| Extracellular matrix comprises approximately 90% of cartilage with colla-gens and proteoglycans making up the bulk of the tissue. Inflammatory arthritis, with destruction and erosion of articular cartilage, is associated with the development of autoimmunity to type II cartilage collagen and to cartilage proteoglycan and this may be related to the immunopathology of the disease. In recent years, several abundant cartilage proteins that are neither collagens nor proteoglycans have been characterized in detail. Cartilage intermediate layer protein (CILP) is a newly identified cartilage component with limited distribution in the middle layer of cartilage. It is cloned and the cDNA in human cartilage codes for pro-forms of two polypeptides, CILP (91. 5 KDa) and a nucleotide pyrophospho-hydrolate (NTPPHase, 51.8 KDa). Moreover, its expression is enhanced during aging and also in the early stages of human osteoarthritis (OA). Based on these reports, it should be investigated whether CILP is involved in physiologic cartilage degeneration and also in the pathogenesis of OA.OA is thought to be merely a noninflammatory degenerative disease. However , there is ample evidence to suggest that the underlying mechanisms of OA, as well as of rheumatoid arthritis (RA) , involve the immune system. First, infiltrating activated T cells and T cells with oligoclonal expansion are often found in the synovia! tissue of patients with OA. Second, autoimmune responses to au-toantigens mainly derived from the cartilage matrix have been detected in OA patients. Third, immunization using these cartilage derived proteins induces arthri-tis in animal models. Taken together, it is likely that locally evoked immune responses to the cartilage matrix and its related proteins are important modulators of cartilage degradation in OA as well as RA.In this study, we investigated whether the recently cloned cartilage specific protein, CILP, was recognized as an autoantigen in patients with OA and RA, using recombinant human CILP fusion proteins. Our results demonstrated the presence of anti - CILP antibodies in a subset of OA and RA patients. Moreover, immunization using the CILP fusion protein induced chronic polyarthritis in mice. These findings strongly suggest the active involvement of the autoimmune response to CILP in the pathogenesis of OA as well as RA.Materials & MethodsI . Sub - cloning and induction of CILP recombinant fusion protein in Escherichia coli.1. Sub - cloning of CILP gene: Based on the previously reported nucleotide sequences of CILP, complementary DNA ( cDNA) fragments encoding the first half (C1: +64 to +999 bp bp) and the second half (C2:+976 to +2031 bp) of the non - NTPPHase - homologous regions of CILP were obtained from mRNA of human articular chondrocytes harvested from an otherwise healthy donor with traumatic fracture by reverse transcription - polymerase chain reaction (RT-PCR). In addition, the C2 region was further subdivided into 3 regions (C2F1 to C2F3) and their respective cDNA fragments were obtained as described above. Nucleotide sequences of the PCR products were confirmed by dideoxy sequencing.2. The construction of CILP recombinant fusion protein; The cDNA fragments that encode the C1 (999 bp) , C2 (1,056 bp), C2F1 (396bp) , C2F2 (399 bp) , and C2F3 (351 bp) regions were subcloned into a plasmid expression vector of pTEX - 2 - eHis, a derivative of a pEX, which carried 6 straight histines at the C - terminus of multiple cloning sites for affinity purification, Then, those plasmids were expressed in the Escherichia coli POP 2136. In orderto study immunodominant epitopes of CILP, those peptides were also fused to maltose binding protein ( MBP) using plasmids of pMAL - eHis which earned six straight histines at the C - terminus of multiple cloning sites and expressed in Escherichia coli strain DH5a. Nucleotide sequences of the insertion genes were confirmed by dideoxy sequencing.3. Purification of CILP recombinant protein; The recombinant proteins were purified from...
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