Collagen chain composition and collagen gene expression in acne keloids.

Keloids are thick scars that grow beyond the boundaries of the original skin damage. Most lesions appear within one year of skin inflammation including burns, surgery and acne vulgaris. They are characterized by an excessive accumulation of extracellular mamx components. Keloid waning results from increased activity of fibroblasts in the dermis with imbalance between synthesis and breakdown of collagen. Previous studies have indicated that collagen types I and I11 are the major components of the lesion [l]. Collagen types V and VI have also been located in keloid fibroblasts [2 and personal observations]. Conflicting results on collagen production within keloids have previously been reported [3, 41. These reports have concerned a variety of lesions irrespective of their precipitating cause, but few have included acne keloids. The aim of this study was to investigate collagen composition and collagen gene expression in early and late lesions of acne keloids. Six patients with unmated truncal keloids secondary to acne were studied. 1-3 month lesions were classified as early keloids (EK) and lesions that presented over 3 months were classified as late keloids (LK). 2-3 mm biopsies were obtained from patients under local anaesthetic using Lignocaine. Biopsies were cultured for 24 h at the &/liquid interface in methionine-free, modified Eagle's medium supplemented with 3%-methionine. The biopsies were then washed, homogenised and lyophilised. Samples were treated with pepsin or with collagenase to degrade non-collagen or collagenous protein respectively. Types I and 111 collagen were separated on 5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions and autoradiographed. Immunoblot analysis was performed with anti-collagen type I (kindly provided by Dr J. Werkmeister, Csiro, Australia) and type Ill (Lab. Impex Ltd. UK) antibody. Densitomeuic scans were used to quantify the collagen bands on autoradiographs. I n siru hybridisation was performed using a modified method of Lewis et al. [ 5 ] . Briefly, 5 mm cryosections were hybridised with biotinylated cDNA probes for procollagen al(I) , a2(I) and procollagen al(II1) (kindly provided by Dr. G. Tromp and Dr Ala-Kokko, Philadelphia, USA). Sections were fixed in 4% formalin, treated with Nonidet + Triton X100, proteinase K, acetic anhydride and post fixed in formalin. Hybridisation was performed at 37OC for 24 h and RNA signal detected using the streptavidinbiotinylated alkaline phosphatase detection system. Fig. 1 shows a typical result of immunoblotting of tissue homogenate with incubated with anti-collagen types I and 111 antibodies. Type 111 collagen was the predominant collagen in keloid tissue (Lane D). Reduced levels of type I collagen was seen in the tissue of normal appearance obtained from the border of the lesion (lane A) and keloid tissue (lane B). Autoradiograph of EK tissue showed higher (6.5 f 1.8 S.D.,%) incorporation of radioactivity into a 1 (I) collagen 330 -