Figure 1.
Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins.
(A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).
Figure 2.
SCGB 1A1 and 1A1A affect neutrophil oxidative burst and phagocytosis.
(A) Blood-derived neutrophils were pre-incubated with endotoxin-free SCGB 1A1 or 1A1A prior to oxidative burst and phagocytosis assays. As seen in the two upper panels, control [0, PBS] or SCGB 1A1 and 1A1A [500 ng/mL] treatment did not induce morphological changes in neutrophils, while PMA stimulation triggered neutrophil activation characterized by larger size, membrane ruffles, and formation of cytoplasmic vacuoles (last panel). (B) Increasing concentrations of SCGB 1A1A (red line) stimulated generation of oxidation-dependent neutrophil fluorescence. Increases at 500 and 1000 ng/mL were significantly different from baseline and 250 ng/mL, and relative to SCGB 1A1 (blue line). (C) Both SCGBs had a tendency to increase neutrophil phagocytic activity, but statistically significant differences from each other or baseline were for SCGB 1A1A at 250 ng/mL. Bars = SEM. * = p<0.05
Figure 3.
Chemotaxis of equine neutrophils after exposure to recombinant SCGB 1A1 and SCGB 1A1A.
Neutrophils were pre-incubated with various concentrations of equine recombinant SCGB 1A1 or 1A1A, and deposited on chemotaxis chambers with IL-8 as chemoattractant. Chemotaxis of untreated neutrophils was assigned 100%. * = p<0.04, ** = p<0.02, bars = SEM; repeated measures ANOVA.
Figure 4.
Ex vivo NET formation was altered by exposure to SCGB 1A1 (A) or 1A1A (B).
Neutrophils were pre-treated with different concentrations of SCGBs and NETosis was induced with calcium ionophore. NET formation was monitored by fluorescence plate reader assay. Bars = SEM. * = p <0.05, ** = p <0.001, repeated measures ANOVA with Bonferroni post tests (0 vs. different concentrations of SCGBs). Background neutrophil control values with no ionophore are included in both graphs.
Figure 5.
SCGB 1A1 and 1A1A inhibit calcium ionophore (A23187) mediated NETosis in equine neutrophils.
Cells obtained from healthy horses were pre-incubated with PBS, SCGB 1A1, or SCGB 1A1A. PBS-treated cells were incubated in the absence (control) or in the presence of calcium ionophore (A23187) to stimulate NETosis. Immunofluorescence analysis revealed discrete nuclei and NETs as string-like structures (green DNA stain). SCGB 1A1 and 1A1A recombinant proteins were detected using SCGB antibody (red, top panel). Neutrophils stimulated with A23187 induced the expression of NETosis markers, including CitH3 and MPO (red, middle and bottom panel). Note reduction of NETs in SCGB 1A1 and 1A1A treated cells. Original magnification 40 ×, Bar 20 µm.
Figure 6.
(A) SCGB1A1 and SCGB1A1A mRNA levels were determined by quantitative RT-PCR in equine bronchoscopic biopsies.
SCGB1A1 expression levels were significantly lower (p = 0.016) in animals with RAO than in control animals. Results were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and 18S ribosomal RNA (RN18S). Experiments were performed in triplicate and results are presented as mean ± SEM of five (RAO) or six (control) animal per group. (B) Total SCGB concentration as measured by ELISA in BAL fluid was lower in horses with RAO than control horses pre- and post-challenge.
Figure 7.
Characterization of NET-derived DNA-protein complexes in BAL fluid.
(A) Nucleic acid-protein complexes were apparent as a high molecular weight band in agarose gels. The complexes were detected in animals with RAO post-challenge but not prior to exacerbation of RAO, and not in healthy control animals. (B) Proteinase K (PK) treatment released the proteins from the complexes and freed the nucleic acid, which migrate as lower molecular weight smear, confirming the presence of nucleic acid-protein complexes in the BAL. Addition of DNAse, but not (D) RNAse (R) abrogated the nucleic acid present in the BAL samples. Therefore, these samples contained DNA. (C) Western blot analysis revealed the presence of CitH3 (15 kDa) in samples that had HMW DNA-protein complexes. Collectively, these data indicate the presence of NETs in these BAL. L = 1 Kb+ DNA ladder; S = Precision plus protein standard; bp = base pair; kDa = kilodaltons.
Table 1.
NETs, neutrophils and total leukocyte concentrations in BAL fluid samples from control and RAO horses.