Limits...
Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis.

Iannaccone A, Giorgianni F, New DD, Hollingsworth TJ, Umfress A, Alhatem AH, Neeli I, Lenchik NI, Jennings BJ, Calzada JI, Satterfield S, Mathews D, Diaz RI, Harris T, Johnson KC, Charles S, Kritchevsky SB, Gerling IC, Beranova-Giorgianni S, Radic MZ, Health ABC stu - PLoS ONE (2015)

Bottom Line: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the targets recognized by circulating AAbs seen on 2D-GE, followed by ELISAs with recombinant proteins to confirm LC-MS/MS results, and quantify autoreactivities.Following immunoprecipitation, 2D-GE and LC-MS/MS, five of the possible autoreactivity targets were conclusively identified: two members of the heat shock protein 70 (HSP70) family, HSPA8 and HSPA9; another member of the HSP family, HSPB4, also known as alpha-crystallin A chain (CRYAA); Annexin A5 (ANXA5); and Protein S100-A9, also known as calgranulin B that, when complexed with S100A8, forms calprotectin.ELISA testing with recombinant proteins confirmed, on average, significantly higher reactivities against all targets in AMD samples compared to controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.

ABSTRACT

Background: We investigated sera from elderly subjects with and without age-related macular degeneration (AMD) for presence of autoantibodies (AAbs) against human macular antigens and characterized their identity.

Methods: Sera were collected from participants in the Age-Related Maculopathy Ancillary (ARMA) Study, a cross-sectional investigation ancillary to the Health ABC Study, enriched with participants from the general population. The resulting sample (mean age: 79.2±3.9 years old) included subjects with early to advanced AMD (n = 131) and controls (n = 231). Sera were tested by Western blots for immunoreactive bands against human donor macular tissue homogenates. Immunoreactive bands were identified and graded, and odds ratios (OR) calculated. Based on these findings, sera were immunoprecipitated, and subjected to 2D gel electrophoresis (GE). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the targets recognized by circulating AAbs seen on 2D-GE, followed by ELISAs with recombinant proteins to confirm LC-MS/MS results, and quantify autoreactivities.

Results: In AMD, 11 immunoreactive bands were significantly more frequent and 13 were significantly stronger than in controls. Nine of the more frequent bands also showed stronger reactivity. OR estimates ranged between 4.06 and 1.93, and all clearly excluded the value. Following immunoprecipitation, 2D-GE and LC-MS/MS, five of the possible autoreactivity targets were conclusively identified: two members of the heat shock protein 70 (HSP70) family, HSPA8 and HSPA9; another member of the HSP family, HSPB4, also known as alpha-crystallin A chain (CRYAA); Annexin A5 (ANXA5); and Protein S100-A9, also known as calgranulin B that, when complexed with S100A8, forms calprotectin. ELISA testing with recombinant proteins confirmed, on average, significantly higher reactivities against all targets in AMD samples compared to controls.

Conclusions: Consistent with other evidence supporting the role of inflammation and the immune system in AMD pathogenesis, AAbs were identified in AMD sera, including early-stage disease. Identified targets may be mechanistically linked to AMD pathogenesis because the identified proteins are implicated in autophagy, immunomodulation, and protection from oxidative stress and apoptosis. In particular, a role in autophagy activation is shared by all five autoantigens, raising the possibility that the detected AAbs may play a role in AMD via autophagy compromise and downstream activation of the inflammasome. Thus, we propose that the detected AAbs provide further insight into AMD pathogenesis and have the potential to contribute to disease biogenesis and progression.

Show MeSH

Related in: MedlinePlus

Plot of odds ratios (ORs) and 95% CIs for immunoreactive (IR) bands detected by Western blot.OR data points are ordered, top to bottom, from the highest χ2 statistic (32.46, p = 0.012 x 10−6) to the lowest (7.30, p = 0.0068) meeting the critical B-H value. All IR bands listed were significantly more intense and/or more common in AMD sera.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696815&req=5

pone.0145323.g001: Plot of odds ratios (ORs) and 95% CIs for immunoreactive (IR) bands detected by Western blot.OR data points are ordered, top to bottom, from the highest χ2 statistic (32.46, p = 0.012 x 10−6) to the lowest (7.30, p = 0.0068) meeting the critical B-H value. All IR bands listed were significantly more intense and/or more common in AMD sera.

Mentions: AMD sera recognized a greater number of IR intervals of human macular antigens and exhibited stronger autoreactivity compared to control sera. More frequent IR was observed in AMD for the dataset as a whole (p = 0.02 x 10−8) and, after applying the B-H multiple test correction method to our data set, specifically for 11 distinct 2-kDa IR intervals. The more frequent IR bands clustered primarily in two areas, between 26 and 32 kDa (n = 3), between 36 and 50 kDa (n = 7), and at the 98–100 kDa interval. By the same method, greater IR intensity was observed for 13 levels of comparisons among the observed IR bands. Of these, 9 of 11 bands coincided with the intervals in which IR was also seen more frequently. Some bands were significantly more intense for various cut-off levels of comparison (e.g., the 40–42 kDa band showed greater IR for the <3 vs. ≥3 and <4 vs. ≥4 IR score comparisons). Two comparisons were significant uniquely for frequency of the IR band (38–40 kDa) and for IR intensity (88–90 kDa, <2 vs. ≥2 comparison), respectively. Fig 1 illustrates these results, by presenting point estimates of ORs and 95% CIs for the IR bands whose χ2 values met the B-H critical value, ranked by the latter. The lowest significance level that met the B-H critical value was a χ2 of 7.85, p = 0.0051. All IR intervals meeting the B-H criteria for inclusion were highly significant, with the 95% CIs excluding clearly the (OR = 1.0) value and OR values ranging between 1.93 and 4.56.


Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis.

Iannaccone A, Giorgianni F, New DD, Hollingsworth TJ, Umfress A, Alhatem AH, Neeli I, Lenchik NI, Jennings BJ, Calzada JI, Satterfield S, Mathews D, Diaz RI, Harris T, Johnson KC, Charles S, Kritchevsky SB, Gerling IC, Beranova-Giorgianni S, Radic MZ, Health ABC stu - PLoS ONE (2015)

Plot of odds ratios (ORs) and 95% CIs for immunoreactive (IR) bands detected by Western blot.OR data points are ordered, top to bottom, from the highest χ2 statistic (32.46, p = 0.012 x 10−6) to the lowest (7.30, p = 0.0068) meeting the critical B-H value. All IR bands listed were significantly more intense and/or more common in AMD sera.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696815&req=5

pone.0145323.g001: Plot of odds ratios (ORs) and 95% CIs for immunoreactive (IR) bands detected by Western blot.OR data points are ordered, top to bottom, from the highest χ2 statistic (32.46, p = 0.012 x 10−6) to the lowest (7.30, p = 0.0068) meeting the critical B-H value. All IR bands listed were significantly more intense and/or more common in AMD sera.
Mentions: AMD sera recognized a greater number of IR intervals of human macular antigens and exhibited stronger autoreactivity compared to control sera. More frequent IR was observed in AMD for the dataset as a whole (p = 0.02 x 10−8) and, after applying the B-H multiple test correction method to our data set, specifically for 11 distinct 2-kDa IR intervals. The more frequent IR bands clustered primarily in two areas, between 26 and 32 kDa (n = 3), between 36 and 50 kDa (n = 7), and at the 98–100 kDa interval. By the same method, greater IR intensity was observed for 13 levels of comparisons among the observed IR bands. Of these, 9 of 11 bands coincided with the intervals in which IR was also seen more frequently. Some bands were significantly more intense for various cut-off levels of comparison (e.g., the 40–42 kDa band showed greater IR for the <3 vs. ≥3 and <4 vs. ≥4 IR score comparisons). Two comparisons were significant uniquely for frequency of the IR band (38–40 kDa) and for IR intensity (88–90 kDa, <2 vs. ≥2 comparison), respectively. Fig 1 illustrates these results, by presenting point estimates of ORs and 95% CIs for the IR bands whose χ2 values met the B-H critical value, ranked by the latter. The lowest significance level that met the B-H critical value was a χ2 of 7.85, p = 0.0051. All IR intervals meeting the B-H criteria for inclusion were highly significant, with the 95% CIs excluding clearly the (OR = 1.0) value and OR values ranging between 1.93 and 4.56.

Bottom Line: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the targets recognized by circulating AAbs seen on 2D-GE, followed by ELISAs with recombinant proteins to confirm LC-MS/MS results, and quantify autoreactivities.Following immunoprecipitation, 2D-GE and LC-MS/MS, five of the possible autoreactivity targets were conclusively identified: two members of the heat shock protein 70 (HSP70) family, HSPA8 and HSPA9; another member of the HSP family, HSPB4, also known as alpha-crystallin A chain (CRYAA); Annexin A5 (ANXA5); and Protein S100-A9, also known as calgranulin B that, when complexed with S100A8, forms calprotectin.ELISA testing with recombinant proteins confirmed, on average, significantly higher reactivities against all targets in AMD samples compared to controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.

ABSTRACT

Background: We investigated sera from elderly subjects with and without age-related macular degeneration (AMD) for presence of autoantibodies (AAbs) against human macular antigens and characterized their identity.

Methods: Sera were collected from participants in the Age-Related Maculopathy Ancillary (ARMA) Study, a cross-sectional investigation ancillary to the Health ABC Study, enriched with participants from the general population. The resulting sample (mean age: 79.2±3.9 years old) included subjects with early to advanced AMD (n = 131) and controls (n = 231). Sera were tested by Western blots for immunoreactive bands against human donor macular tissue homogenates. Immunoreactive bands were identified and graded, and odds ratios (OR) calculated. Based on these findings, sera were immunoprecipitated, and subjected to 2D gel electrophoresis (GE). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the targets recognized by circulating AAbs seen on 2D-GE, followed by ELISAs with recombinant proteins to confirm LC-MS/MS results, and quantify autoreactivities.

Results: In AMD, 11 immunoreactive bands were significantly more frequent and 13 were significantly stronger than in controls. Nine of the more frequent bands also showed stronger reactivity. OR estimates ranged between 4.06 and 1.93, and all clearly excluded the value. Following immunoprecipitation, 2D-GE and LC-MS/MS, five of the possible autoreactivity targets were conclusively identified: two members of the heat shock protein 70 (HSP70) family, HSPA8 and HSPA9; another member of the HSP family, HSPB4, also known as alpha-crystallin A chain (CRYAA); Annexin A5 (ANXA5); and Protein S100-A9, also known as calgranulin B that, when complexed with S100A8, forms calprotectin. ELISA testing with recombinant proteins confirmed, on average, significantly higher reactivities against all targets in AMD samples compared to controls.

Conclusions: Consistent with other evidence supporting the role of inflammation and the immune system in AMD pathogenesis, AAbs were identified in AMD sera, including early-stage disease. Identified targets may be mechanistically linked to AMD pathogenesis because the identified proteins are implicated in autophagy, immunomodulation, and protection from oxidative stress and apoptosis. In particular, a role in autophagy activation is shared by all five autoantigens, raising the possibility that the detected AAbs may play a role in AMD via autophagy compromise and downstream activation of the inflammasome. Thus, we propose that the detected AAbs provide further insight into AMD pathogenesis and have the potential to contribute to disease biogenesis and progression.

Show MeSH
Related in: MedlinePlus