MMP-9 cleaves SP-D and abrogates its innate immune functions in vitro

Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype

Prospectus, April 29, 1974

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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