Cleaved SP-D Fails to Increase Phagocytosis by MH-S Cells.

<p>Examination and quantitation of phagocytosis of <i>E. coli</i> by MH-S cells was performed using (A) a gentamicin protection assay and (B and C) flow cytometry. For (A), all conditions are significantly different (p<0.05) from 1 µg/mL intact SP-D. An asterisk (*) denotes p≤0.001 when compared to 1 µg/mL intact SP-D. Columns marked with # are significantly different when compared to 0.5 µg/mL intact SP-D (p<0.05). For (B), intact SP-D causes MH-S cells to have significantly higher mean fluorescence intensity (MFI) than all other conditions (p≤0.001). Cleaved SP-D is significantly different when compared to both MMP-9 and PBS controls (p≤0.001). For (C), flow data was gated on forward and side scatter to select for MH-S cells. For both (A) and (B), error bars represent standard deviation.</p

MMP-9 Cleaves SP-D in a Dose- and Time-dependent Manner.

<p>(A) Dose and (B) time course digestions of SP-D by MMP-9. For the dose course (A), the reaction loaded onto lane 2 contained 62 ng/mL MMP-9 and the concentration was raised 3-fold serially to 5 µg/mL MMP-9 at lane 6. Reactions were incubated for 4 hours. For the time course (B), the length of digestion in minutes is listed above the lane. SP-D concentration was 20 µg/mL for both experiments, and MMP-9 concentration was 5 µg/mL for the time course. For the Native PAGE (C), lane 1 contains intact SP-D and lane 2 contains cleaved SP-D. An arrow indicates the band corresponding to MMP-9.</p

Cleaved SP-D Retains its Ability to Bind <i>E. coli</i> LPS.

<p>Examination of the ability of SP-D to bind to LPS-coated plates. In (A), the ELISA was performed using 2-fold serially diluted SP-D samples. In (B), 1 µg/mL intact or cleaved SP-D was analyzed in triplicate with MMP-9 as a control and NE-cleaved SP-D as a negative control. The average of PBST alone ran in triplicate was subtracted from all values. While intact and MMP-9-cleaved SP-D in PBST are significantly different from PBST alone (p≤0.001 and p<0.05, respectively) and NE-cleaved SP-D (p≤0.001 and p<0.05, respectively), MMP-9 along with intact and cleaved SP-D in PBST with maltose were not significantly different from PBST or PBST with maltose. For (B), error bars represent standard deviation.</p