A Major B Cell Epitope Present on the Apoptotic but Not the Intact Form of the U1-70-kDa Ribonucleoprotein Autoantigen

Abstract

Apoptotically modified forms of autoantigens have been hypothesized to participate in lupus immunopathogenesis. This study identifies a major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein lupus autoantigen (70k). Human autoimmune sera with strong recognition of apoptotic 70k and minimal recognition of intact 70k were identified and tested for reactivity to truncated forms of 70k by immunoblot and ELISA. Patient sera that preferentially recognized apoptotic 70k were specific for an epitope dependent on residues 180–205 of the protein. This epitope was also recognized by 19 of 28 (68%) intact anti-70k-positive autoimmune human sera with Abs also recognizing apoptotic but not the intact form 70k, but only 1 of 9 (11%) intact 70k-positive sera without such Abs (Fisher’s exact, p = 0.0055). Immunization of HLA-DR4-transgenic C57BL/6 mice with a peptide containing this epitope induced anti-70k immunity in 13 of 15 mice, including Abs recognizing apoptotic but not intact forms of autoantigens in 12 of 15 mice. Anti-70k responder mice also developed spreading of immunity to epitopes on the endogenous form of 70k, and proliferative lung lesions consistent with those described in patients with anti-70k autoimmunity. Thus, a major epitope in the B cell response to U1-70 kDa localizes to the RNA binding domain of the molecule, overlaps with the most common T cell epitope in the anti-70k response, and is not present on the intact form of the 70k molecule. Immunization of mice against this epitope induces an immune response with features seen in human anti-70k autoimmune disease.

A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen

“…As we have previously reported (12), C57BL/6 mice transgenic for HLA-DR4 that were immunized with the p205 70K fusion protein plus CFA (70K ϩ CFA) produced an anti-RNP immune response to 70K and developed additional autoantibodies and interstitial plus perivascular lung disease ( Figure 1) similar to that seen in humans with MCTD (20). Also consistent with reports of human MCTD-like lung disease (20), no fibrosis was observed by Masson's trichrome staining (results not shown).…”

“…For immunization studies, the p205 peptide of the 70K protein, spanning amino acids 63-205, was expressed as a maltose binding protein (MaBP) fusion protein in Escherichia coli, affinity purified over amylose columns, as previously described (12), and gel purified. For enzyme-linked immunosorbent assay (ELISA) studies, MaBP was cleaved from the 70K peptide with Genenase (New England Biolabs, Ipswich, MA), and the resulting 70K product was gel purified.…”

“…Anti-70K and anti-MaBP assays were performed as previously described (12). Briefly, 96-well flat-bottomed microtiter plates were incubated overnight at 4°C with purified antigen in phosphate buffered saline (PBS).…”

“…(17,18). Lungs were inflated with fixative at the time of harvest, as previously described (12). Paraffin-embedded sections were stained using hematoxylin and eosin, and imaged with a CoolPix 990 camera (Nikon, Melville, NY) mounted on a BH2 microscope (Olympus, Lake Success, NY).…”

“…Moreover, anti-RNP antibodies have been found to interact with lung tissue in ways that could mediate MCTD lung pathology (11). In a small series of mice, we have previously found that direct immunization of a susceptible animal with RNP antigen could induce MCTD-like serologic responses and clinical manifestations (12). However, reactivity to RNP components has recently been shown to correlate with the development of severe disease in lupus, including the development of GN (13).…”