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Novel Autoantigens Associated with Lupus Nephritis.

Onishi S, Adnan E, Ishizaki J, Miyazaki T, Tanaka Y, Matsumoto T, Suemori K, Shudou M, Okura T, Takeda H, Sawasaki T, Yasukawa M, Hasegawa H - PLoS ONE (2015)

Bottom Line: To obtain other autoantibodies associated with LN, we screened autoantigens reacting with the sera of LN patients by using an N-terminal biotinylated protein library created from a wheat cell-free protein production system.Both proteins are cationic, and their respective antibodies did not cross-react with dsDNA.These autoantibodies may be useful for prediction of LN in subsets of SLE patients who are negative for anti-dsDNA antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Ehime, Japan.

ABSTRACT
Systemic lupus erythematosus (SLE) is characterized by production of a variety of autoantibodies. Although anti-double-stranded DNA (anti-dsDNA) antibodies contribute to the pathogenesis of lupus nephritis (LN), they are not sufficient for diagnosis and evaluation of disease activity. To obtain other autoantibodies associated with LN, we screened autoantigens reacting with the sera of LN patients by using an N-terminal biotinylated protein library created from a wheat cell-free protein production system. We screened 17 proteins that showed higher positive signals in the active phase than in the inactive phase of SLE, and higher positive signals in the serum of SLE patient with nephritis than in that of patient without nephritis. Of these, two LN-associated autoantigens, ribosomal RNA-processing protein 8 (RRP8) and spermatid nuclear transition protein 1 (TNP1) were identified by immunoprecipitation and immunofluorescence of renal tissues. Circulating anti-RRP8 and anti-TNP1 autoantibodies were recognized and deposited as an immune complex (IC) in glomeruli. IC was deposited preferentially in glomeruli rather than in other organs in C57BL/6 mice injected with RRP8 or TNP1. ELISA analysis of sera from patients with various rheumatic diseases demonstrated reactivity for RRP8 and TNP1 in 20% and 14.7% of SLE patients, respectively, whereas there was little or no reactivity in patients with other rheumatic diseases. Among SLE patients, 63.6% and 45.5% of those with LN were positive for anti-RRP8 and anti-TNP1 antibodies, compared with 12.5% and 9.4% of SLE patients without nephritis, respectively. Both proteins are cationic, and their respective antibodies did not cross-react with dsDNA. These proteins released from apoptotic cells form ICs with each autoantibody, and their ICs may become trapped at anionic sites in the glomerular basement membrane, leading to deposition in glomeruli. These autoantibodies may be useful for prediction of LN in subsets of SLE patients who are negative for anti-dsDNA antibodies.

No MeSH data available.


Related in: MedlinePlus

Double immunofluorescence of RRP8 or TNP1 and IgG or C3 in renal sections from patients with LN.Formalin-fixed, paraffin-embedded sections of biopsy or autopsy specimens from LN patients were processed for double immunofluorescence staining. Representative photographs are shown. The specimens were stained with Alexa 546-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with FITC-conjugated anti-IgG or anti-C3 antibodies (green). (A) The same glomeruli were stained with periodic acid-Schiff. (B) Co-presence of RRP8 and C3 was detected along the sub-epithelial area of the glomeruli showing a dotted pattern (arrowhead) in LN1. Also in Autopsy1, both RRP8 and IgG signals were detected in the mesangial area as well as in the sub-epithelial area (arrowhead). (C) Co-presence of TNP1 and IgG was revealed along the basement membrane showing a linear pattern in LN2, as was the case for TNP1 and C3 in Autopsy2. The bold and strong autofluorescence in each of the panels is mainly due to red blood cells.
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pone.0126564.g002: Double immunofluorescence of RRP8 or TNP1 and IgG or C3 in renal sections from patients with LN.Formalin-fixed, paraffin-embedded sections of biopsy or autopsy specimens from LN patients were processed for double immunofluorescence staining. Representative photographs are shown. The specimens were stained with Alexa 546-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with FITC-conjugated anti-IgG or anti-C3 antibodies (green). (A) The same glomeruli were stained with periodic acid-Schiff. (B) Co-presence of RRP8 and C3 was detected along the sub-epithelial area of the glomeruli showing a dotted pattern (arrowhead) in LN1. Also in Autopsy1, both RRP8 and IgG signals were detected in the mesangial area as well as in the sub-epithelial area (arrowhead). (C) Co-presence of TNP1 and IgG was revealed along the basement membrane showing a linear pattern in LN2, as was the case for TNP1 and C3 in Autopsy2. The bold and strong autofluorescence in each of the panels is mainly due to red blood cells.

