INTERLEUKIN (IL)-1-BETA AND IL-1-BETA MESSENGER-RNA EXPRESSION IN NORMAL AND DISEASED SKELETAL-MUSCLE ASSESSED BY IMMUNOCYTOCHEMISTRY, IMMUNOBLOTTING AND REVERSE TRANSCRIPTASE-NESTED POLYMERASE CHAIN-REACTION

To confirm the production of IL-1 beta and to optimize detection and s emiquantitation of IL-1 beta mRNA by polymerase chain reaction (PCR) t echniques in skeletal muscle tissue, immunocytochemistry, immunoblotti ng and several procedures of RNA extraction and reverse transcription (RT)-PCR amplification were used on muscle samples from 12 patients wi th conditions associated with local production of IL-1 beta (AZT myopa thy: 6 patients; sarcoid myopathy: 6 patients) and from 9 patients wit h normal muscle used as controls. Abundant IL-1 beta immunoreactivitie s, corresponding to both pro IL-1 beta and mature IL-1 beta as assesse d by immunoblotting, were observed in all diseased muscles, either in inflammatory cells (sarcoid myopathy) or in atrophic muscle fibers (AZ T myopathy). Acid guanidinium isothiocyanate phenol-chloroform extract ion of RNA appeared less efficient for IL-1 beta mRNA detection by RT- PCR than proteinase K digestion followed by phenolchloroform extractio n. Even using the latter procedure, RT-single PCR for IL-1 beta mRNA w as puzzlingly negative in all cases but one; in contrast, RT-nested PC R specified by DNA enzyme immunoassay yielded detection of IL-1 beta m RNA in all diseased muscles and in occasional controls, including the expected PCR product of 391 bp, but also another product of 935 bp, co rresponding to IL-1 beta mRNA with unsplicing of the fourth intron. Se mi-quantitative PCR showed that production of IL-1 beta mRNA was highe r in sarcoid myopathy than in AZT myopathy, and in AZT myopathy than i n controls. In conclusion, IL-1 beta expression can be reliably studie d using immunocytochemistry, but assessment of IL-1 beta mRNA producti on in muscle tissue requires optimized extraction and RT-PCR procedure s.