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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 in renal sections from RRP8-injected or TNP1-injected mice.Cryostat kidney sections from mice were analyzed by double immunofluorescence staining with a combination of anti-human RRP8 or anti-human TNP1 antibody and anti-mouse IgG antibody. The specimens were stained with Alexa 568-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with Alexa 488-conjugated anti-murine IgG antibody (green). (A) Photomicrograph of representative glomeruli of RRP8- and TNP1-injected mice (periodic acid-Schiff staining). Both manifested the slight enlargement of glomeruli with thickening of capillary wall (arrows) as well as the endocapillary proliferation with leukocytes (arrow head). (B) RRP8 signals coexisted with IgG signals in the sub-endothelial (arrows) and sub-epithelial (arrowhead) regions, exhibiting a granular pattern. On the other hand, TNP1 signals coexisted with IgG signals in the sub-epithelial region (arrows).
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pone.0126564.g007: Double immunofluorescence of RRP8 or TNP1 and IgG in renal sections from RRP8-injected or TNP1-injected mice.Cryostat kidney sections from mice were analyzed by double immunofluorescence staining with a combination of anti-human RRP8 or anti-human TNP1 antibody and anti-mouse IgG antibody. The specimens were stained with Alexa 568-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with Alexa 488-conjugated anti-murine IgG antibody (green). (A) Photomicrograph of representative glomeruli of RRP8- and TNP1-injected mice (periodic acid-Schiff staining). Both manifested the slight enlargement of glomeruli with thickening of capillary wall (arrows) as well as the endocapillary proliferation with leukocytes (arrow head). (B) RRP8 signals coexisted with IgG signals in the sub-endothelial (arrows) and sub-epithelial (arrowhead) regions, exhibiting a granular pattern. On the other hand, TNP1 signals coexisted with IgG signals in the sub-epithelial region (arrows).

Mentions: Next, we examined whether IC would be formed by injection of human RRP8 or TNP1 into C57BL/6 mice and whether glomerulonephritis would occur predominantly when IC was formed with RRP8 or TNP1. C57BL/6 mice were injected repeatedly with recombinant human RRP8 or TNP1 protein. C57BL/6 mice are not prone to autoimmune disease, but after 7–9 injections with RRP8 or TNP1, they demonstrated proteinuria (Fig 6A). In the sera of these injected mice, a high titer of anti-RRP8 or anti-TNP1 antibody was evident, but anti-dsDNA antibodies were negative (Fig 6B–6D). This indicates that RRP8 and TNP1 do not cross-react with anti-dsDNA antibodies. In kidneys, some glomeruli of RRP8-injected mice showed a slight enlargement of glomeruli with thickening of capillary wall and endocapillary proliferation with infiltrating leukocytes as same as those of TNP1-injected mice (Fig 7A). IC deposits in cryostat kidney sections were analyzed by double immunofluorescence using a combination of anti-RRP8 or anti-TNP1 antibody and anti-IgG antibody. As shown in Fig 7B, in RRP8-injected mice, co-presence of RRP8 and IgG was evident in sub-endothelial and sub-epithelial regions with a coarse granular pattern. In TNP1-injected mice, co-presence of TNP1 and IgG was also demonstrated as a granular pattern in the sub-endothelial regions of glomeruli.


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 in renal sections from RRP8-injected or TNP1-injected mice.Cryostat kidney sections from mice were analyzed by double immunofluorescence staining with a combination of anti-human RRP8 or anti-human TNP1 antibody and anti-mouse IgG antibody. The specimens were stained with Alexa 568-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with Alexa 488-conjugated anti-murine IgG antibody (green). (A) Photomicrograph of representative glomeruli of RRP8- and TNP1-injected mice (periodic acid-Schiff staining). Both manifested the slight enlargement of glomeruli with thickening of capillary wall (arrows) as well as the endocapillary proliferation with leukocytes (arrow head). (B) RRP8 signals coexisted with IgG signals in the sub-endothelial (arrows) and sub-epithelial (arrowhead) regions, exhibiting a granular pattern. On the other hand, TNP1 signals coexisted with IgG signals in the sub-epithelial region (arrows).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476694&req=5

pone.0126564.g007: Double immunofluorescence of RRP8 or TNP1 and IgG in renal sections from RRP8-injected or TNP1-injected mice.Cryostat kidney sections from mice were analyzed by double immunofluorescence staining with a combination of anti-human RRP8 or anti-human TNP1 antibody and anti-mouse IgG antibody. The specimens were stained with Alexa 568-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with Alexa 488-conjugated anti-murine IgG antibody (green). (A) Photomicrograph of representative glomeruli of RRP8- and TNP1-injected mice (periodic acid-Schiff staining). Both manifested the slight enlargement of glomeruli with thickening of capillary wall (arrows) as well as the endocapillary proliferation with leukocytes (arrow head). (B) RRP8 signals coexisted with IgG signals in the sub-endothelial (arrows) and sub-epithelial (arrowhead) regions, exhibiting a granular pattern. On the other hand, TNP1 signals coexisted with IgG signals in the sub-epithelial region (arrows).
Mentions: Next, we examined whether IC would be formed by injection of human RRP8 or TNP1 into C57BL/6 mice and whether glomerulonephritis would occur predominantly when IC was formed with RRP8 or TNP1. C57BL/6 mice were injected repeatedly with recombinant human RRP8 or TNP1 protein. C57BL/6 mice are not prone to autoimmune disease, but after 7–9 injections with RRP8 or TNP1, they demonstrated proteinuria (Fig 6A). In the sera of these injected mice, a high titer of anti-RRP8 or anti-TNP1 antibody was evident, but anti-dsDNA antibodies were negative (Fig 6B–6D). This indicates that RRP8 and TNP1 do not cross-react with anti-dsDNA antibodies. In kidneys, some glomeruli of RRP8-injected mice showed a slight enlargement of glomeruli with thickening of capillary wall and endocapillary proliferation with infiltrating leukocytes as same as those of TNP1-injected mice (Fig 7A). IC deposits in cryostat kidney sections were analyzed by double immunofluorescence using a combination of anti-RRP8 or anti-TNP1 antibody and anti-IgG antibody. As shown in Fig 7B, in RRP8-injected mice, co-presence of RRP8 and IgG was evident in sub-endothelial and sub-epithelial regions with a coarse granular pattern. In TNP1-injected mice, co-presence of TNP1 and IgG was also demonstrated as a granular pattern in the sub-endothelial regions of glomeruli.

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