Allergy Asthma Immunol Res.  2012 Nov;4(6):332-340. 10.4168/aair.2012.4.6.332.

Most Highly Cytokinergic IgEs Have Polyreactivity to Autoantigens

• Kashiwakura Ji
• Okayama Y
• Furue M
• Kabashima
• Shimada S
• Ra C
• Siraganian RP
• Kawakami Y
• Kawakami T

• KMID: 2167018
• DOI: http://doi.org/10.4168/aair.2012.4.6.332

Abstract

PURPOSE
Monomeric IgE molecules, when bound to the high-affinity receptor, exhibit a vast heterogeneity in their ability to induce survival promotion and cytokine production in mast cells. At one end of this spectrum, highly cytokinergic (HC) IgEs can induce potent survival promotion, degranulation, cytokine production, migration, etc., whereas at the other end, poorly cytokinergic (PC) IgEs can do so inefficiently. In this study, we investigated whether IgEs recognize autoantigens and whether IgEs' binding of autoantigens correlates with difference s in HC versus PC properties.
METHODS
Enzyme-linked immunosorbent assays were performed to test whether IgEs bind antigens. Histamine-releasing factor in human sera was quantified by western blotting. Cultured mast cells derived from human cord blood were used to test the effects of human sera on cytokine production.
RESULTS
Most (7/8) of mouse monoclonal HC IgEs exhibited polyreactivity to double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), beta-galactosidase, thyroglobulin and/or histamine-releasing factor. By contrast, mouse PC IgEs failed to react with these antigens. A human monoclonal HC IgE also showed polyreactivity to histamine-releasing factor, dsDNA and ssDNA. Interestingly, sera from atopic dermatitis patients showed increased reactivity to ssDNA and beta-galactosidase and increased levels of histamine-releasing factor. Some atopic dermatitis patients, but not healthy individuals, had substantial serum levels of HRF-reactive IgE. Sera from atopic dermatitis patients with high titers of DNA-reactive IgE could induce several fold more IL-8 secretion in human mast cells than sera from healthy individuals.
CONCLUSIONS
The results show that most HC, but not PC, IgEs exhibit polyreactivity to autoantigens, supporting the autoimmune mechanism in the pathogenesis of atopic dermatitis.

Keyword

Atopic dermatitis; mast cell; cytokine; IgE; autoantigen; histamine-releasing factor

MeSH Terms

Animals
Autoantigens
beta-Galactosidase
Blotting, Western
Dermatitis, Atopic
DNA
DNA, Single-Stranded
Enzyme-Linked Immunosorbent Assay
Fetal Blood
Humans
Immunoglobulin E
Interleukin-8
Mast Cells
Mice
Population Characteristics
Thyroglobulin
Autoantigens
DNA
DNA, Single-Stranded
Immunoglobulin E
Interleukin-8
Thyroglobulin
beta-Galactosidase

Figure

• 

  Fig. 1 Mouse HC, but not PC, IgEs react with multiple antigens. ELISAs were performed on the indicated mouse IgE mAbs using wells coated with β-galactosidase, thyroglobulin, HRF, dsDNA, ssDNA, insulin or LPS. Bound IgE was detected by biotinylated anti-mouse IgE mAb, followed by streptavidin-HRP and color development. Arbitrary cutoff values for protein ligands and DNA ligands were 0.1 and 0.01, respectively. A representative result was shown from two or three independent experiments.

• 

  Fig. 2 A human IgE preparation can induce IL-8 and IL-6 production and promote survival in human mast cells. (A-C) Human CBMCs were incubated with 0 (PBS, [-]), 0.5 (white bar), or 5 (gray or black bar) µg/mL of the indicated human IgEs for 2 hr. (A,C) IL-8, IL-6, and GAPDH mRNA levels were measured by quantitative RT-PCR. The amount of cytokine mRNA was normalized against GAPDH mRNA and the relative value in PBS-treated cells was set at 1. (B) IL-8 protein was measured by ELISA. *P<0.05 vs PBS control by Student's t-test. (D) Human CBMCs (1×105 cells) were incubated with 0 (PBS), 0.5, or 5 µg/mL of the indicated human IgEs in the absence of SCF and IL-6 for 72 hr. As controls, CBMCs were pre-incubated with 1 µg/mL of PS IgE in the presence of IL-4 for 24 hr, and washed cells were incubated with 0 or 1 µg/mL anti-IgE for 72 hr. Trypan blue excluding live cell numbers were counted. *P<0.05 vs control (no IgE sensitization/no IgE stimulation) by Student's t-test.

• 

  Fig. 3 A human HC IgE HE1 reacts with dsDNA, ssDNA and HRF. ELISAs were performed on the indicated human IgE samples, as shown in Fig. 1 except that anti-human IgE was used in place of anti-mouse IgE.

• 

  Fig. 4 AD patients have higher serum levels of ssDNA- or β-galactosidase-reactive IgE and HRF. ELISAs were performed on human sera using wells coated with ssDNA (A), dsDNA (B), β-galactosidase (C) and HRF (E). Sera were taken from cohorts of AD patients and healthy individuals. Note that low OD450 values, compared to those in Fig. 3, were obtained with sera diluted tenfold. (D) Serum HRF levels were quantified by immunoblotting. (A-D) n=27, AD patients; n=23, healthy subjects; (E) n=34, AD patients; n=25, healthy subjects. (F) Human CBMCs were pretreated with 10 ng/mL IL-4 for 24 hr and stimulated with sera (5% or 10%) from normal subjects (n=2) or AD patients (n=8) for 24 hr. Supernatants were collected and IL-8 was quantified by ELISA.

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