Primers and probes.

1<p>All genes are human.</p

MMP7, MMP25, Trypsin1 and RPL-19 expression in sulindac sulfide-treated cells.

<p>(<b>A</b>) PCR product of RT-PCR of RNA from HT-29 cells after 24 hours of treatment with DMSO or sulindac sulfide. Lane 1, 50 bp DNA ladder; lane 2, <i>MMP25</i> after sulindac sulfide treatment; lane 3, <i>MMP25</i> after DMSO treatment; lane 4, <i>Trypsin1</i> after sulindac sulfide treatment; lane 5, <i>Trypsin1</i> after DMSO treatment; lane 6, <i>MMP7</i> after sulindac sulfide treatment; lane 7, <i>MMP7</i> after DMSO treatment; lane 8 <i>RPL-19</i> after sulindac sulfide treatment; lane 9, <i>RPL-19</i> after DMSO treatment. Results demonstrate that only <i>MMP7</i> expression is affected by sulindac sulfide treatment. (<b>B</b>) Quantitative RT-PCR of <i>MMP25</i>, <i>Trypsin1</i>, and <i>MMP7</i> after DMSO or sulindac sulfide treatment. The results were obtained using the ΔΔCt method with <i>hRPL-19</i> as the housekeeping gene. Results indicate quantitative differences in <i>MMP7</i> expression after sulindac sulfide treatment relative to DMSO treatment. (<b>C</b>) Western blot of secreted fraction of HT-29 cells demonstrating that the protein levels of MMP25 in the membrane fraction are similar between sulindac sulfide and DMSO treated cells.</p

Enzymatic immunoassay for LTB4.

<p>Secreted proteomes of DMSO-treated and sulindac sulfide-treated cells were analyzed for LTB4 levels using an enzymatic immunoassay.</p

Percent cell death of HT-29 human colon cancer cells.

<p>At 80% confluency, cells were treated with increasing concentrations of sulindac (grey), sulindac sulfide (white), and sulindac sulfone (black).</p

Activity based proteomics and LTA4H in HT-29 cells.

<p>Metallohydrolase activity-based labeling of HT-29 cells secreted proteomes. MMP2 and MMP7 standards were added to cell proteomes (arrows). The band displaying a strong decrease (*) after sulindac sulfide treatment was extracted and analyzed by mass spectrometry. MS/MS spectra of LVVDLTDIDPDVAYSSVPYEK and LTYTAEVSVPK peptides corresponded to amino acids 366–386 and 155–165, respectively, of the LTA4H protein. Protein content across all samples was adjusted to 1 mg/ml and the equivalent of 15 µg of proteome was added per lane. MW denotes molecular weight; STDs, standards, Sd, sulindac sulfide-treated and D, DMSO-treated.</p

MMP7 expression in sulindac-treated cells.

<p>(<b>A</b>) Real time RT-PCR of RNA from HT-29 cells after 24 hours of treatment with DMSO, sulindac (S), sulindac sulfide (Sd) and sulindac sulfone (Sn). The results were obtained using the ΔΔCt method with <i>hRPL-19</i> as the housekeeping gene. C_T values between 20.10 and 20.56 across all samples show constant levels of <i>hRPL-19</i> expression. (<b>B</b>) The expression of <i>hMMP7</i> after 24 hours of sulindac treatment. The downregulation of <i>hMMP7</i> was significant at all concentrations. The results were obtained using the ΔΔCt method with <i>hRPL-19</i> as the housekeeping gene. C_T values between 23.22 and 23.56 across all samples show constant levels of <i>hRPL-19</i> expression. (<b>C</b>) Cytosolic, membrane, and secreted proteome fractions extracted from DMSO- and sulindac sulfide-treated HT-29 cells were subjected to western blot analysis using an antibody which does not differentiate between active and inactive MMP7 (top), and an antibody that only recognizes the active site (bottom). The expected band for MMP7 appears at 28 kDa, however a band at 45 kDa is often reported, possibly a dimer. Cyto denotes cytosolic fraction; Mem, membrane fraction, Secr, secreted fraction, and aMMP7, active MMP7.</p

Demographic and Clinical Characteristics of the Study Cohort.

<p>All these subjects had ≥10 pack yr smoking histories. ICS  =  inhaled corticosteroid use; past oral steroids  =  oral steroid use within the preceding six months (none were currently taking oral steroids); FEV₁% predicted  =  forced expiratory volume in the first second of expiration as percentages of predicted values; FEV₁/FVC  =  ratio of FEV₁ to forced vital capacity; DLCO% predicted  =  diffusing capacity for carbon monoxide as percentages of predicted values; Obstructed  =  subjects with spirometric findings of expiratory airflow obstruction (i.e., COPD); Emphysema  =  subjects with emphysema on CT scan; p values are for nonparametric comparisons of smokers who do not have anti-GRP78 IgG autoantibodies (GRP78⁻) vs. subjects who have these autoantibodies (GRP78⁺); NS  =  not significant. * denotes data are depicted as mean ± standard errors.</p><p>Demographic and Clinical Characteristics of the Study Cohort.</p

Cellular effects of autoantibodies to GRP78 on macrophages.

<p><i>A</i>.) Mean fluorescence intensity (MFI) for phosphorylated NFkB among paired, concurrent autologous CD14⁺ derived macrophages was increased in all 10 normal specimens after incubation with patient-derived autoantibodies to GRP78 (α-GRP78), relative to control cells treated with normal human IgG. Patient derived anti-GRP78 autoantibodies also increased macrophage production of IL-8 (<i>B</i>.), CCL-2 (<i>C</i>.) and MMP9 (<i>C</i>.). Population means are denoted with a horizontal line.</p

Anti-GRP78 autoantibody association with osteoporosis, bone metabolism markers, and concurrent low bone density and emphysema.

<p><i>A</i>.) T scores (left panel) and osteoporosis prevalence (at either/both hip or spine) (right panel) among the smoking cohort. <i>B</i>.) Serum levels of bone turnover metabolite collagen type 1 cross-linked C-telopeptide (CTX) were greatest among smokers with anti-GRP78 autoantibodies. The lowest, second lowest, middle, second highest, and highest lines represent 10^th, 25^th, median, 75^th, and 90^th percentiles, respectively. Means are denoted by solid squares. <i>C</i>.) Serum levels of bone turnover metabolite type 1 (N-terminal) procollagen (P1NP) were greatest among smokers with anti-GRP78 autoantibodies. <i>D</i>.) The relationship between GRP78 autoantibody positivity and the concurrent co-existences of low BMD and emphysema in smokers is significant in both genders, but greatest in males.</p

GRP78 expression in lung, bronchoalveolar lavage fluid, alveolar macrophages, and bone marrow derived osteoclasts.

<p><i>A</i>.) Compared to normal lung (Left panel), emphysematous lungs (middle and right panels) demonstrated increased immunostaining in macrophages (yellow arrows) and alveolar epithelial cells (blue arrows). Magnification x100 left and middle panels, and magnification x400 in the right panel, n = 3. <i>B</i>.) GRP78 was also greater in bronchoalveolar lavage fluid (BALF) from emphysematous lungs compared to normal preparations. Lanes 1 and 8 are rGRP78 standards. Lanes 2–7 are BALF from individual emphysematous lung explants; lanes 9–14 are BALF from normal lung explants. All specimen lanes were loaded with equal amounts of BALF proteins. <i>C</i>.) Indirect immunofluorescent assays showed anti-GRP78 IgG isolated from patients bind to alveolar macrophages from normal lung explants (panel a), and osteoclasts derived from bone marrow (panel b). Normal human IgG control is illustrated in panel c.</p