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Mechanism of the antigen-independent cytokinergic SPE-7 IgE activation of human mast cells in vitro.

Bax HJ, Bowen H, Dodev TS, Sutton BJ, Gould HJ - Sci Rep (2015)

Bottom Line: However, certain monoclonal IgE antibodies have been shown to elicit mast cell activation in an antigen-independent cytokinergic manner, and DNP-specific murine SPE-7 IgE is the most highly cytokinergic antibody known.Using SPE-7 IgE, a non-cytokinergic human IgE and a poorly cytokinergic murine IgE, we reveal that interaction of the Fab region of 'free' SPE-7 IgE with the Fab of FcεRI-bound SPE-7 IgE is the basis of its cytokinergic activity.We rule out involvement of IgE Fc, Cε1 and Cλ/κ domains, and propose that 'free' SPE-7 IgE binds to FcεRI-bound SPE-7 IgE by an Fv-Fv interaction.

View Article: PubMed Central - PubMed

Affiliation: 1] Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom [2] MRC &Asthma UK Centre in Allergic Mechanisms of Asthma, London, United Kingdom.

ABSTRACT
Release of pro-inflammatory mediators by mast cells is a key feature of allergic disease. The 'dogma' is that IgE molecules merely sensitise mast cells by binding FcεRI prior to cross-linking by multivalent allergen, receptor aggregation and mast cell activation. However, certain monoclonal IgE antibodies have been shown to elicit mast cell activation in an antigen-independent cytokinergic manner, and DNP-specific murine SPE-7 IgE is the most highly cytokinergic antibody known. We show that both monovalent hapten and recombinant SPE-7 IgE Fab inhibit its cytokinergic activity as measured by mast cell degranulation and TNF-α release. Using SPE-7 IgE, a non-cytokinergic human IgE and a poorly cytokinergic murine IgE, we reveal that interaction of the Fab region of 'free' SPE-7 IgE with the Fab of FcεRI-bound SPE-7 IgE is the basis of its cytokinergic activity. We rule out involvement of IgE Fc, Cε1 and Cλ/κ domains, and propose that 'free' SPE-7 IgE binds to FcεRI-bound SPE-7 IgE by an Fv-Fv interaction. Initial formation of a tri-molecular complex (one 'free' IgE molecule cross-linking two receptor-bound IgE molecules) leads to capture of further 'free' and receptor-bound IgEs to form larger clusters that trigger mast cell activation.

No MeSH data available.


Related in: MedlinePlus

Human Mast Cells are Activated by a Combination of FcεRI-Bound and ‘Free’ SPE-7 IgE.Percentage degranulation of LAD-2 cells, induced by indicated nM concentration of SPE-7, 102.1F10 and 27–74 IgE antibodies, in non-sensitised cells (A, n = 2–6) and those sensitised with SPE-7 IgE (B, n = 2–5), 27–74 IgE (C, n = 2–3) or 102.1F10 IgE (D, n = 2–6). Statistically significant difference to PBS background control was determined by one-way ANOVA with Dunnett's post-test; *** p < 0.001, ** p = 0.001 to 0.01. Comparison of degranulation in non-sensitised cells or cells pre-sensitised with SPE-7 IgE (E). Statistically significant difference was analysed by one-way ANOVA with Tukey's post-test. All data are shown as mean ± SEM.
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f4: Human Mast Cells are Activated by a Combination of FcεRI-Bound and ‘Free’ SPE-7 IgE.Percentage degranulation of LAD-2 cells, induced by indicated nM concentration of SPE-7, 102.1F10 and 27–74 IgE antibodies, in non-sensitised cells (A, n = 2–6) and those sensitised with SPE-7 IgE (B, n = 2–5), 27–74 IgE (C, n = 2–3) or 102.1F10 IgE (D, n = 2–6). Statistically significant difference to PBS background control was determined by one-way ANOVA with Dunnett's post-test; *** p < 0.001, ** p = 0.001 to 0.01. Comparison of degranulation in non-sensitised cells or cells pre-sensitised with SPE-7 IgE (E). Statistically significant difference was analysed by one-way ANOVA with Tukey's post-test. All data are shown as mean ± SEM.

