Modulation of CXC Chemokine Receptor Expression and Function in Human Neutrophils during Aging In Vitro Suggests a Role in Their Clearance from Circulation
Figure 2
Effects of SDF-1 and IL-8 on transendothelial migration and p44/42 MAP kinase phosphorylation in human PMN in vitro.
(a) Migration of freshly isolated PMN toward SDF-1 (* versus control) and IL-8 (*** versus control) across microvascular endothelium is shown, . The specific antibody clone 12G5 was used for functional blocking of CXCR4. Control: spontaneous migration without addition of cytokines
(b) SDF-1- and IL-8-induced transendothelial migration of neutrophils cultured for 18 hours at 37°C compared to cells kept for the same time period at 4°C to prevent aging. The influence of aging in vitro on the migratory response to the chemokines was statistically significant (SDF-1 ***, 37°C versus 4°C and IL-8*, 37°C versus 4°C). For functional blocking of CXCR4 in aged PMN (18 hours/37°C), the 12G5 antibody was used. Control: spontaneous migration without chemokines
(c) Phosphorylation of p44/42 MAP kinase (Erk1/2) in human PMN induced by SDF-1. Cells were cultured in serum-free medium for 18 hours at 37°C (aged in vitro) and 4°C, respectively. After stimulation with SDF-1 at 37°C for the time intervals indicated, whole cell lysates were prepared and analyzed by Western blot for phosphorylated p44/42 MAPK (lower lane). As a control, total p44/42 MAPK is shown in the upper lane
(d) Quantitive analysis of the Western blots by densitometry (, **, aged PMN at 37° versus cells kept at 4°C to prevent aging)