h. A control antibody (+mIgG2a) was used at the same concentration and incubated for the same period of time. Data represent mean±s.e.m. of three independent experiments done in duplicate, *P⩽0.05. (b) ELISA analysis for soluble L-selectin in U-937 cell-free supernatants. Neutralizing anti-ANXA1 mAb (20 μg ml−1) or its isotype-matched control antibody (+mIgG2a) was incubated with PMA-treated cells 10 min before adding hrANXA1 for 30 min. Soluble L-selectin was trapped with a polyclonal L-selectin antibody and detected with a monoclonal L-selectin antibody (Dreg-56), as described in Methods. Data represent mean±s.e.m. of three independent experiments done in triplicate, P≤0.05.
Figure 3
Figure 3
Coimmunoprecipitation experiments suggest an interaction between ANXA1 and L-selectin. (a) Immunoprecipitation analysis of ANXA1. Upper panel: total protein lysates from HEK-293 and nontreated (C), PMA (P) or PMA+dexamethasone (P+D)-treated U-937 cells were extracted, immunoprecipitated with specific antibodies for ANXA1 (IP) and immunoblotted for L-selectin or for its isotype-matched antibody, hIgG1 (WB). Lower panel: relative intensities of L-selectin immunolabeling in the samples. Bars indicate mean±s.e.m. of three independent experiments, *P⩽0.05. (b) Presence of ANXA1 protein in the immunoprecipitation was confirmed by reprobing the membrane with specific ANXA1 antibody. Internal control for the immunoprecipitation was demonstrated by using nonspecific control antibody (rabbit IgG), and blotting with ANXA1 and L-selectin-specific antibodies, (c) Specificity of the interaction is shown by reversion of the immunoprecipitation–immunoblot experiment in U-937-treated cells. All data shown are representative of three independent experiments giving similar results.
Figure 4
Figure 4
Chymotrypsin-induced loss of L-selectin reduced ANXA1 interaction with L-selectin. (a) Effect of different concentrations of chymotrypsin on U-937 expression of L-selectin. Cells were incubated with different doses of chymotrypsin (U (1 × 106)−1 cells) for 15 min at 37°C. The expression of L-selectin antigen was analyzed by flow cytometry using the FITC-DREG-56 antibody. Chymotrypsin induced a dose-dependent decrease in L-selectin expression with a maximal effect achieved at 10 U per 1 × 106 cells (−80%). Bars indicate mean±s.e.m. of three independent experiments, (b) Loss of L-selectin antigen by chymotrypsin is associated with a decrease in the interaction between ANXA1 and L-selectin. U-937 cells (C) were stimulated with 1 nM PMA (P) for 24 h and then with 10 U per 1 × 106 cells of chymotrypsin for 15 min at 37°C, before performing the immunoprecipitation with a specific ANXA1 antibody, as described above. L-selectin immunoreactivity was detected with DREG-56 (WB). Data shown are representative of three independent experiments giving similar results.
Figure 5
Figure 5
L-selectin binding to ANXA1 is calcium-dependent and involves the N-terminal domain of ANXA1. (a) Comparison of the amounts of ANXA1 detected after presentation of the protein by a specific mAb or after direct binding to plastic. A monoclonal ANXA1 antibody was coated onto microtiter plates (3 μg ml−1) to capture varying concentrations of ANXA1 (mAB1-A+ANXA1). This binding was compared to the binding for the same range of ANXA1 recombinant protein concentrations directly coated on extra-high binding capacity plastic (ANXA1). In both cases, ANXA1 was detected with a polyclonal ANXA1 antibody and showed no major difference between the two methods, (b–d) Direct binding between annexins and L-selectin chimeric protein (Lec-IgM) in the presence or absence of calcium, (b) Increasing concentrations of Lec-IgM (black square) were immobilized on plastic and then incubated with 9.4 μg ml−1 of ANXA1. Internal control for nonspecific binding was determined in the presence of human IgM (hIgM) (open square), (c) Lec-IgM or human IgM were added to increasing concentrations of ANXA1, either directly coated on the plastic (ANXA1) or presented by the monoclonal antibody mAB1-A (mAB-1A+ANXA1). Presence of the ANXA1 in the assay was detected with a polyclonal antibody (mAB1-A+ANXA1) as in (a), (d) Effect of the presence of calcium of the N-terminal domain of ANXA1 on the interaction between ANXA1 and Lec-IgM. Binding of Lec-IgM (0.125 μg ml−1) to increasing concentrations of ANXA1 was compared to the binding to the same concentration range of the chimeric protein ANXA1-5 (N-terminal domain of ANXA1 (1–26) and core domain of ANXA5) or to an unrelated protein, ANXA5. Experiments were done in PBS+1 mM Ca2++1 mM Mg2+ with or without 10 mM EDTA (+EDTA). Each point represents the mean±s.e.m. of three independent experiments done in duplicates.
Figure 6
Figure 6
Colocalization of ANXA1 and L-selectin on U-937 cell surface by confocal microscopy. ANXA1 (panels I in red) and L-selectin (panels II in green) cell-surface immunostaining were performed on nonstimulated U-937 cells (a) or after PMA+dexamethasone treatment (b). Column III (a, b) shows the distribution of fluorescence signal in merged images. To show the distribution of the colocalization, successive Z-sections through the same cell are illustrated in (b). A monoclonal anti-α-tubulin antibody was used to confirm that the plasma membrane was not permeabilized during immunofluorescence analysis and showed no staining (data not shown). Pictures show representative pattern of polarity and colocalization observed in four different high-power fields (× 100 lens) per condition. Results were obtained from five separate experiments. Scale bars, 20 μm.
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References

    1. ALLPORT J.R., DING H.T., AGER A., STEEBER D.A., TEDDER T.F., LUSCINSKAS F.W. L-selectin shedding does not regulate human neutrophil attachment, rolling, or transmigration across human vascular endothelium in vitro. J. Immunol. 1997;158:4365–4372. - PubMed
    1. ARRIBAS J., COODLY L., VOLLMER P., KISHMOTO T.K., ROSE-JOHN S., MASSAGUE J. Diverse cell surface protein ectodomains are shed by a system sensitive to metalloprotease inhibitors. J. Biol. Chem. 1996;271:11376–11382. - PubMed
    1. BLACKWELL G.J., CARNUCCIO R., DI R.M., FLOWER R.J., LANGHAM C.S., PARENTE L., PERSICO P., RUSSEL S.N., STONE D. Glucocorticoids induce the formation and release of anti-inflammatory and anti-phospholipase proteins into the peritoneal cavity of the rat. Br. J. Pharmacol. 1982;76:185–194. - PMC - PubMed
    1. BLACKWELL G.J., CARNUCCIO R., DI ROSA M., FLOWER R.J., PARENTE L., PERSICO P. Macrocortin: a polypeptide causing the anti-phospholipase effect of glucocorticoids. Nature. 1980;287:147–149. - PubMed
    1. BRUEHL R.E., BERTOZZI C.R., ROSEN S.D. Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody. J. Biol. Chem. 2000;275:32642–32648. - PubMed

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