Mentions: Earlier studies in the rodent system showed that removal of ‘free’ IgE ablates cytokinergic mast cell activation715. We therefore investigated the necessity for SPE-7 IgE to be bound to FcεRI and/or be ‘free’ in solution. LAD-2 human cells that were primed with IL-4 and stem cell factor alone, or in combination with an FcεRI-saturating concentration of SPE-7 IgE below the threshold (6 nM; 1 μg/ml) for cytokinergic activity, were activated by addition of increasing concentrations of SPE-7 IgE (Figures 4A and B). There was no significant difference between the cytokinergic activity elicited by SPE-7 IgE, whether or not cells were pre-sensitised with SPE-7 IgE (Figures 4A, B and E). However, cells that had been sensitised and FcεRI-saturated with either a poorly cytokinergic murine IgE 27–74 (Figure 4C), non-cytokinergic human IgE 102.1F10 (Figure 4D), or human IgE Fcε2–4 (constant-region fragment, data not shown), were not activated upon addition of SPE-7 IgE, even at 30 nM (5 μg/ml). The non-cytokinergic and poorly cytokinergic nature of 102.1F10 and 27–74 IgE antibodies, respectively, were demonstrated in non-sensitised LAD-2 human mast cells, primed with IL-4 and stem cell factor alone, and in all sensitisation conditions (Figures 4A–D). These studies indicate that SPE-7 IgE must bind FcεRI and also be ‘free’ in solution, and that either of these IgEs alone is insufficient for cytokinergic activity. The data also show that ‘free’ SPE-7 IgE does not act by directly cross-linking non-sensitised FcεRI, as observed for anti-FcεRI IgG antibodies262728. If this were the case mast cell activation would be observed regardless of the IgE used to saturate the receptor.


Mechanism of the antigen-independent cytokinergic SPE-7 IgE activation of human mast cells in vitro.

Bax HJ, Bowen H, Dodev TS, Sutton BJ, Gould HJ - Sci Rep (2015)

Human Mast Cells are Activated by a Combination of FcεRI-Bound and ‘Free’ SPE-7 IgE.Percentage degranulation of LAD-2 cells, induced by indicated nM concentration of SPE-7, 102.1F10 and 27–74 IgE antibodies, in non-sensitised cells (A, n = 2–6) and those sensitised with SPE-7 IgE (B, n = 2–5), 27–74 IgE (C, n = 2–3) or 102.1F10 IgE (D, n = 2–6). Statistically significant difference to PBS background control was determined by one-way ANOVA with Dunnett's post-test; *** p < 0.001, ** p = 0.001 to 0.01. Comparison of degranulation in non-sensitised cells or cells pre-sensitised with SPE-7 IgE (E). Statistically significant difference was analysed by one-way ANOVA with Tukey's post-test. All data are shown as mean ± SEM.
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getmorefigures.php?uid=PMC4402612&req=5

