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. 2020 Jan 8;11(1):120.
doi: 10.1038/s41467-019-13992-8.

IgA Subclasses Have Different Effector Functions Associated With Distinct Glycosylation Profiles

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Free PMC article

IgA Subclasses Have Different Effector Functions Associated With Distinct Glycosylation Profiles

Ulrike Steffen et al. Nat Commun. .
Free PMC article

Abstract

Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. IgA2 activates neutrophils and macrophages more potently than IgA1.
ac Human neutrophils were stimulated with 200 µg/ml of monomeric or heat aggregated (=HAA) IgA1 and IgA2, or with human serum albumin (HSA). Neutrophil extracellular trap (NET) formation was evaluated by staining extracellular DNA with Sytox Green. a NET formation over time. b Relative amount of extracellular DNA at 280 min after stimulation normalized on HSA treatment; n = 5 donors. c Representative images at 280 min after stimulation. Scale bar = 50 µm. df NET formation of human neutrophils with 200 µg/ml HAA1 or HAA2 in the presence of 10 µg/ml blocking antibody against FcαRI (=αCD89) or isotype control (=iso). d NET formation over time. e Relative amount of extracellular DNA at 260 min after stimulation normalized on HSA treatment; n = 4 donors. f Representative images at 35 min after stimulation. Scale bar = 20 µm. g IL-8 released by neutrophils 4 h after stimulation with HSA, HAA1, or HAA2; n = 6 donors. h Cytokines released by macrophages 6 h after seeding in wells coated with HSA, IgA1, or IgA2; n = 4 donors. Significances were tested with paired one-way ANOVA followed by Bonferroni correction for selected pairs of columns b, d or Dunnet’s correction g, h. *p < 0,05; **p < 0,01; and ***p < 0,001. Data are presented as mean ± s.e.m. a, d or scatter blots with bars showing mean ± s.e.m. b, e, g, h. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. IgA2 induces stronger binding and activating signaling to neutrophils than IgA1.
a, b Human neutrophils were stimulated with 100 µg/ml of heat aggregated IgA1 and IgA2 (=HAA1 and HAA2) or with human serum albumin (=HSA) in the presence of inhibitors for Syk (=R406), Erk (=PD98059) or Shp-2 (=SHP099) at the indicated concentrations. Neutrophil extracellular trap (NET) formation was evaluated by staining extracellular DNA with Sytox Green. a NET formation over time and b relative amount of extracellular DNA at 265 min after stimulation normalized on treatment without inhibitors; n = 5–6 donors. c Binding of HAA1 and HAA2 to neutrophils measured by flow cytometry; n = 4 donors. d Representative blots of flow cytometry analysis showing neutrophil gating and fluorescence intensity. FSC, forward scatter; SSC, side scatter. Significances were tested with paired one-way ANOVA followed by Dunnett’s correction b or Bonferroni correction for selected pairs of columns c. *p < 0,05; **p < 0,01; and ***p < 0,001. Data are presented as mean ± s.e.m. a, c or scatter blots with bars showing mean ± s.e.m. b. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. IgA1 and IgA2 are differentially glycosylated.
a Schematic overview of glycosylation sites in IgA1 and IgA2. Bold letters indicate glycosylation sites present in both IgA subclasses. b Representative lectin blots of IgA1 and IgA2 isolated from sera of healthy donors using antibodies against IgA or lectins against the core structure of N-glycans (=lens culinaris agglutinin), terminal α2,6-linked sialic acid (=sambuccus nigra agglutinin), and terminal galactose (=erythrina christagalli lectin). c Quantification of the lectin blots; n = 6 donors. df Mass spectrometric quantification of sialyation, galactosylation, bisection, fucosylation, and the presence of noncomplex structures for the glycosylation sites N144/N131 d, N340/N327 e, N41 f, and N205 g; n = 12 donors. Significances were tested with paired two-sided Student’s t-test cg or paired one-way ANOVA followed by Bonferroni correction for selected pairs of columns dg. *p < 0,05; **p < 0,01; and ***p < 0,001. Data are presented as scatter plots with mean ± s.e.m. c or box plots with medians and inter-quartile ranges + whiskers ranging from min to max dg. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Removal of sialic acid or all N-glycans increases pro-inflammatory activity of IgA1.
a Human neutrophils were stimulated with 100 µg/ml of human serum albumin (=HSA) and native or desialylated (=-ds) heat aggregated IgA1 and IgA2 (=HAA1 and HAA2). NET formation was evaluated by staining extracellular DNA with Sytox Green. Shown is NET formation over time and the relative amount of extracellular DNA at 280 min after stimulation normalized on HSA treatment; n = 6 donors. b IL-8 released by macrophages 6 h after seeding in wells coated with HSA, IgA1, desialylated IgA1 (=IgA1-ds), IgA2 or desialylated IgA2 (=IgA2-ds); n = 7 donors. c Human neutrophils were stimulated with 100 µg/ml of human serum albumin (=HSA) and native or deglycosylated (=-dg) heat aggregated IgA1 and IgA2 (=HAA1 and HAA2); n = 5 donors. d IL-8 released by macrophages 6 h after seeding in wells coated with HSA, IgA1, deglycosylated IgA1 (=IgA1-dg) or IgA2; n = 6 donors. e Binding of heat aggregated IgA1 and IgA2 (=HAA1 and HAA2) that has been desialylated (=-ds) or deglycosylated (=-dg) on neutrophils measured by flow cytometry; n = 7 donors. Significances were tested with paired one-way ANOVA followed by Bonferroni correction for selected pairs of columns a, c, e or Dunnet’s correction b, d. *p < 0,05; **p < 0,01; and ***p < 0,001. Data are presented as mean ± s.e.m. a, c or scatter blots with bars showing mean ± s.e.m. ae. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. A high IgA2 percentage in ACPA correlates with higher RA disease activity.
a Amount of serum IgA, IgA1, and IgA2 as well as IgA2 percentage in patients with RA (n = 48) and healthy controls (n = 32). b IgA2 percentage of total IgA compared to ACPA in the serum of ACPA-positive RA patients (n = 38). c Correlation of the IgA1 and IgA2 amount, and IgA2 percentage in total serum IgA and IgA-ACPA with the disease activity score (DAS) 28 score of ACPA positive RA patients (n = 38). Significances were tested with two-sided Student’s t-test using Welch’s correction a, paired two-sided Student’s t-test b, or Pearson c. *p < 0,05; **p < 0,01; and ***p < 0,001. Data are presented as scatter plots with mean ± s.e.m. Source data are provided as a Source Data file.

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