Background Tumour necrosis factor alpha (TNF-a) is found in both, autoimmune and normal conditions and may therefore not mediate inflammation by overexpression alone. Upregulation of polymorph TNF-a peptides caused by single base pair (bp) exchanges in mRNA transcripts might modify inflammatory events in arthritis.
Objectives We wished to examine variants of TNF-a transcripts expressed in collagen-induced arthritis (CIA) as a classical model of autoimmune diseases.
Methods Severe osteodestructive CIA was induced in male Dark Agouti rats, the nature of arthritis was confirmed by radiology. Total articular RNA from diseased animals was used for cDNA synthesis. PCR was performed for the 705 bp TNF-a propeptide and the amplified bands were cloned in E. coli. Two clones were sequenced and the results compared to the NIH gene bank. To confirm polymorph expressions ribonuclease protection assays (RPA) were performed with RNA from normal spleen tissue and joints from healthy and arthritic animals.
Results Radiology showed severe osteodestruction in CIA animals. Two clones with a complete open reading frame for the 705 bp TNF-a propeptide transcript were obtained by RT-PCR. Single base pair mismatches were found at position 365 and 568 leading to amino acid exchanges of proline versus leucine and lysine versus glutamic acid, respectively. Based on the NIH database four previously published rat TNF-a sequences showed up to twelve single base pair mismatches. In RPA analyses we found protected bands at 705 bp size in all conditions. In addition, smaller bands indicated the presence of transcripts with several single base pair mismatches. Some fragments were not observed in healthy control joints.
Conclusion The two novel TNF-a transcripts described here are expressed in arthritic and control animals displaying a nonspecific polymorphism. The additional presence of smaller bands in the RPA suggest the coexpression of similar transcripts which may in part correspond to previously reported sequences. The absence of some fragments in healthy control joints is indirect evidence for differentially upregulated TNF-a peptide variants in arthritis.