The terminal pathway regulators vitronectin and clusterin when bound to Lpd are functionally active.

<p>Both vitronectin (<b>A</b>) and clusterin (<b>B</b>) when bound to immobilized Lpd inhibits C5b-9 deposition. <b>A</b>, Vitronectin (10–50 μg/ml) or Factor H (10–50 μg/ml) was bound to immobilized Lpd and after extensive washing C5b-6 and C7 were added. After 10 min incubation C8 and C9 were added and C5b-9 deposition was detected with mouse anti-C5b-9 mAb and HRP-conjugated anti-mouse pAb. <b>B</b>, Clusterin (2.5–20 μg/ml) or Factor H (2.5–20 μg/ml) was bound to immobilized Lpd and after extensive washing C5b-6 and C7 were added. After 10 min incubation C8 and C9 were added and C5b-9 deposition was detected with mouse anti-C5b-9 mAb and HRP-conjugated anti-mouse pAb. The mean values of three independent experiments and SD are presented. Statistical significance of differences was estimated using Student’s t test. ***, <i>p</i>≤ 0.001.</p

Vitronectin bind to <i>P</i>. <i>aeruginosa</i> via amino acids 354–363.

<p>A, Localization of the region within vitronectin that mediates binding to intact <i>P</i>. <i>aeruginosa</i>. Vitronectin uses one contact region i.e. aa 354–363 to contact <i>P</i>. <i>aeruginosa</i>. Vitronectin ^80–396 (Vn^80-396) and seven deletion mutants were expressed in HEK cells and purified. The numbers refer to amino acids residues that are included in each construct (<i>left panel</i>). Black indicates the heparin binding regions of vitronectin (<i>left panel</i>). B, Binding of serum purified full-length vitronectin and vitronectin deletion mutants (5 μg/ml) to immobilized bacteria was assayed by ELISA (<i>right panel</i>). Bound vitronectin was detected with polyclonal vitronectin antiserum followed by HRP-conjugated anti-rabbit. <b>C</b>, Heparin inhibits binding of vitronectin to <i>P</i>. <i>aeruginosa</i> strain SG137 and to Lpd, the effect was dose-dependent. The effect of heparin (0.01–5 mg/ml) on vitronectin binding to immobilized <i>P</i>. <i>aeruginosa</i> strain SG137 was assayed. Bound vitronectin was detected with polyclonal vitronectin antiserum and HRP-conjugated anti-rabbit pAb. The mean values of three independent experiments and SD are presented. Statistical significance of differences was estimated using Student’s t test. **, <i>p</i>≤ 0.01; ***, <i>p</i>≤ 0.001.</p

<i>P</i>. <i>aeruginosa</i> binds the terminal complement regulator vitronectin.

<p><b>A</b>, Binding of vitronectin to <i>P</i>. <i>aeruginosa</i> strain SG137 was assayed by flow cytometry. Bacteria were incubated with vitronectin (10–50 μg/ml) and bound vitronectin was detected with polyclonal vitronectin antiserum and Alexa488-labeled rabbit antiserum. Bacteria incubated with vitronectin specific antiserum and Alexa488-labeled rabbit antiserum served as controls. <b>B</b>, Vitronectin binds to <i>P</i>. <i>aeruginosa</i> and binding was dose-dependent. Four laboratory strains of <i>P</i>. <i>aeruginosa</i> were analyzed for vitronectin binding using a whole cell ELISA. Whole bacteria were immobilized onto microtiter plates and vitronectin (1–5 μg/ml) was added. Bound vitronectin was detected with polyclonal vitronectin antiserum followed by HRP-conjugated anti-rabbit. <b>C</b>, Vitronectin bind to <i>P</i>. <i>aeruginosa</i>. <i>P</i>. <i>aeruginosa</i> strains SG137, ATCC 27853, NCTC 10662 and PAO1 were incubated with NHS. Bacteria were washed, lysed, separated by SDS-PAGE and analysed by Western blotting. Bound vitronectin was detected with polyclonal vitronectin antiserum and HRP-conjugated anti-rabbit. A representative experiment of three is shown. <b>D</b>, Vitronectin bound to both laboratory and clinical <i>P</i>. <i>aeruginosa</i> strains. Binding of vitronectin (5 μg/ml) to immobilized bacteria (0.5x10⁷) was assayed by ELISA. Bound vitronectin was detected with polyclonal vitronectin antiserum followed by HRP-conjugated anti-rabbit pAb. The mean values of three independent experiments and standard deviations (SD) are presented. Statistical significance of differences was estimated using Student’s t test. **, <i>p</i>≤ 0.01; ***, <i>p</i>≤ 0.001.</p

