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

Factors influencing the measurement of plasma/serum surfactant protein D levels by ELISA.

Extensive variations in human surfactant protein D (SP-D) levels in circulation as measured by ELISA exist in the published literature. In order to determine the source of these variations, factors influencing the measurement by ELISA were explored.Peripheral blood from healthy individuals was collected into various vacutainers during the same blood draw. Recombinant SP-D was diluted into different matrices and used for a standard curve. Samples were analyzed by capture ELISA using one of two distinct detection antibodies.The type of matrix had some effects on detection of recombinant SP-D. The type of anticoagulant used and dilution factor had very little effect, except for in plasma collected in EDTA vacutainers. The extent of variation in published values seemed to be due to the ELISA configuration employed, and, in agreement with this, we found that by switching the detection antibody, there was a 50% decrease in the extrapolated SP-D value of serum and plasma samples. Storage of samples resulted in slight changes in measured SP-D levels.The ELISA configuration employed to measure circulating levels of SP-D has a significant effect on the extrapolated values. In both configurations tested, the use of EDTA as a coagulant resulted in inconsistent values, and we, therefore, suggest the avoidance of this anticoagulant when assaying for SP-D by ELISA. While the demonstrated effects of several factors on measurement of SP-D may not account for all the disparities amongst the previous studies, they stress that variations in methodologies for measuring the same protein can result in very inconsistent results

Characterization and Prevention of the Adsorption of Surfactant Protein D to Polypropylene

<div><p>Surfactant Protein D (SP-D) is a multifunctional protein present in the lung and in respiratory secretions. In the process of developing new experimental approaches to examine SP-D function, we observed that SP-D adsorbs to polypropylene tubes to a great extent, thereby depleting SP-D from the solution. Although it is well known that proteins adsorb nonspecifically to plastic, this effect is usually diminished by treatments to make the plastic “low-retention” or “low-binding”. However, these treatments actually increased the binding of SP-D to the plastic. In addition, this adsorption affected the results of several assays, including proteolytic cleavage assays. In order to block SP-D from adsorbing to polypropylene and the effects caused by this adsorption, we coated the tubes with bovine serum albumin (BSA), as is commonly performed for ELISAs. This coating greatly diminished the amount of SP-D sticking to the plastic, providing an inexpensive and effective method for preventing adsorption and the artifacts resulting from this adsorption.</p></div

Detection of SP-D in serum and plasma collected using various anticoagulants.

<p>a) SP-D concentrations were measured in samples collected into four different vacutainers during a single blood draw. b) Measured values of samples were normalized to the SP-D concentration in serum for each patient in order to compare the effect of the anticoagulant on the measured SP-D concentration. An asterisk (*) denotes values are significantly different by Wilcoxon signed rank test (for EDTA, p = 0.0156, and for Citrate, p = 0.0078).</p

Influence of calcium concentration on detection of recombinant SP-D and native SP-D in serum and plasma.

<p>a) Recombinant SP-D (rSP-D), serum, and EDTA plasma were assayed in twofold dilutions of [Ca²⁺] or [EDTA] (concentrations are displayed in mM). For the inclusion of calcium with recombinant SP-D, Hank's Balanced Salt Solution (HBSS) was used as a buffer instead of PBS. Recombinant SP-D was assayed at 5 ng/mL while serum and plasma were diluted twofold. Each line represents an independent experiment. b) ELISAs were performed with detection reagents diluted in HBSS without calcium chloride (-CaCl²) or with 5 mM calcium chloride (+CaCl²). Wash buffer also either lacked or included calcium chloride. Recombinant SP-D was assayed at 10 ng/mL and Heparin plasma from four subjects was pooled, and sample values were extrapolated from a single standard curve under the conditions lacking calcium. n = 3 samples per group.</p

Comparison of SP-D values in samples stored in various conditions.

<p>Serum and heparin plasma samples collected during a single draw were either analyzed fresh or aliquoted and stored at 4°C, −20°C, or −80°C.</p

Adsorption of SP-D to Polypropylene Pipet Tips.

<p>5 µl aliquots were taken from a serially aspirated/dispensed solution of SP-D after each round of pipetting, analyzed by Western blot (representative blot shown in A), and quantified using ImageJ software (B). The control consisted of 6 aliquots taken without pipetting. The means were found to be significantly different by One-way ANOVA (p = 0.044).</p

Depletion of SP-D during Extended Incubations.

<p>5 µl aliquots were taken from SP-D solutions after incubating at the indicated time/temperature, analyzed by Western blot, and quantified using ImageJ software (A). Values were normalized to the average of the “4 hours @ RT” group. The SP-D content of tubes was analyzed and compared to the total amount of SP-D in the supernatant and tube (B). An asterisk (*) denotes p≤0.05 when comparing columns.</p

SP-D Adsorption to Polypropylene is Greatly Diminished by Coating with BSA.

<p>Western blotting detects the 43-D stripped from polypropylene tubes using Laemmli Buffer (A). 1 through 6 denotes the tube order during serial transfer. The amount of SP-D detected on uncoated tubes was quantified using ImageJ software (B). Columns with an asterisk (*) are significantly different (p≤0.05), as are columns labeled with the number sign (#, p≤0.01).</p