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Blood, Vol. 104, Issue 10, 3205-3213, November 15, 2004
Mouse CD99 participates in T-cell recruitment into inflamed skin
Blood Bixel et al.
104: 3205
Supplemental materials for: Bixel et al, Vol 104, Issue 10, 3205-3213
Files in this Data Supplement:
- Figure S1. Immunofluorescence staining of CD99 at cell contacts in mixed cell cultures containing CHO-CD99 and CHO cells (PDF, 178 KB) -
Mock-transfected CHO cells and CD99-transfected CHO cells were co-cultured and stained for CD99 by indirect immunofluorescence. The bottom panel is a phase contrast image of the same cells depicted in the top panel. Green arrows mark cell contact areas between CD99-transfected and mock-transfected cells lacking CD99 staining, whereas the blue arrow marks a CD99-containing cell contact zone between CD99 and mock-transfected cells. The corresponding positions in the phase contrast micrograph are marked with black arrows. In most cases, CD99 is enriched between CD99-expressing CHO cells and weakly or not expressed at cell contacts between mock- and CD99-transfected CHO cells. Bar = 20 µm.
- Figure S2. Binding of SJL.PLP7 T lymphocytes to ICAM-1 on CHO cells cannot be affected by anti-CD99 antibodies (PDF, 33 KB) -
SJL-PLP7 cells were allowed to bind to mouse ICAM-1—transfected CHO cells cultured in 16-well chamber slides. Adhesion was performed either in the absence of antibodies (co), or in the presence of the mAb against ICAM-1 (anti—ICAM-1) or affinity-purified antibodies against CD99 (anti-CD99). Adhesion was dependent largely on ICAM-1 (inhibition by anti—ICAM-1) and was neither reduced nor enhanced by anti-CD99 antibodies. In order to test whether the activation status of ICAM-1—binding
 2 integrins on SJL-PLP7 is already maximal or whether it can further be enhanced, we performed the assays in the presence of Mn2+ (allowing the integrin to obtain a conformation of maximal activity) and compared adhesion to standard conditions (Mg2+/Ca2+). As depicted, adhesion was strongly enhanced in the presence of Mn2+, indicating that the lack of an enhancing effect of anti-CD99 on 2 integrin–mediated adhesion indeed demonstrates that these antibodies do not activate 2 integrins on these cells. In addition, anti-CD99 antibodies did not inhibit adhesion of SJL-PLP7 cells at low activation levels of 2 integrin (Mg2+/Ca2+) or under conditions of high activation levels. P < .0005, anti-ICAM-1 versus co- Mg2+/Ca2+ or anti-CD99 Mg2+/Ca2+; P < .0001, co-Mn2+ versus co- Mg2+/Ca2+; P < .0001, anti-CD99 Mn2+ versus anti-CD99 Mg2+/Ca2+. Assays in the presence of Mn2+ were done in adhesion medium containing 400 µM Mn2+ instead of Mg2+/Ca2+. ICAM-1—transfected CHO cells have been described by Blanks et al.48
- Figure S3. Anti-CD99 antibodies do not inhibit lymphocyte homing into secondary lymphatic tissue (PDF, 46 KB) -
Lymphocytes isolated from lymph nodes of female CD1 mice were labeled with 51Cr. Two × 106 living cells were resuspended in 250 µL PBS and were injected into the tail vein with the following antibodies: 150 µg control IgG from pre-immune serum (co-IgG), 350 µg of the mAb MECA79 against the peripheral node addressin (anti-PNAd), or 150 µg affinity-purified antibodies against CD99 (anti-CD99). Mice were killed 3 hours after injection, and the following lymphatic organs were counted: peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), Peyer plaques (PPs), and spleen. Note that the mAb against PNAd specifically blocked homing into peripheral and mesenteric lymph nodes and consequently enhanced migration into the spleen, as shown before (Hamann A, Andrew DP, Jablonski-Westrich D, Holtzmann B, Butcher EC. Role of
 4-integrins in lymphocyte homing to mucosal tissues in vivo. J Immunol. 1994;152:3282-3293). In contrast, anti-CD99 antibodies did not affect lymphocyte homing. pLN: P < .01, anti-PNAd versus co-IgG or anti-CD99; mLN: P < .02, anti-PNAd versus co-IgG or anti-CD99; spleen: P < .005, anti-PNAd versus co-IgG.
Isolation and labeling of lymphocytes was done as described by Hamann and Jonas.49
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