Figure 2: Degranulating agonists rapidly up-regulate surface TIMP-1 levels on human PMNs:
Human PMNs were incubated for 30 min at 37°C with (grey bars) or without (open bar) 100 U/ml TNF-α, 10−7 M fMLP, 300 nM phorbol myristate acetate (PMA) or 5 μM calcium ionophore (A23187) in A, or varying concentrations of fMLP in B, or 10−7 M fMLP for 1–90 min in C. In D, human PMNs were incubated for 30 min at 37°C without (open bar) or with (grey bars) LPS (100 ng/ml), PAF (10−7 M), TNF-α (100 U/ml), IL-8 (10−7 M) and fMLP (10−7 M) alone, or PMN were primed for 15 min at 37°C with and without the same concentrations of LPS or PAF and then activated for 30 min with 10−7 M fMLP. In E, human PMNs were incubated for 30 min at 37°C without or with 10−7 M fMLP, 10−7 M IL-1β, or 10−7 M IL-6, or varying concentrations of IL-8 in F. In A-F, cells were then fixed and immunostained for surface-bound TIMP-1 and surface TIMP-1 levels were quantified using image analysis software as described in Methods. Data are mean + SEM; n = 150–200 cells per group. In A-F, data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with unstimulated cells or the group indicated. The results shown are representative of at least 3 independent experiments.