Mentions: To reveal whether anti-RRP8 and anti-TNP1 antibodies are deposited in glomeruli of LN patients, double immunofluorescence was performed using a combination of anti-RRP8 or anti-TNP1 antibodies and anti-IgG or anti-C3 antibodies. Kidney sections obtained by renal biopsy and autopsy from LN patients were analyzed in this study. Paraffin sections, and not frozen sections, were used as no frozen sections had been stocked. Therefore, nonspecific fluorescence due to red blood cells was evident (S1 Fig). As shown in Fig 2, in LN1 and Autopsy1, coexistence of RRP8 and IgG or C3 was detected in the sub-epithelial area of the glomeruli, showing a dotted pattern, and in the mesangial and sub-epithelial areas, respectively. On the other hand, in LN2 and Autopsy2, coexistence of TNP1 and IgG or C3 was revealed along the basement membrane, exhibiting a linear pattern. Anti-RRP8 antibody was deposited in glomeruli of 3 LN patients, while anti-TNP1 antibody was deposited in glomeruli of 3 LN patients (Table 2). These findings confirmed that anti-RRP8 and anti-TNP1 autoantibodies were deposited as IC in the glomeruli of some LN patients.


Novel Autoantigens Associated with Lupus Nephritis.

Onishi S, Adnan E, Ishizaki J, Miyazaki T, Tanaka Y, Matsumoto T, Suemori K, Shudou M, Okura T, Takeda H, Sawasaki T, Yasukawa M, Hasegawa H - PLoS ONE (2015)

Double immunofluorescence of RRP8 or TNP1 and IgG or C3 in renal sections from patients with LN.Formalin-fixed, paraffin-embedded sections of biopsy or autopsy specimens from LN patients were processed for double immunofluorescence staining. Representative photographs are shown. The specimens were stained with Alexa 546-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with FITC-conjugated anti-IgG or anti-C3 antibodies (green). (A) The same glomeruli were stained with periodic acid-Schiff. (B) Co-presence of RRP8 and C3 was detected along the sub-epithelial area of the glomeruli showing a dotted pattern (arrowhead) in LN1. Also in Autopsy1, both RRP8 and IgG signals were detected in the mesangial area as well as in the sub-epithelial area (arrowhead). (C) Co-presence of TNP1 and IgG was revealed along the basement membrane showing a linear pattern in LN2, as was the case for TNP1 and C3 in Autopsy2. The bold and strong autofluorescence in each of the panels is mainly due to red blood cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476694&req=5

