Prevention of leukocyte migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen - PubMed (original) (raw)

Prevention of leukocyte migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen

Charles J Dimitroff et al. J Clin Invest. 2003 Oct.

Abstract

E-selectin and P-selectin on dermal postcapillary venules play critical roles in the migration of effector T cells into inflamed skin. P-selectin glycoprotein ligand-1 (PSGL-1) modified by alpha1,3-fucosyltransferase is the principal selectin ligand on skin-homing T cells and is required for effector T cell entry into inflamed skin. We have previously shown that a fluorinated analog of N-acetylglucosamine peracetylated-4-fluorinated-d-glucosamine (4-F-GlcNAc), inhibits selectin ligand expression on human T cell PSGL-1. To analyze 4-F-GlcNAc efficacy in dampening effector T cell migration to inflamed skin, we elicited allergic contact hypersensitivity (CHS) reactions in mice treated with 4-F-GlcNAc. We also investigated 4-F-GlcNAc efficacy on lymphocyte E-selectin ligand expression in LNs draining antigen-sensitized skin and on other immunological processes requisite for CHS responses. Our results showed that 4-F-GlcNAc treatment attenuated lymphocyte E-selectin ligand expression in skin-draining LNs and prevented CHS reactions. Significant reductions in inflammatory lymphocytic infiltrate were observed, while pathways related to antigenic processing and presentation and naive T cell recognition within skin-draining LNs were unaffected. These data indicate that 4-F-GlcNAc prevents CHS by inhibiting selectin ligand activity and the capacity of effector T cells to enter antigen-challenged skin without affecting the afferent phase of CHS.

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Figures

Figure 1

Parallel-plate flow chamber analysis of E- and P-selectin binding of murine Th cells following 4-F-GlcNAc treatment. Murine Th1 cells were generated from spleens of C57BL/6 wild-type and FucTVII-deficient mice. To assess the effects of 4-F-GlcNAc on the de novo synthesis of selectin ligands, Th1 cell cultures were first treated with neuraminidase (0.1 U/ml for 1 hour at 37°C) and then regrown for 30 hours in fresh medium containing PBS (diluent), 0.05 mM 4-F-GlcNAc, 0.23 mM swainsonine, or 1 mM GlcNAc (negative control). Cells were harvested, suspended in HBSS with 2 mM CaCl2 and 10 mM HEPES, and perfused over human recombinant E-selectin–Ig chimera (a) or P-selectin–Ig chimera (b) in the parallel-plate flow chamber. Assessments of cell rolling were made at 1.5 dynes/cm2 from the midpoint of the chamber viewing field (mean ± SEM from four fields per selectin spot and three different experiments). Rolling Th1 cells on E- or P-selectin–Ig were inhibitable with 0.5 mM EDTA, and rolling adhesions on human IgG–coated plastic were absent. *P < 0.001, Student’s paired t test. Neur, neuraminidase.

Figure 2

Allergic CHS in mice treated with 4-F-GlcNAc. (a) Wild-type mice were sensitized on the abdomen with 0.5% DNFB in vehicle (acetone/olive oil, 4:1) on days 0 and 1. Mice were given 0.9% saline (diluent), 50, 100, or 250 mg/kg 4-F-GlcNAc, 5 mg/kg swainsonine, or 250 mg/kg GlcNAc (negative drug control) intraperitoneally daily from day 1 to day 6. FucTIV/VII–null mice were also sensitized with 0.5% DNFB and treated with diluent control. Mice were then challenged on the right ear on day 6 with 0.25% DNFB and on the left ear with vehicle alone. After 24 hours, ear swelling was determined by measurement of ear thickness, which was subtracted from base-line ear-thickness measurements. *P < 0.0002, Student’s paired t test. ND, not determined. (b) Wild-type mice were sensitized on the abdomen with 0.5% DNFB on days 0 and 1. Mice were given diluent control or 50 or 100 mg/kg 4-F-GlcNAc intraperitoneally daily either from day 1 to day 6 or from day 6 to day 11. Mice were then challenged on the right ear on day 11 with 0.25% DNFB and on the left ear with vehicle alone. After 24 hours, ear swelling was determined. *P < 0.007, Student’s paired t test. Data in a and b represent mean ear swelling ± SEM from eight mice per group and three experiments. (c) Representative photomicrographs of H&E-stained sections from ears of mice sensitized and challenged with 0.5% DNFB illustrate differences in ear thickness between mice treated with diluent control and with 4-F-GlcNAc. ×200 magnification. Bar: 100 μm.

