1. The 130-kDa Glycoform of CD43 Functions as an E-Selectin Ligand for Activated Th1 Cells In Vitro and in Delayed-Type Hypersensitivity Reactions In Vivo 
Pilar Alcaide, Sandra L. King, Charles J. Dimitroff, Yaw-Chyn Lim, Robert C. Fuhlbrigge, Francis W. Luscinskas 
Journal of Investigative Dermatology 
Volume 127, Issue 8, Pages (August 2007)
DOI: /sj.jid
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Th1 cells express an E-selectin ligand independent of PSGL-1 that is functional under flow conditions in vitro. The data represent the accumulation of wild-type (WT) and PSGL-1−/− Th1 cells on glass coverslips coated with recombinant (a) E-selectin (10μg/ml) or (b) P-selectin (2μg/ml) under three different conditions of shear stress. Data are mean±SD values from six separate experiments.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
E-selectin-mediated interactions with cellular proteins from PSGL-1−/− Th1 cells under flow conditions. (a) PSGL-1−/− Th1 lysates (50μg) were separated by molecular mass under non-reducing conditions, immobilized on Western blots, and stained with 1B11 antibody to detect glycoCD43. High-range (left) and full-range (right) molecular-mass markers are included in the flanking lanes of each blot. The blot was assayed for functional E-selectin ligand activity by blot rolling analysis. (b) Screening of the blot showing cells per unit area (× 10 objective) in overlapping fields of view extending from 250 to 40kDa (identified alongside blot image in (a)) at 0.4dyn/cm2 of shear stress. A protein of 130-kDa (field of view 7) has high E-selectin-specific rolling activity, and a protein of 100-kDa (field of view 9) has minor E-selectin-specific rolling activity. (c) E-selectin ligand activity observed on the 130-kDa band (field of view 7) at increasing shear stress (black bars) was calcium dependent (EDTA, white bars) and specific (P-selectin, gray bars). Data represent mean±SD of three separate experiments and are representative of 3–4 separate determinations on each band.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
E-selectin-specific mediated rolling on immunoprecipitated GlycoCD43. (a) Aliquots of lysate (300–450μg) from PSGL-1−/− Th1 cells were immunoprecipitated with 1B11 plus sc-7054 antibody to glycoCD43 or the isotype control (data not shown) using protein A-G PLUS Agarose beads. Three rounds of immunoprecipitation resulted in complete clearance of CD43 from the lysate (data not shown). Immune complexes were subjected to SDS-PAGE and Western blotting for glycoCD43 under standard non-reduced conditions as described in Materials and Methods. Molecular-mass markers are included in the flanking lanes of each blot. Representative Western blot stained for glycoCD43 is shown. (b) CHO-E (in the presence or absence of EDTA) or CHO-P cells were drawn and shear stress was increased sequentially. CHO-E cells (black bars) showed specific rolling activity in the immunoprecipitated glycoCD43 at the different assayed shear stresses. CHO-P or CHO-E in the presence of EDTA did not roll on glycoCD43. (c) Immune complexes obtained as in (a) were directly immobilized on plastic and the flow chamber was placed on top and filled with CHO-E (in the presence or absence of EDTA) or CHO-P cells that were drawn at increasing shear stresses. Cell number is represented after substracting the background corresponding to cells rolling on lysates immunoprecipitated with the isotype control rat-Ig-G (10±6cells/mm2). Results shown are the mean±SD of four different experiments and two separate determinations on each blot or antigen captured in plastic.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Lack of E-selectin- and P-selectin-mediated rolling in Th1 protein lysates from CD43−/− mice cleared of PSGL-1 by immunoprecipitation. (a) Aliquots of lysate (300–450μg) from CD43−/− Th1 cells were immunoprecipitated with 2PH1 antibody to PSGL-1 and immune complexes were subjected to SDS-PAGE and Western blotting to detect PSGL-1. (b) The supernatant corresponding to the residual soluble material from the immunoprecipitation was also subjected to SDS-PAGE and Western blotting for PSGL-1. This material does not contain PSGL-1 and simulates Th1 cells that lack both CD43 and PSGL-1. (a and b) Molecular-mass markers are included in the flanking lanes of each blot. (c) Screening of the blots from (a) and (b) (× 10 objective) in fields of view extending from 250 to 45kDa by blot rolling analysis. Both CHO-P and CHO-E cells showed calcium-dependent rolling activity on the immunoprecipitated PSGL-1 band (field of view 2) and not on other areas of the blot, including field of view 4 ~130kDa (left panel). Neither CHO-E nor CHO-P cells rolled on any region of the supernatant (right panel). Results are the mean of two separate determinations on each blot and are representative of two separate immunoprecipitation experiments.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Impaired interactions of PSGL-1−/−/CD43−/− DKO T-cell subsets with E-selectin and P-selectin under flow conditions. (a and b) Th1 cells or (c and d) Tc-1 cells from WT, PSGL-1−/−, CD43−/−, and DKO mice were drawn across coverslips coated with (a and c) E-selectin or (b and d) P-selectin within a flow chamber at decreasing levels of shear stress. Th1 and Tc-1 DKO cells (checkered bars) showed reduced E-selectin binding as compared with WT (black bars), PSGL-1−/− (white bars), and CD43−/− (gray bars), and no binding to P-selectin. CD43−/− Th1 cells did not show reduced E-selectin binding but did exhibit an enhanced binding to P-selectin, whereas Tc-1 cells showed normal E-selectin and slightly elevated binding to E-selectin. Data are represented as percentage of WT control, and show the mean±SD of triplicate determinations from two separate experiments. P-values comparing the different groups versus WT are indicated: *P≤0.05, **P≤0.01, and ***P≤0.001, and nonsignificant (NS).
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
DNFB-induced DTH is reduced only in mice unable to express both PSGL-1 and CD43. WT, PSGL-1−/−, CD43−/−, and DKO mice were sensitized with DNFB and challenged on the right ear after 5 days. Left ears were challenged with vehicle only. Ear thickness was measured 24h later. (a) The difference in the ear thickness (right ear–left ear) is shown. Data are the mean±SD of two different experiments (n=8 mice per group). P-values are indicated comparing DKO with WT: **P≤0.01, P>0.05 was considered nonsignificant (NS); with PSGL-1−/−: P≤0.05 (#); or with CD43−/−: P≤0.05 (!!). (b and c) Detection of infiltrated cells is shown by detection of CD3, CD4, and MPO in Western blot. Ear tissues from DTH-induced mice were lysed and subjected to Western blot for CD3 detection (b), MPO, and CD4 detection (c). A typical Western blot is shown for each antigen. The graphs represent numeric values obtained by densitometry analysis that indicate the relative absorbance of CD3, MPO, and CD4 antigens with respect to β-actin and are the mean±SD of four different blots of tissue lysates from four different mice challenged in two separate experiments. P-value for DKO versus WT is indicated: *P≤0.05 (d). Ears were fixed with 10% formalin, mounted in paraffin, and 6μm sections were stained with hematoxylin and eosin for detection of infiltrates. Bar=10mm, pictures show original magnification with × 4 objective.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions