An abnormality in basement membrane metabolism has been postulated to play an important role in the pathogenesis of experimental murine AA amyloidosis. The potential contribution of the structural basement membrane proteins laminin, type IV collagen and entactin to amyloidogenesis in this model was investigated with a kinetic analysis of the expression of the corresponding genes during amyloid formation. Splenic AA amyloid deposition was stimulated by the concomitant administration of subcutaneous silver nitrate, as an inflammatory stimulus, and intravenous amyloid enhancing factor. Using a reverse transcription-polymerase chain reaction assay, a differential pattern of expression of these genes was observed at the mRNA level. Whereas laminin B1 mRNA levels did not change at any time during amyloidogenesis, a 2.2 to 3 fold induction of laminin B2, entactin and alpha 1-type IV collagen mRNAs coincided with the initial detection of splenic amyloid deposits at 48 hours post-stimulation, as detected by immunohistochemistry. Temporal and spatial codeposition of laminin and type IV collagen with amyloid was demonstrated by immunohistochemistry. A 1.4, 2.3 and 2.2-fold increase in laminin B2, entactin and alpha 1-type IV collagen mRNA levels, respectively, was detected at 24 hours post-stimulation, a point at which amyloid deposits could not be detected. Neither inflammation nor amyloid enhancing factor alone influenced laminin, entactin or type IV collagen expression at the protein or mRNA level. These observations suggest that the laminin B2 chain and alpha 1-type IV collagen chain account, at least in part, for the observed laminin and collagen IV immunoreactivity in AA amyloid deposits and that entactin may also be a component of the amyloid deposit. The onset of the induction of laminin B2, entactin and alpha 1-type IV collagen gene expression prior to the appearance of amyloid deposits, and our previous data with the heparan sulfate proteoglycan, perlecan, suggests these basement membrane proteins may play a role in the initial stages of AA fibrillogenesis.

Nonisotopic methods of mRNA in situ hybridization have distinct advantages over isotopic techniques. Nonisotopically labeled probes are stable and nontoxic, have short detection times, demonstrate excellent spatial resolution of their signals and have sensitivities comparable to radiolabeled probes. We developed a simple method of generating nonisotopically labeled cRNA probes which is based on the reverse transcription polymerase chain reaction (RT-PCR) and used it to synthesize a panel of probes for various murine extracellular matrix genes. Engelbreth-Holm-Swarm (EHS) tumor RNA was reverse transcribed and PCR was used to amplify defined regions of multiple extracellular matrix protein genes from the resulting first strand cDNAs. Bacteriophage promoters which had been incorporated into the PCR products were then used to generate digoxigenin-conjugated antisense and sense cRNAs. The antisense probes were employed to detect the specific extracellular matrix protein mRNAs in the EHS tumor by in situ hybridization. This technique provides a rapid and efficient alternative to conventional transcription systems which use plasmid vectors for the synthesis of digoxigenin-labeled cRNA probes.

No abstract