pone.0126564.g002: Double immunofluorescence of RRP8 or TNP1 and IgG or C3 in renal sections from patients with LN.Formalin-fixed, paraffin-embedded sections of biopsy or autopsy specimens from LN patients were processed for double immunofluorescence staining. Representative photographs are shown. The specimens were stained with Alexa 546-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with FITC-conjugated anti-IgG or anti-C3 antibodies (green). (A) The same glomeruli were stained with periodic acid-Schiff. (B) Co-presence of RRP8 and C3 was detected along the sub-epithelial area of the glomeruli showing a dotted pattern (arrowhead) in LN1. Also in Autopsy1, both RRP8 and IgG signals were detected in the mesangial area as well as in the sub-epithelial area (arrowhead). (C) Co-presence of TNP1 and IgG was revealed along the basement membrane showing a linear pattern in LN2, as was the case for TNP1 and C3 in Autopsy2. The bold and strong autofluorescence in each of the panels is mainly due to red blood cells.
Mentions: To reveal whether anti-RRP8 and anti-TNP1 antibodies are deposited in glomeruli of LN patients, double immunofluorescence was performed using a combination of anti-RRP8 or anti-TNP1 antibodies and anti-IgG or anti-C3 antibodies. Kidney sections obtained by renal biopsy and autopsy from LN patients were analyzed in this study. Paraffin sections, and not frozen sections, were used as no frozen sections had been stocked. Therefore, nonspecific fluorescence due to red blood cells was evident (S1 Fig). As shown in Fig 2, in LN1 and Autopsy1, coexistence of RRP8 and IgG or C3 was detected in the sub-epithelial area of the glomeruli, showing a dotted pattern, and in the mesangial and sub-epithelial areas, respectively. On the other hand, in LN2 and Autopsy2, coexistence of TNP1 and IgG or C3 was revealed along the basement membrane, exhibiting a linear pattern. Anti-RRP8 antibody was deposited in glomeruli of 3 LN patients, while anti-TNP1 antibody was deposited in glomeruli of 3 LN patients (Table 2). These findings confirmed that anti-RRP8 and anti-TNP1 autoantibodies were deposited as IC in the glomeruli of some LN patients.

Bottom Line: To obtain other autoantibodies associated with LN, we screened autoantigens reacting with the sera of LN patients by using an N-terminal biotinylated protein library created from a wheat cell-free protein production system.Both proteins are cationic, and their respective antibodies did not cross-react with dsDNA.These autoantibodies may be useful for prediction of LN in subsets of SLE patients who are negative for anti-dsDNA antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Ehime, Japan.

ABSTRACT
Systemic lupus erythematosus (SLE) is characterized by production of a variety of autoantibodies. Although anti-double-stranded DNA (anti-dsDNA) antibodies contribute to the pathogenesis of lupus nephritis (LN), they are not sufficient for diagnosis and evaluation of disease activity. To obtain other autoantibodies associated with LN, we screened autoantigens reacting with the sera of LN patients by using an N-terminal biotinylated protein library created from a wheat cell-free protein production system. We screened 17 proteins that showed higher positive signals in the active phase than in the inactive phase of SLE, and higher positive signals in the serum of SLE patient with nephritis than in that of patient without nephritis. Of these, two LN-associated autoantigens, ribosomal RNA-processing protein 8 (RRP8) and spermatid nuclear transition protein 1 (TNP1) were identified by immunoprecipitation and immunofluorescence of renal tissues. Circulating anti-RRP8 and anti-TNP1 autoantibodies were recognized and deposited as an immune complex (IC) in glomeruli. IC was deposited preferentially in glomeruli rather than in other organs in C57BL/6 mice injected with RRP8 or TNP1. ELISA analysis of sera from patients with various rheumatic diseases demonstrated reactivity for RRP8 and TNP1 in 20% and 14.7% of SLE patients, respectively, whereas there was little or no reactivity in patients with other rheumatic diseases. Among SLE patients, 63.6% and 45.5% of those with LN were positive for anti-RRP8 and anti-TNP1 antibodies, compared with 12.5% and 9.4% of SLE patients without nephritis, respectively. Both proteins are cationic, and their respective antibodies did not cross-react with dsDNA. These proteins released from apoptotic cells form ICs with each autoantibody, and their ICs may become trapped at anionic sites in the glomerular basement membrane, leading to deposition in glomeruli. These autoantibodies may be useful for prediction of LN in subsets of SLE patients who are negative for anti-dsDNA antibodies.

No MeSH data available.


Related in: MedlinePlus