Figure 6
Figure 6. Antibodies against CD99L2 and CD99 block transendothelial migration of neutrophils in vitro. (A) Antibodies against CD99 or CD99L2 inhibit transendothelial migration of PMNs through a bEnd.5 monolayer. Bone-marrow derived PMNs were allowed to transmigrate for 30 minutes through a monolayer of bEnd.5 cells grown on transwell filters in the presence of 100 ng/mL KC in the lower chamber. Endothelial cells were incubated for 30 minute with 30 μg/mL of antibody before the start of the assay as indicated. Left panel: with preimmune control IgG (co-IgG) or affinity purified anti-CD99 IgG (anti-CD99) or F(ab′)2 fragments of control IgG (co-IgG F(ab′)2) or anti-CD99 IgG (anti-CD99 F(ab′)2) or a monoclonal antibody against ICAM-1 (anti-ICAM-1). **, P < .01 anti-CD99 versus co-IgG; *, P < .05 anti-CD99 F(ab′)2 versus co-IgG F(ab′)2. Right panel: with preimmune control IgG (co-IgG) or affinity purified anti-CD99L2 IgG (anti-CD99L2) or F(ab′)2 fragments of control IgG (co-IgG F(ab′)2) or anti-CD99L2 IgG (anti-CD99L2 F(ab′)2) or a monoclonal antibody against ICAM-1 (anti-ICAM-1). **, P < .01 anti-CD99 versus co-IgG; *, P < .05 anti-CD99 F(ab′)2 versus co-IgG F(ab′)2. Antibodies remained present during the assay. (B) After preincubation of the endothelial cells with the indicated antibodies, the antibodies either remained present during the assay (first 4 bars) or were washed away before adding the PMNs (indicated by “pre-inc.” underneath the last 4 bars). ***, P < .001 anti-CD99 versus co-IgG; **, P < .01 anti-CD99L2 versus co-IgG; ***, P < .001 anti-CD99 pre-inc. versus co-IgG pre-inc. *, P < .05 anti-CD99L2 pre-inc. versus co-IgG pre-inc. (C) PMNs were preincubated with medium without antibody (w/o Ab) or with F(ab′)2 fragments of control IgG (co-IgG F(ab′)2), or anti-CD99 IgG (anti-CD99 F(ab′)2) or anti-CD99L2 IgG (anti-CD99L2 F(ab′)2). Antibodies were washed away before adding the PMNs into the assay. Each panel is representative of at least 3 experiments; each measurement was done in triplicate. (D) No additive effect of anti-CD99 and anti-CD99L2 F(ab′)2-fragments: experiments were done as in (A), endothelial cells were preincubated with F(ab′)2 fragments of control IgG (co-IgG F(ab′)2), anti-CD99L2 F(ab′)2 (anti-CD99L2 F(ab′)2), anti-CD99 F(ab′)2 (anti-CD99 F(ab′)2), or a mixture of anti CD99L2 and CD99 F(ab′)2 (anti CD99L2/CD99 F(ab′)2), or with monoclonal antibody against ICAM-1 (anti-ICAM-1). (E) Binding of PMNs to endothelial cells is not affected by antibodies against CD99 or CD99L2. Bone-marrow derived PMNs were allowed to adhere to a monolayer of bEnd.5 cells in the absence of antibody (w/o Ab) or in the presence of control antibodies (co-IgG) or antibodies against LFA-1 (anti-LFA-1), affinity-purified antibodies against CD99 (anti-CD99) or CD99L2 (anti-CD99L2). ***, P < .001 anti-LFA-1 vs. co-IgG. Statisitical analysis was done by Student t test. Each panel is representative of at least 3 experiments; each measurement was done in triplicate.

Antibodies against CD99L2 and CD99 block transendothelial migration of neutrophils in vitro. (A) Antibodies against CD99 or CD99L2 inhibit transendothelial migration of PMNs through a bEnd.5 monolayer. Bone-marrow derived PMNs were allowed to transmigrate for 30 minutes through a monolayer of bEnd.5 cells grown on transwell filters in the presence of 100 ng/mL KC in the lower chamber. Endothelial cells were incubated for 30 minute with 30 μg/mL of antibody before the start of the assay as indicated. Left panel: with preimmune control IgG (co-IgG) or affinity purified anti-CD99 IgG (anti-CD99) or F(ab′)2 fragments of control IgG (co-IgG F(ab′)2) or anti-CD99 IgG (anti-CD99 F(ab′)2) or a monoclonal antibody against ICAM-1 (anti-ICAM-1). **, P < .01 anti-CD99 versus co-IgG; *, P < .05 anti-CD99 F(ab′)2 versus co-IgG F(ab′)2. Right panel: with preimmune control IgG (co-IgG) or affinity purified anti-CD99L2 IgG (anti-CD99L2) or F(ab′)2 fragments of control IgG (co-IgG F(ab′)2) or anti-CD99L2 IgG (anti-CD99L2 F(ab′)2) or a monoclonal antibody against ICAM-1 (anti-ICAM-1). **, P < .01 anti-CD99 versus co-IgG; *, P < .05 anti-CD99 F(ab′)2 versus co-IgG F(ab′)2. Antibodies remained present during the assay. (B) After preincubation of the endothelial cells with the indicated antibodies, the antibodies either remained present during the assay (first 4 bars) or were washed away before adding the PMNs (indicated by “pre-inc.” underneath the last 4 bars). ***, P < .001 anti-CD99 versus co-IgG; **, P < .01 anti-CD99L2 versus co-IgG; ***, P < .001 anti-CD99 pre-inc. versus co-IgG pre-inc. *, P < .05 anti-CD99L2 pre-inc. versus co-IgG pre-inc. (C) PMNs were preincubated with medium without antibody (w/o Ab) or with F(ab′)2 fragments of control IgG (co-IgG F(ab′)2), or anti-CD99 IgG (anti-CD99 F(ab′)2) or anti-CD99L2 IgG (anti-CD99L2 F(ab′)2). Antibodies were washed away before adding the PMNs into the assay. Each panel is representative of at least 3 experiments; each measurement was done in triplicate. (D) No additive effect of anti-CD99 and anti-CD99L2 F(ab′)2-fragments: experiments were done as in (A), endothelial cells were preincubated with F(ab′)2 fragments of control IgG (co-IgG F(ab′)2), anti-CD99L2 F(ab′)2 (anti-CD99L2 F(ab′)2), anti-CD99 F(ab′)2 (anti-CD99 F(ab′)2), or a mixture of anti CD99L2 and CD99 F(ab′)2 (anti CD99L2/CD99 F(ab′)2), or with monoclonal antibody against ICAM-1 (anti-ICAM-1). (E) Binding of PMNs to endothelial cells is not affected by antibodies against CD99 or CD99L2. Bone-marrow derived PMNs were allowed to adhere to a monolayer of bEnd.5 cells in the absence of antibody (w/o Ab) or in the presence of control antibodies (co-IgG) or antibodies against LFA-1 (anti-LFA-1), affinity-purified antibodies against CD99 (anti-CD99) or CD99L2 (anti-CD99L2). ***, P < .001 anti-LFA-1 vs. co-IgG. Statisitical analysis was done by Student t test. Each panel is representative of at least 3 experiments; each measurement was done in triplicate.

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