Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells

Abstract

Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

Figure 1. Cleavage of fluorescent phospholipid by nmPLA₂ and inhibition by lactadherin.

Sonicated phospholipid vesicles, 10 µM, of composition PS:PE:PC:bbPC 4∶20:75∶1 were pre-incubated for 15 minutes in PBS, pH 7.2 at 22°C with, or without, pure lipid-free bovine lactadherin before addition to a quartz cuvette of 3×3×45 mm. Vesicles were incubated for 5 minutes at 4°C in the Peltier-thermostatted sample chamber of the fluorometer before adding nmPLA₂ to a final concentration of 0.06 U/ml. Reaction curves are normalized to baseline fluorescence intensity before addition of phospholipase as per Eq. 2.

Figure 2. Relationship between PLA₂ concentration, phospholipid concentration, and phospholipase activity.

(A) Varying concentrations of sonicated vesicles of composition PS:PE:PC:bbPC 4∶20:75∶1 were allowed to equilibrate at 4°C for 5 minutes before adding 0.125 U/ml nmPLA₂. The resulting curve replicates were standardized and fitted to a two-phase exponential association model (Eq. 1) and solved for global rate constants (fitted curves). (B) Total Y_max obtained from fitted curves was plotted against phospholipid concentrations and fitted to a Michaelis-Menten equation. (C) The initial reaction rate was obtained from the original datasets using linear regression from 0–5 seconds and plotted against phospholipid concentration. A Michaelis-Menten equation was fitted to the data and of high fit quality. The results indicate a saturable dose-response relationship. Experiments at each phospholipid or phospholipase concentration were performed a minimum of two times, and values averaged.

Figure 3. Lactadherin inhibition of hsPLA₂ vs. nmPLA₂.

(A) Various concentrations of lactadherin were added to 10 µM phospholipid vesicles with composition as described for Fig. 2. Phospholipid vesicles were preincubated for 15 minutes with bovine lactadherin before transfer to a quartz cuvette and cooling for 5 minutes at 4°C. hsPLA₂-V was added to a final concentration of 0.06 U/ml and the activity monitored continuously as fluorescence emission at 515 nm. Data were fitted to a double exponential model and fitted globally as described in Fig. 2. Addition of lactadherin diminished both components of enzyme activity. (B) Normalized net phospholipase activity on sonicated PLVs after 400 s ± SD are plotted as a function of lactadherin concentration with uninhibited Y_{max total} as reference. (C) Experiments like those in panel 3A were performed utilizing extruded PLV rather than sonicated vesicles (not shown). Normalized phospholipase activity after 400 s ± SD are plotted as function of lactadherin concentration. As seen in all panels, the PS sensitive hsPLA₂-V is more readily blocked by lactadherin. All experiments were performed at least twice and results averaged for data displayed in panels B and C.

Figure 4. Inhibitory activity of lactadherin toward sPLA₂’s on human leukemia cells.

(A) NB4 cells were treated with 6 µM A23187 for 10 minutes at 22°C prior to addition of phospholipases. This treatment resulted in 61.6% pre-apoptotic cells. Phospholipase activity was detected as release of free fatty acids using the ADIFAB reagent. Each curve was run as a set in quadruplicates using a single mixture of ADIFAB and PLA₂. Curves were run in separate sets. The initial reaction rates for nmPLA₂ and hsPLA₂-V were calculated by linear regression to the first 5 and 15 seconds respectively, were r² = 0.85–0.95. (B) 0.06 U/ml nmPLA₂ phospholipase activity on quiescent, stressed cells and stressed with 300 nM lactadherin were measured. Lactadherin was added to the cell mix immediately before adding the ionophore, proceeding with 10 minute incubation. Control curves from similar treated cells without added enzyme were subtracted as background and extrapolated Y values at injection point found using Eq. 3. Sum curves of quadruplicate sets are displayed (C) The experiment was repeated as in panel B using 0.06 U/ml hsPLA₂-V. The initial reaction rate was calculated the same way as panel B. See panel 4D and 4E for quadruplicate results of nmPLA₂ and hsPLA₂-V respectively, SD displayed with *denoting p<0.05 and **denoting p<0.001. To discount any adverse interactions between A23187 and lactadherin, cells were stressed using 40 µM etoposide as described in materials and methods. As seen in Figure 4F , inhibition of hsPLA₂-V to near quiescent levels by addition of 300 nM lactadherin was observed, producing very similar inhibition ratios as found when using the quick, A23187 and lactadherin co-incubation protocol. Statistical significance using a one-tailed T-test assuming unequal variance showed a significance of p<0.03.