gp130-RAPS, a novel soluble form of gp130, is a new autoantigen in RA. Its autoantibodies, α-RAPC15 Ab’s, are produced specifically and in association with disease activity in RA. Furthermore, α-RAPC15 Ab’s from patients with RA neutralized the inhibitory function of gp130-RAPS against IL-6. Thus, the α-RAPC15 Ab can not only serve as a clinical indicator, but also provide an important clue to the pathology of RA.
Our cloned gp130-RAPS cDNA had an 83-bp fragment of gp130 cDNA missing. Cloning artifacts of a partial sequence deletion can originate when hairpin structure–forming mRNA templates are skipped during RT (23
). Genomic gp130 DNA, however, had the introns adjacent to both the 5′ and 3′ ends of the deleted 83-bp fragment, which had a donor-acceptor structure compatible with the GT-AG rule. This shows that the 83-bp fragment is an exon and that mRNA splicing with loss of the entire fragment containing such introns and the 83-bp exon can produce gp130-RAPS mRNA. In addition, gp130-RAPS mRNA was actually identified by RT-PCR in human cells. gp130-RAPS cDNA-transfected COS-7 cells produced its translated products. gp130-RAPS protein with a predicted molecular weight of 50 kDa was actually found in synovial fluids and plasma by different specific Ab’s. On the basis of these analyses of DNA, mRNA, and protein levels, we can conclude that gp130-RAPS is not a cloning artifact, but a splicing variant of gp130.
sgp130s are supposed to be generated by two mechanisms, proteolytic cleavage (shedding) and alternative mRNA splicing (24
), although cDNA of sgp130 had not been isolated. We succeeded in the cloning of sgp130 cDNA. sgp130s have been identified as three different molecules of about 50, 90, and 110 kDa (10
). Considering the similarity of molecular size, it is possible that gp130-RAPS is identical to the 50-kDa species, which have not been cloned but whose existence was described while this study was under way (25
). IL-6 upregulated the expression of gp130-RAPS in SF-1 cells. This suggests that gp130-RAPS production is a negative feedback to IL-6 stimulation.
In the Hep G2 fibrinogen production assay, rabbit α-RAPC15 Ab, raised by immunization of RAPC15 peptides and affinity purified, inhibited the function of gp130-RAPS. Similarly, affinity-purified α-RAPC15 Ab’s from patients with RA impaired gp130-RAPS activity. These results show that α-RAPC15 Ab’s actually associate with native forms of gp130-RAPS molecules and exert a blocking effect. Therefore, α-RAPC15 Ab’s from patients with RA can be considered autoantibodies to gp130-RAPS. It is not clear why the same amounts of affinity-purified α-RAPC15 Ab’s had different activities for gp130-RAPS. It is probably due to the difference in the antibody reactivity to RAPC15 peptides covalently conjugated with agarose beads, to those on ELISA plates, and to the native form of gp130-RAPS. In fact, in Western blot analyses, affinity-purified α-RAPC15 Ab’s from patients with RA did not react with eukaryotic gp130-RAPS molecules bound to membranes after SDS-PAGE, but reacted with prokaryotic ones (data not shown). In any case, α-RAPC15 Ab’s, antibodies to plate-attached RAPC15 peptides, seem to have biologic activity for gp130-RAPS under physiological conditions.
It was revealed that signals of gp130-related cytokines are transduced by dimerization of the cytoplasmic domains of gp130 after the coupling of two ligand-receptor complexes consisting of a ligand, its receptor, and gp130 (26
). The known 90- and 110-kDa sgp130s are thought to interfere with the ligand signaling by forming nonfunctional ligand-receptor complexes due to a lack of cytoplasmic domains. gp130-RAPS appears to inhibit IL-6 activity in the same manner. α-RAPC15 Ab’s, therefore, seem to recover IL-6 activity by disturbing such nonfunctional ligand-receptor complex formation.
α-RAPC15 Ab’s, neutralizing antibodies to gp130-RAPS, promote IL-6 activity whereby gp130-RAPS is induced by negative feedback to IL-6. It remains to be clarified whether this phenomenon has significance in vivo and whether α-RAPC15 Ab production is a cause or result in RA. It is well documented that some autoantibodies are pathogenic, modifying or inhibiting the activities of their target molecules in autoimmune and malignant diseases, e.g., autoantibody to β2-glycoprotein-I in antiphospholipid syndrome, autoantibody to thyroid-stimulating hormone receptor in Graves disease, and autoantibody to acetylcholine receptor in myasthenia gravis. In RA, blocking autoantibody to calpastatin was reported to aid calpain activity in destructive joint inflammation (27
). Autoantibody to follistatin-related protein, which we reported previously (3
), is another candidate and under investigation. Given its IL-6–supporting effect, and high prevalence and correlation with disease activity in RA, α-RAPC15 Ab, autoantibody to gp130-RAPS, seems to play an important role in disease development in RA.
Detection of α-RAPC15 Ab’s may well be a more valuable clinical finding than known clinical parameters in RA because it has high sensitivity and specificity (73.0% and 96.9%, respectively, in Figure ) and also reflects disease activity as already described here. In addition, ELISA with RAPC15 peptides is a promising tool for the detection of α-RAPC15 Ab’s, because of its simplicity and reproducibility due to the usage of synthetic peptides, rather than recombinant proteins or tissue extracts.
Why do α-RAPC15 Ab’s appear in patients with RA? One possibility is that α-RAPC15 Ab’s are generated directly against gp130-RAPS that is overexpressed in pathological states. Another possibility is that α-RAPC15 Ab’s are originally produced against infectious agents containing an NIASF or NIASF-like sequence and then cross-react to gp130-RAPS. The latter speculation seems more plausible than the former because the upregulated expression of gp130-RAPS was not specific to RA and observed in synovial fluids from patients with osteoarthritis as well as those with RA. In the pathogenesis of RA, infection with various organisms including viruses, bacteria, and mycoplasmas has been suggested to be the trigger of disease development (1
). By using the FASTA program on the GenomeNet service (29
), we found NIASF or NIASF-like sequences in several organic proteins; -NIASF- in human monocyte/macrophage serine esterase (A49816), yeast glutamate dehydrogenase (A25275), simian rotavirus SA11 glycoprotein VP7 precursor (VGXR1S), Streptococcus pyogenes
exotoxin type B precursor (A37768), Escherichia coli
dnaC protein (XMECNC), Haemophilus influenzae
probable membrane protein HI0608 (I64080), Bacillus subtilis
cheV protein (A55592); -NI-SF- in saimiriine herpesvirus 1 gene 34 protein (QQBEN3); -NI—F- in Escherichia coli
ribonuclease T2 (S32940); and -I-SF- in Staphylococcus aureus
cell division protein FtsZ (S58814) (accession numbers of the PIR protein sequence database are in parentheses). Among them, we could not find any protein containing COOH-terminal NIASF or NIASF-like sequences. However, interestingly, Streptococcus pyogenes
, whose exotoxin precursor contains the NIASF sequence, has been reported to induce arthritis both in humans (30
) and in an animal model (31
). To identify pathogenic molecules in RA, α-RAPC15 Ab’s and synovial fluid and/or sera from patients with recent-onset RA may be useful materials as probes and antigen sources.
The α-RAPC15 Ab, the autoantibody to gp130-RAPS, provides a novel parameter for the clinical characterization of RA. However, more intensive studies including follow-up surveys about the correlation between its titers and clinical manifestations are necessary to prove its advantages over available clinical tests and to establish its definitive significance in RA. In any case, gp130-RAPS and its autoantibody may provide the tools with which to elucidate the pathogenesis of and to create new therapies for RA.