Collagen Metabolism in Cutis Laxa Fibroblasts: Increased Collagenase Gene Expression Associated with Unaltered Expression of Type I and Type III Collagen

Collagen metabolism was studied in cutis laxa by analyzing collagen and collagenase gene expression in three dermal fibroblast strains from patients with congenital cutis laxa and comparing them with fibroblasts obtained from age-matched healthy subjects. Normal collagen synthetic activity was observed in the cutis laxa fibroblasts. An increased level of collagenase mRNA and unaltered levels of α1(I) and α1(III) collagen mRNA were found in all cutis laxa cell strains by dot blot hybridization. Reduced levels of elastin mRNA were also detected in these strains. However, no qualitative differences in these mRNA transcripts were detected between the control and cutis laxa fibroblasts by Northern blot analysis. Collagenase activity in fibroblast culture supernatants was then measured using fluorescein isothiocyanate (FITC)-labeled type I collagen. Increased collagenolytic activity in cutis laxa fibroblast culture supernatants was also found. These data suggest that increased collagenase expression of fibroblasts is related to the structural abnormality of dermal connective tissue in cutis laxa

<i>Propionibacterium acnes</i>-derived insoluble immune complexes in sinus macrophages of lymph nodes affected by sarcoidosis

<div><p>Background</p><p><i>Propionibacterium acnes</i> is thought to be a causative agent of sarcoidosis. Patients with sarcoidosis have circulating immune complexes. We attempted to detect <i>P</i>. <i>acnes</i>-derived immune complexes in sarcoid lesions.</p><p>Methods</p><p>We evaluated formalin-fixed and paraffin-embedded lymph node samples from 38 sarcoidosis patients and 90 non-sarcoidosis patients (27 patients with necrotizing lymphadenitis, 28 patients with reactive lymphadenitis, 16 patients with colon cancer, 19 patients with gastric cancer) by immunohistochemistry using anti-human immunoglobulins (IgG, IgA, and IgM) and complement (C1q and C3c) antibodies, and a <i>P</i>. <i>acnes</i>-specific monoclonal antibody (PAB antibody) that reacts with the membrane-bound lipoteichoic acid of <i>P</i>. <i>acnes</i>.</p><p>Results</p><p>Small round bodies (SRBs) bound to IgA, IgM, or IgG were detected in sinus macrophages, in 32 (84%), 32 (84%), or 11 (29%) sarcoid samples, respectively, and in 19 (21%), 26 (29%), or no (0%) control samples, respectively. Some of these insoluble immune complexes (IICs) also bound to C1q and C3c. We developed a microwave treatment followed by brief trypsin digestion (MT treatment) to detect PAB-reactive SRBs bound to immunoglobulins (IIC-forming <i>P</i>. <i>acnes</i>). MT treatment revealed abundant IIC-forming <i>P</i>. <i>acnes</i> in most (89%) of the sarcoid samples and sparse distribution in some (20%) of the control samples with lymphadenitis, but no IIC-forming <i>P</i>. <i>acnes</i> was detected in control samples without inflammation. IIC-forming <i>P</i>. <i>acnes</i> were mostly bound to both IgA and IgM. The PAB-reactive antigen and immunoglobulins were both located at the peripheral rim of the IIC-forming <i>P</i>. <i>acnes</i>. Conventional electron microscopy identified many SRBs (0.5–2.0 μm diameter) in sinus macrophages of sarcoid lymph nodes with many IIC-forming <i>P</i>. <i>acnes</i>, some of which were in phagolysosomes with a degraded and lamellar appearance.</p><p>Conclusions</p><p><i>P</i>. <i>acnes</i>-derived IICs in sinus macrophages were frequent and abundant in sarcoid lymph nodes, suggesting a potential etiologic link between sarcoidosis and this commensal bacterium.</p></div

Immuno-electron microscopy images of SRBs in sinus macrophages of sarcoid lymph nodes.

<p>A and B: IHC with PAB antibody after MT treatment, C: IHC with anti-IgA antibody, and D: IHC with anti-IgM antibody. Note that dense black-colored reaction products by each antibody were located along the peripheral rim of the SRBs. A similar distribution of PAB-reactivity was observed in a large spherical-shaped HW body (A).</p

Frequency of PAB-reactive granuloma cells, HW-bodies, and SRBs in sarcoid and control lymph nodes.

<p>Frequency of PAB-reactive granuloma cells, HW-bodies, and SRBs in sarcoid and control lymph nodes.</p

Insoluble immune complexes in sinus macrophages of control lymph nodes from patients with reactive lymphadenitis.

<p>In a representative case of control lymph nodes with IICs from patients with reactive lymphadenitis, identical areas of the lymphatic sinus are shown in semi-serial sections; HE stain (A), IHC with anti-human IgG (B), IgA (C), and IgM (D) antibody, IHC with PAB antibody (E), and IHC with PAB antibody after MT treatment (F). In the sinus macrophages, IgA- and IgM-positive SRBs were detected (C and D, respectively), and PAB-reactive SRBs were also detected with a small increase in the number after MT treatment (F). Scale bar: 20 μm.</p

Higher magnification of small round bodies detected in the lymphatic sinus of sarcoid lymph nodes by IHC with each antibody.

<p>A: IHC with PAB antibody after MT treatment, B: IHC with anti-IgG antibody, C: IHC with anti-IgA antibody, D: IHC with anti-IgM antibody, E: IHC with anti-C1q antibody, and F: IHC with anti-C3c antibody. Note that the dark brown-colored reaction products produced by each antibody are all located along the peripheral rim of the small round bodies. Scale bar: 5 μm.</p

Co-localization of PAB-reactive PLTA antigen, IgA, IgM, and C3c detected by double fluorescence immunohistochemistry.

<p>A: Anti-IgA antibody (red) vs PAB antibody (green) after MT treatment, B: anti-IgM antibody (red) vs PAB antibody (green) after MT treatment, C: anti-IgM antibody (red) vs anti-IgA antibody (green), and D: anti-C3c antibody (red) vs PAB antibody (green) after MT treatment. Many PAB-reactive SRBs were also positive for IgA, IgM, and C3c, showing yellow-colored double-positive signals (A, B, and D, respectively). Both IgA and IgM colocalized with these SRBs, indicated by yellow-colored double-positive signals (C).</p

PAB-reactivity blocking experiment in tissue sections of P. acnes-infected rat liver and a sarcoid lymph node containing many IICs.

<p>Identical areas of serial sections from <i>P</i>. <i>acnes</i>-infected rat liver (A-C) and from a sarcoid lymph node containing many IICs in sinus macrophages (D-F). A and D: IHC with the PAB antibody, B and E: IHC with the PAB antibody after MT treatment, and C and F: IHC with the PAB antibody after MT treatment and subsequent reaction with a human plasma sample from a healthy adult volunteer. Scale bar: 20 μm.</p