Role of interleukin-6 in a glucan-induced model of granulomatous vasculitis.

The role of interleukin-6 (IL-6) in granulomatous vasculitis is not well understood. To investigate its involvement in this type of vasculitis a model of glucan-induced pulmonary vasculitis employed interleukin-6 deficient (IL-6-/-) mice. Briefly, IL-6-/- mice and C57B/J6 wild type (IL-6+/+) mice were injected intravenously with a suspension of glucan isolated from the cell wall of bakers yeast which results in a granulomatous vasculitis primarily in the pulmonary vasculature. Histological examination demonstrated no significant difference in the number of infiltrating leukocytes between the IL-6+/+ and IL-6-/- glucan-injured mice. Similar numbers of granulomas were noted in both the IL-6+/+ and IL-6-/- injured animals, while no granulomas were seen in saline injected control mice. Cells recovered from the bronchoalveolar lavage (BAL) fluid were differentially stained and counted. While there was a significant increase in infiltrating leukocytes recovered from the BAL following glucan-induced injury, there was no significant difference between the IL-6+/+ and IL-6-/- mice. In addition, no difference was demonstrated in total protein content in the BAL fluid between IL-6+/+ and IL-6-/- mice. However, myeloperoxidase (MPO) activity in the lungs of the IL-6-/- mice was less than in their IL-6+/+ counterparts suggesting that these animals have a partial defect in their ability to recruit neutrophils in this model. Studies done to look for levels of other cytokines/chemokines in these animals to compensate for the loss of IL-6 revealed that only IL-10 in the sera (p<0.016) and BAL fluid (p<0.05) of IL-6-/- mice was significantly higher then their IL-6+/+-injured counterparts. These studies suggest that IL-6, while possibly involved in early neutrophil accumulation in this model does not appear critical to the development of the TH-2 mediated granulomatous vasculitis.

Screening of serum samples from Wegener's granulomatosis patients using antibody microarrays.

Wegener's Granulomatosis (WG) is an idiopathic granulomatosis autoimmune vasculitis that primarily affects small vessels and is associated with glomerulonephritis and pulmonary granulomatous vasculitis. Anti-neutrophil cytoplasmic auto-antibodies (cANCA) against proteinase-3 are used to identify WG, but ANCA titers are not present in some patients with the localized disease. The objective of this study was to develop an antibody array to help identify protein expression patterns in serum from patients with WG as compared to normals. The arrays were tested for limits of detection, background, and cross reactivity using standard proteins. The arrays were hybridized with either normal patient serum (n = 30) or with serum samples from a population of WG patients (n = 26) that were age and sex matched. Data analysis and curve fitting of the standard dilution series calculated r(2) values and determined a sensitivity of <50 pg/mL for the majority of proteins. A total of 24 proteins were assessed. Several statistically significant increases (p<0.05) were seen in the expression of: angiotensin converting enzyme-I, IFN-γ, IL-8, s-ICAM-1 and s-VCAM in WG patients as compared to controls. Utilizing the antibody microarray technology has led to the identification of potential biomarkers of vascular injury in the serum of WG patients.

Matrix metalloproteinase-9 is an important factor in hepatic regeneration after partial hepatectomy in mice.

Partial hepatectomy triggers hepatocyte proliferation, hepatic matrix remodeling, and hepatocyte apoptosis, all of which are important processes in the regenerating liver. Previous studies have shown an increase in the levels of matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) after partial hepatectomy. The goal of this study was to investigate the role of MMP-9 in liver regeneration after partial hepatectomy. A 70% hepatectomy or sham laparotomy was performed in wild-type or MMP-9-deficient (MMP-9-/-) mice. Hepatic regeneration was determined by liver weight/total body weight ratios and BrdU staining, which was used to a calculate mitotic index at several times postoperatively. Cytokine and growth factor expression was evaluated by Luminex bead-based ELISA and Western blots. Finally, the effect of MMP-9 on apoptosis was measured using TUNEL and caspase expression. The MMP-9-/- animals had a delayed hepatic regenerative response when compared with wild-type controls. The MMP-9-deficient animals expressed significantly less VEGF, HGF, and TNF-alpha between days 2 and 3 post-hepatectomy. Apoptosis, as measured by TUNEL staining and caspase expression, was decreased in the MMP-9-/-. In conclusion, MMP-9 plays an important role in liver regeneration after partial hepatectomy by affecting matrix remodeling, as well as cytokine, growth factor, and caspase expression.