Vitronectin and clusterin bind simultaneously to Lpd.

<p><b>A,</b> Effect of increasing clusterin levels in the presence of a constant concentration of vitronectin. Binding of clusterin (used at the indicated concentrations) and vitronectin (5 μg/ml) to immobilized Lpd was analysed by ELISA. Bound clusterin was detected with anti-clusterin mAb (■) and bound vitronectin was detected with anti-vitronectin mAb (◇). <b>B,</b> In a reverse setting, the clusterin concentration was kept constant (2.5 μg/ml) and binding of vitronectin (used at the indicated concentrations) was evaluated. The mean values of three independent experiments and SD are presented.</p

The binding of vitronectin is inhibited by heparin and high ionic strength.

<p>Heparin and NaCl inhibited the binding of [¹²⁵I]-labeled vitronectin to the microbial pathogens. The microbial pathogens were incubated with [¹²⁵I]-labeled vitronectin and 10 μM heparin (A) or 1 M NaCl (B), followed by washing and determination of radioactivity associated with the pellet. The vitronectin binding of each microbe in the absence of competitor was defined as 100%. The mean values of three experiments are shown with error bars indicating SD. Statistical significance of differences was estimated using Student’s <i>t</i> test. **, <i>p</i>≤ 0.01; ***, <i>p</i>≤ 0.001.</p

The microbial binding site is located within the third HBD.

<p>(A) Schematic representation of the different truncated vitronectin (Vn) fragments and deletion mutants used for analysis of vitronectin binding. (B) Eight different microbes, including Gram-negative and Gram-positive bacteria and <i>C</i>. <i>albicans</i> were incubated with truncated recombinant vitronectin fragments and three vitronectin deletion mutants. NHS was run in parallel as a positive control. Microbes were washed and total proteins were separated by SDS-PAGE gels, transferred to a membrane and bound vitronectin fragments were detected using an anti- vitronectin polyclonal antiserum. One representative experiment of three independent ones performed is shown. (C) Microbes were immobilized on microtiter plates and incubated with vitronectin fragments including deletion mutants. Bound fragments were detected with rabbit anti-vitronectin pAb and secondary HRP-conjugated goat anti-rabbit pAb. The mean values of three experiments are shown with error bars indicating SD. Statistical significance of differences was estimated using Student’s <i>t</i> test. *, <i>p</i>≤ 0.05; **, <i>p</i>≤ 0.01; ***, <i>p</i>≤ 0.001.</p

Pathogenic microbes bind the terminal complement pathway inhibitor vitronectin.

<p>A series of Gram-negative and Gram-positive bacterial species and <i>Candida albicans</i> were grown overnight and incubated with [¹²⁵I]-labeled vitronectin purified from plasma (A) or recombinant vitronectin^80-396 (B). After washing, bound vitronectin was determined by liquid scintillation counting. The mean values of three experiments are shown. In C, the relative binding of [¹²⁵I]-labeled vitronectin to selected microbes are shown. The mean values of three experiments are shown with error bars indicating standard deviations (SD).</p

Vitronectin bound to intact bacteria inhibits C5b-9 deposition.

