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, 60 (3), 848-59

Early Targets of Nuclear RNP Humoral Autoimmunity in Human Systemic Lupus Erythematosus

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Early Targets of Nuclear RNP Humoral Autoimmunity in Human Systemic Lupus Erythematosus

Brian D Poole et al. Arthritis Rheum.

Abstract

Objective: The U1 small nuclear RNPs are common targets of autoantibodies in lupus and other autoimmune diseases. However, the etiology and progression of autoimmune responses directed against these antigens are not well understood. The aim of this study was to use a unique collection of serial samples obtained from patients before and after the development of nuclear RNP (nRNP) antibodies to investigate early humoral events in the development of anti-nRNP autoimmunity.

Methods: Lupus patients with sera available from both before and after the development of nRNP antibody precipitin were identified from the Oklahoma Clinical Immunology Serum Repository. Antibodies in the serial samples were analyzed by enzyme-linked immunosorbent assay, Western blotting, solid-phase epitope mapping, and competition assays.

Results: The first-detected nRNP antibodies targeted 6 common initial epitopes in nRNP A, 2 in nRNP C, and 9 in nRNP 70K. The initial epitopes of nRNP A and nRNP C were significantly enriched for proline and shared up to 95% sequence homology. The initial nRNP 70K humoral epitopes differed from those of nRNP A and nRNP C. The initial antibodies to nRNP A and nRNP C were cross-reactive with the SmB'-derived peptide PPPGMRPP. Antibody binding against all 3 nRNP subunits diversified significantly over time.

Conclusion: Autoantibodies to nRNP A and nRNP C initially targeted restricted, proline-rich motifs. Antibody binding subsequently spread to other epitopes. The similarity and cross-reactivity between the initial targets of nRNP and Sm autoantibodies identifies a likely commonality in cause and a focal point for intermolecular epitope spreading.

Figures

Figure 1
Figure 1. Autoantibody development to nRNP proteins began by targeting a limited number of epitopes and then expands
Serial serum samples from 6 patients collected concurrent with and after the first observation of nRNP A-specific antibodies, from 6 patients concurrent with and after nRNP-C-specific antibodies, and from 3 patients concurrent with and after the development of nRNP 70K-specific antibodies were analyzed by solid-phase peptide mapping to identify initial humoral epitopes. Serum antibody binding was measured to each overlapping octapeptide in the sequence of nRNP A, and each overlapping decapeptide in the sequences of nRNP C and nRNP 70K. Representative antibody binding profiles of nRNP A (first column), nRNP C (middle column), and nRNP 70K (last column) from pre-nRNP antibody-positive (top row), first nRNP antibody-positive (middle row) and late nRNP antibody-positive samples (bottom row) are shown.
Figure 2
Figure 2. Identification of sequential humoral epitopes in early and late nRNP A humoral autoimmunity
Solid-phase peptide octamers were used to map the antibody binding pattern to nRNP A. Antibody binding to the sequential overlapping peptides of nRNP A in the first nRNP-A antibody-positive sera is shown. Bars represent the number of standard deviations that the mean absorbance (OD) of the first positive samples is above the control mean (n=6) (A). Epitopes, defined as overlapping significantly bound peptide sequences, recognized by the first nRNP A-positive serum antibodies are shaded (A) and described (B). The amount of antibody binding to nRNP accounted for by the dominant first epitope was quantified by blocking binding of epitope 4 using 2 peptides representing epitope 4 and then detecting residual binding in Western blots. Significant inhibition was detected when either the peptide nRNP A 164-172 (p=0.026) or a mixture of both peptides (p=0.011) (n=9) were incubated with the patient sera. Error bars indicate standard error (C). Antibody binding in the last available sera after nRNP antibodies have already developed is shown as for panel A. Shaded areas and numbers show the antibody binding pattern in the first positive samples for comparison (D).
Figure 3
Figure 3. Identification of key epitopes in the early and mature nRNP C humoral immune response
Two epitopes were significantly recognized by using solid-phase mapping in the first samples positive for nRNP C autoantibodies. Antibody binding is shown as the difference between the mean of the nRNP-positive samples and the mean of the negative controls in terms of standard deviations of the negative control mean (n=6). Epitopes bound by antibodies from the first positive sera are highlighted (A) and described (B). (C) Inhibition of antibody binding by incubation with peptides corresponding to the dominant epitopes is shown. Significant inhibition was seen after incubation with peptides representing aa 117-125 (mean 46.1% p=0.034) and with a mixture of peptides representing both epitopes (mean 40.3% p=0.034). (D) The mature anti-nRNP C antibody binding pattern in sera collected a mean 2.5 years after the initial development of nRNP C antibodies is shown (n=6). Shaded portions indicate areas of antibody binding in the initial nRNP-positive samples for comparison.
Figure 4
Figure 4. Epitopes in nRNP 70K humoral immunity
The nRNP 70K epitopes bound by antibodies in the first nRNP 70K-positive and late nRNP 70K-positive samples are shown in terms of the number of standard deviations above the negative control mean absorbance (n=3) (A). Epitopes exhibiting an increase in absorbance of at least 3 standard deviations were listed in (B). Decapeptides that were significantly bound by autoantibodies from sera collected 2 years after the initial development of nRNP 70K antibodies are shown in (C) (n=3).
Figure 5
Figure 5. Inhibition of binding to nRNP A and C by an Sm B′-derived peptide
Sera with the first observable anti-nRNP A or -nRNP C autoantibodies were incubated with the Sm B′-derived peptide PPPGMRPP or the negative control peptide poly-proline. Inhibition of binding to recombinant proteins was quantified by using Western blotting. PPPGMRPP significantly inhibited serum antibody binding to recombinant nRNP A (n=9, p=0.0068) (A) and recombinant nRNP C (n=6, p=0.0087) (B). Representative Western blots are shown for each protein. Shown is the mean (SE) of three independent experiments for each sample.

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