Comparison of antibody array substrates and the use of glycerol to normalize spot morphology.

Antibody microarrays are a high-throughput proteomic technology used to examine the expression of multiple proteins in complex solutions. Antibody microarrays can be manufactured on a variety of commercially available activated glass or coated slides. The goal of this study was to compare Hydrogeltrade mark, nitrocellulose, aldehyde-silane and epoxy-silane slides to determine the amount of antibody bound. The optimal substrate was defined as one that bound the greatest amount of antibody with minimal background. Our studies found that epoxy-silane enhanced surface (ES) slides gave the greatest degree of binding along with a minimal background. However, larger antibody microarrays showed variability in spot size, high intra-spot coefficient of variation and drying artifacts. Increasing the amount of glycerol in the spotting buffer caused a dose-dependent improvement in overall spot morphology. Glycerol was tested on 128 different antibodies and showed decreased: mean spot diameter, intra-spot coefficient of variation and drying artifacts. These studies revealed that the optimal slide substrate was epoxy-silane ES microarray slides. Furthermore, glycerol could normalize spot size, decrease intra-spot coefficient of variability, decrease drying artifacts and increase antibody-spotting density.

Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation.

BACKGROUND: Neutrophils may be an important source of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), two matrix-degrading enzymes thought to be critical in the formation of an abdominal aortic aneurysm (AAA). The purpose of this investigation was to test the hypothesis that neutrophil depletion would limit experimental AAA formation by altering one or both of these enzymes. METHODS AND RESULTS: Control, rabbit serum-treated (RS; n=27) or anti-neutrophil-antibody-treated (anti-PMN; n=25) C57BL/6 mice underwent aortic elastase perfusion to induce experimental aneurysms. Anti-PMN-treated mice became neutropenic (mean, 349 cells/microL), experiencing an 84% decrease in the circulating absolute neutrophil count (P<0.001) before elastase perfusion. Fourteen days after elastase perfusion, control mice exhibited a mean aortic diameter (AD) increase of 104+/-14% (P<0.0001), and 67% developed AAAs, whereas anti-PMN-treated mice exhibited a mean AD increase of 42+/-33%, with 8% developing AAAs. The control group also had increased tissue neutrophils (20.3 versus 8.6 cells per 5 high-powered fields [HPFs]; P=0.02) and macrophages (6.1 versus 2.1 cells per 5 HPFs, P=0.005) as compared with anti-PMN-treated mice. There were no differences in monocyte chemotactic protein-1 or macrophage inflammatory protein-1alpha chemokine levels between groups by enzyme-linked immunosorbent assay. Neutrophil collagenase (MMP-8) expression was detected only in the 14-day control mice, with increased MMP-8 protein levels by Western blotting (P=0.017), and MMP-8-positive neutrophils were seen almost exclusively in this group. Conversely, there were no statistical differences in MMP-2 or MMP-9 mRNA expression, protein levels, enzyme activity, or immunostaining patterns between groups. When C57BL/6 wild-type (n=15) and MMP-8-deficient mice (n=17) were subjected to elastase perfusion, however, ADs at 14 days were no different in size (134+/-7.9% versus 154+/-9.9%; P=0.603), which suggests that MMP-8 serves only as a marker for the presence of neutrophils and is not critical for AAA formation. CONCLUSIONS: Circulating neutrophils are an important initial component of experimental AAA formation. Neutrophil depletion inhibits AAA development through a non-MMP-2/9-mediated mechanism associated with attenuated inflammatory cell recruitment.

Role of interleukin-6 in immune complex induced models of vascular injury.