<p>Vitronectin (25–50 μg/ml) was bound to Hib (<i>A</i>), NTHi (<i>B</i>), <i>M</i>. <i>catarrhalis</i> (<i>C</i>) or <i>P</i>. <i>aeruginosa</i> (<i>D</i>), and after extensive washing C5b-6 and C7 were added. After incubation for 10 min, C8 and C9 were added, and thereafter C5b-9 deposition on the microbial surface was detected with a mouse anti-C5b-9 mAb and Alexa 647-conjugated anti-mouse pAb by flow cytometry. E, vitronectin (50 μg/ml) was bound to immobilized proteins, and after extensive washing C5b-6 and C7 were added. After 10 min incubation, C8 and C9 were added, and C5b-9 deposition was detected with a mouse anti-C5b-9 mAb and an HRP-conjugated anti-mouse polyclonal antiserum. The mean values from three independent experiments are shown with error bars indicating SD. **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001.</p

Image_6_The Pulmonary Extracellular Matrix Is a Bactericidal Barrier Against Haemophilus influenzae in Chronic Obstructive Pulmonary Disease (COPD): Implications for an in vivo Innate Host Defense Function of Collagen VI.PDF

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human commensal commonly residing in the nasopharynx of preschool children. It occasionally causes upper respiratory tract infection such as acute otitis media, but can also spread to the lower respiratory tract causing bronchitis and pneumonia. There is increasing recognition that NTHi has an important role in chronic lower respiratory tract inflammation, particularly in persistent infection in patients suffering from chronic obstructive pulmonary disease (COPD). Here, we set out to assess the innate protective effects of collagen VI, a ubiquitous extracellular matrix component, against NTHi infection in vivo. In vitro, collagen VI rapidly kills bacteria through pore formation and membrane rupture, followed by exudation of intracellular content. This effect is mediated by specific binding of the von Willebrand A (VWA) domains of collagen VI to the NTHi surface adhesins protein E (PE) and Haemophilus autotransporter protein (Hap). Similar observations were made in vivo specimens from murine airways and COPD patient biopsies. NTHi bacteria adhered to collagen fibrils in the airway mucosa and were rapidly killed by membrane destabilization. The significance in host-pathogen interplay of one of these molecules, PE, was highlighted by the observation that it confers partial protection from bacterial killing. Bacteria lacking PE were more prone to antimicrobial activity than NTHi expressing PE. Altogether the data shed new light on the carefully orchestrated molecular events of the host-pathogen interplay in COPD and emphasize the importance of the extracellular matrix as a novel branch of innate host defense.</p

Image_3_The Pulmonary Extracellular Matrix Is a Bactericidal Barrier Against Haemophilus influenzae in Chronic Obstructive Pulmonary Disease (COPD): Implications for an in vivo Innate Host Defense Function of Collagen VI.PDF

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human commensal commonly residing in the nasopharynx of preschool children. It occasionally causes upper respiratory tract infection such as acute otitis media, but can also spread to the lower respiratory tract causing bronchitis and pneumonia. There is increasing recognition that NTHi has an important role in chronic lower respiratory tract inflammation, particularly in persistent infection in patients suffering from chronic obstructive pulmonary disease (COPD). Here, we set out to assess the innate protective effects of collagen VI, a ubiquitous extracellular matrix component, against NTHi infection in vivo. In vitro, collagen VI rapidly kills bacteria through pore formation and membrane rupture, followed by exudation of intracellular content. This effect is mediated by specific binding of the von Willebrand A (VWA) domains of collagen VI to the NTHi surface adhesins protein E (PE) and Haemophilus autotransporter protein (Hap). Similar observations were made in vivo specimens from murine airways and COPD patient biopsies. NTHi bacteria adhered to collagen fibrils in the airway mucosa and were rapidly killed by membrane destabilization. The significance in host-pathogen interplay of one of these molecules, PE, was highlighted by the observation that it confers partial protection from bacterial killing. Bacteria lacking PE were more prone to antimicrobial activity than NTHi expressing PE. Altogether the data shed new light on the carefully orchestrated molecular events of the host-pathogen interplay in COPD and emphasize the importance of the extracellular matrix as a novel branch of innate host defense.</p