Figure 3

Effects of 4-F-GlcNAc on effector lymphocyte E-selectin ligand and PSGL-1 expression. Lymphocytic lysates were prepared from inguinal LNs draining DNFB-sensitized skin after day 7 of DNFB sensitization. Lysates (40 μg/lane) were separated on reducing 4–20% SDS-PAGE gradient gels, transferred to PVDF membrane, and blotted with mouse E-selectin–Ig (1 μg/ml) (a) or rabbit anti-sera against mouse PSGL-1 (0.5 μg/ml) (b) and relevant AP-conjugated secondary Ab’s (1:2,000) and AP substrate solution. Negative control blots, E-selectin–Ig staining in the presence in 5 mM EDTA or AP-conjugated secondary Ab’s alone, did not show any signal. Please note the induction of lymphocyte E-selectin ligand principally by the 120- and 220-kDa isoforms of PSGL-1, as well as by a 190-kDa glycoprotein from skin-draining LNs of DNFB-sensitized mice (a). Also, though PSGL-1 levels were relatively unchanged (b), lymphocyte E-selectin ligand induction from skin-draining LNs of DNFB-sensitized FucTIV/VII–/– mice or of DNFB-sensitized mice treated with 100 mg/kg 4-F-GlcNAc was absent (a).

Figure 4

Effects of 4-F-GlcNAc on antigen presentation in regional skin-draining LNs. Following a 4-day intraperitoneal pretreatment with diluent control or with 50, 100, or 250 mg 4-F-GlcNAc, mice were sensitized on the right ear with 0.5% FITC (acetone/dibutyl phthalate, 1:1) and on the left ear with vehicle alone. After 30 hours, DC-enriched cells from ipsilateral cervical/auricular nodes from three mice were stained with PE–anti-CD11c mAb for flow cytometry. Representative histograms of green fluorescence (FITC) of CD11c+ cells from DC-enriched cells prepared from skin-draining LNs of the left side or from contralateral skin-draining LNs of the right side illustrate the increased level of FITC-expressing DCs from LNs draining right FITC-sensitized ears. Data show a lack of dose response due to 4-F-GlcNAc treatment, though a slight increase in DCs staining positive for FITC was observed in all 4-F-GlcNAc–treated mice. Percent positive FITC expression is indicated in diluent control by subtracting gates R8 from R7 (8%); in 50 mg/kg 4-F-GlcNAc by subtracting gates R10 from R9 (12%); in 100 mg/kg 4-F-GlcNAc by subtracting gates R12 from R11 (13%); and in 250 mg/kg 4-F-GlcNAc by subtracting gates R14 from R13 (10%). These data are representative of three independent experiments.

Figure 5

Effects of 4-F-GlcNAc on lymphocyte binding to skin-draining LN HEVs. To examine L-selectin ligand activity of HEVs in inguinal LNs draining antigen-sensitized skin from mice treated with 4-F-GlcNAc, Stamper-Woodruff assays were performed. Lymphocyte suspensions (107 per milliliter) were overlaid onto glass slides containing glutaraldehyde-fixed LN sections (8 μm) and incubated for 30 minutes at 4°C under a constant rotation of 80 rpm. Slides were rinsed in cold PBS and incubated in glutaraldehyde for 10 minutes. In representative photomicrographs, lymphocytes (black dots) bound in an L-selectin–dependent manner to LN HEVs (outlined in red). At least three slides per experiment were used, and three independent experiments were performed. ×100 magnification. Bar: 100 μm.

Comment in

Selectin and selectin ligand binding: a bittersweet attraction.
Zollner TM, Asadullah K. Zollner TM, et al. J Clin Invest. 2003 Oct;112(7):980-3. doi: 10.1172/JCI19962. J Clin Invest. 2003. PMID: 14523033 Free PMC article.

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Prevention of leukocyte migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen - PubMed (original) (raw)