Previous studies have suggested that Interleukin-6 (IL-6) acts as a marker of vasculitis. To determine the role of IL-6 in vasculitis we utilized two models of immune complex induced vascular injury (dermal Arthus and acute pulmonary alveolitis) in IL-6 deficient (IL-6(-/-)) and IL-6 sufficient (IL-6(+/+)) mice. Plasma and bronchoalveolar lavage (BAL) levels of IL-6 were elevated in the injured IL-6(+/+) mice with acute alveolitis and in the plasma of IL-6(+/+) mice with dermal Arthus vasculitis. While, IL-6 levels in IL-6(-/-) mice were near or below the levels of detection. Histological examination of the intensity of vascular injury response demonstrated no significant differences between IL-6(-/-) and IL6(+/+) mice. More specifically, lung permeability (total protein in the BAL) in the lung injury model in IL-6(-/-) mice was the same as injured IL-6(+/+) mice. As a corollary, assessment of vascular permeability in both models was the same in the IL-6(-/-) as the IL-6(+/+) mice. Quantification of leukocyte influx into the injured tissues in both models also revealed no differences between the IL-6(-/-) and IL-6(+/+) mice. These data demonstrate that while IL-6 is upregulated in acute vascular injury it does not appear to be critical in the development of the vascular inflammatory response.

Early MT-1 MMP expression following elastase exposure is associated with increased cleaved MMP-2 activity in experimental rodent aortic aneurysms.

OBJECTIVE: The objective of this study was to determine the significance of membrane type 1 matrix metalloproteinase (MT1-MMP) activation of MMP-2 in experimental abdominal aortic aneurysms. METHODS: Rat aortas were perfused with either saline as a control or elastase, and harvested on 2, 4, or 7 days after perfusion (n = 5 per treatment group/day). Aortic MT1-MMP and MMP-2 expression and protein were determined by real time polymerase chain reaction and Western blotting, respectively. Aortic explants were used to measure MMP-2 activity by zymography. Rat aortic smooth muscle cells in vitro were exposed to increasing doses of elastase and analyzed for MT-1 MMP expression. RESULTS: Aneurysms formed in 80% of the elastase-perfused aortas at 7 days, whereas none formed in the saline-perfused aortas. Significantly increased MT1-MMP expression was observed only on day 4, when levels were 6.5-fold higher in elastase-perfused aortas compared with saline-perfused aortas (P < .01). By day 7, MT1-MMP protein was present only in the elastase-perfused aortas (P = .02). By immunohistochemistry, MT1-MMP was detectable only in the elastase-perfused group at day 7. Cleaved MMP-2 activity (P = .045) was increased in elastase-perfused aortas compared with saline perfused aortas at day 7. In rat aortic smooth muscle cells, MT-1 MMP expression increased in response to elastase (P = .02). CONCLUSION: The rodent aortic aneurysm model exhibits upregulation of MT1-MMP expression and protein with subsequent increased conversion of MMP-2 from the latent to the cleaved form.

Increased MMP-9 expression and activity by aortic smooth muscle cells after nitric oxide synthase inhibition is associated with increased nuclear factor-kappaB and activator protein-1 activity.

OBJECTIVE: To determine the mechanism underlying increased expression and activity of matrix metalloproteinase 9 (MMP-9) by rat aortic smooth muscle cells (RA-SMC) after inhibition of inducible nitric oxide synthase (iNOS). METHODS AND RESULTS: Treatment of interleukin-1beta-stimulated RA-SMC with aminoguanidine led to an increase of 96% in MMP-9 activity (P = 0.003) by gelatin zymography, a 40% increase in pro-MMP-9 protein (P = 0.018) by Western blot, and a 155% increase in MMP-9 mRNA (P = 0.06) by reverse transcription polymerase chain reaction. Aminoguanidine also caused a 26% decrease in cytosolic IkappaB levels (P = 0.014) by Western blot, as well as a 97% increase in nuclear factor-kappaB binding and a 216% increase in activator protein-1 binding as measured by electrophoretic mobility shift assay. No significant changes were noted in MMP-2 or TIMP-1 expression, protein levels, or activity after aminoguanidine administration. CONCLUSIONS: MMP-9 expression and activity is increased in cytokine stimulated RA-SMCs after iNOS inhibition, coincident with activation of the nuclear factor-kappaB and activator protein-1 pathways. We speculate that local derangements in iNOS may favor MMP-9-dependent vessel wall damage in vivo via an inflammatory cascade mechanism.