f4: Human Mast Cells are Activated by a Combination of FcεRI-Bound and ‘Free’ SPE-7 IgE.Percentage degranulation of LAD-2 cells, induced by indicated nM concentration of SPE-7, 102.1F10 and 27–74 IgE antibodies, in non-sensitised cells (A, n = 2–6) and those sensitised with SPE-7 IgE (B, n = 2–5), 27–74 IgE (C, n = 2–3) or 102.1F10 IgE (D, n = 2–6). Statistically significant difference to PBS background control was determined by one-way ANOVA with Dunnett's post-test; *** p < 0.001, ** p = 0.001 to 0.01. Comparison of degranulation in non-sensitised cells or cells pre-sensitised with SPE-7 IgE (E). Statistically significant difference was analysed by one-way ANOVA with Tukey's post-test. All data are shown as mean ± SEM.
Mentions: Earlier studies in the rodent system showed that removal of ‘free’ IgE ablates cytokinergic mast cell activation715. We therefore investigated the necessity for SPE-7 IgE to be bound to FcεRI and/or be ‘free’ in solution. LAD-2 human cells that were primed with IL-4 and stem cell factor alone, or in combination with an FcεRI-saturating concentration of SPE-7 IgE below the threshold (6 nM; 1 μg/ml) for cytokinergic activity, were activated by addition of increasing concentrations of SPE-7 IgE (Figures 4A and B). There was no significant difference between the cytokinergic activity elicited by SPE-7 IgE, whether or not cells were pre-sensitised with SPE-7 IgE (Figures 4A, B and E). However, cells that had been sensitised and FcεRI-saturated with either a poorly cytokinergic murine IgE 27–74 (Figure 4C), non-cytokinergic human IgE 102.1F10 (Figure 4D), or human IgE Fcε2–4 (constant-region fragment, data not shown), were not activated upon addition of SPE-7 IgE, even at 30 nM (5 μg/ml). The non-cytokinergic and poorly cytokinergic nature of 102.1F10 and 27–74 IgE antibodies, respectively, were demonstrated in non-sensitised LAD-2 human mast cells, primed with IL-4 and stem cell factor alone, and in all sensitisation conditions (Figures 4A–D). These studies indicate that SPE-7 IgE must bind FcεRI and also be ‘free’ in solution, and that either of these IgEs alone is insufficient for cytokinergic activity. The data also show that ‘free’ SPE-7 IgE does not act by directly cross-linking non-sensitised FcεRI, as observed for anti-FcεRI IgG antibodies262728. If this were the case mast cell activation would be observed regardless of the IgE used to saturate the receptor.

Bottom Line: However, certain monoclonal IgE antibodies have been shown to elicit mast cell activation in an antigen-independent cytokinergic manner, and DNP-specific murine SPE-7 IgE is the most highly cytokinergic antibody known.Using SPE-7 IgE, a non-cytokinergic human IgE and a poorly cytokinergic murine IgE, we reveal that interaction of the Fab region of 'free' SPE-7 IgE with the Fab of FcεRI-bound SPE-7 IgE is the basis of its cytokinergic activity.We rule out involvement of IgE Fc, Cε1 and Cλ/κ domains, and propose that 'free' SPE-7 IgE binds to FcεRI-bound SPE-7 IgE by an Fv-Fv interaction.

View Article: PubMed Central - PubMed

Affiliation: 1] Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom [2] MRC &Asthma UK Centre in Allergic Mechanisms of Asthma, London, United Kingdom.

ABSTRACT
Release of pro-inflammatory mediators by mast cells is a key feature of allergic disease. The 'dogma' is that IgE molecules merely sensitise mast cells by binding FcεRI prior to cross-linking by multivalent allergen, receptor aggregation and mast cell activation. However, certain monoclonal IgE antibodies have been shown to elicit mast cell activation in an antigen-independent cytokinergic manner, and DNP-specific murine SPE-7 IgE is the most highly cytokinergic antibody known. We show that both monovalent hapten and recombinant SPE-7 IgE Fab inhibit its cytokinergic activity as measured by mast cell degranulation and TNF-α release. Using SPE-7 IgE, a non-cytokinergic human IgE and a poorly cytokinergic murine IgE, we reveal that interaction of the Fab region of 'free' SPE-7 IgE with the Fab of FcεRI-bound SPE-7 IgE is the basis of its cytokinergic activity. We rule out involvement of IgE Fc, Cε1 and Cλ/κ domains, and propose that 'free' SPE-7 IgE binds to FcεRI-bound SPE-7 IgE by an Fv-Fv interaction. Initial formation of a tri-molecular complex (one 'free' IgE molecule cross-linking two receptor-bound IgE molecules) leads to capture of further 'free' and receptor-bound IgEs to form larger clusters that trigger mast cell activation.

No MeSH data available.


Related in: MedlinePlus