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IgG4 autoantibodies induce dermal-epidermal separation.

Mihai S, Chiriac MT, Herrero-González JE, Goodall M, Jefferis R, Savage CO, Zillikens D, Sitaru C - J. Cell. Mol. Med. (2007 Sep-Oct)

Bottom Line: In these diseases, IgG1 and IgG3 isotypes, but not the IgG4 subclass, are thought to trigger inflammatory pathways resulting in tissue damage.Purified IgG4 did not fix complement, but, interestingly, like IgG1, activated leucocytes and induced dermal-epidermal separation.The potential of IgG4 autoantibodies to induce Fc-dependent dermal-epidermal separation was significantly lower compared to IgG1.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Lübeck, Lübeck, Germany.

ABSTRACT
Bullous pemphigoid (BP) is a sub-epidermal autoimmune blistering disease associated with autoantibodies to the dermal-epidermal junction (DEJ). Patients' autoantibodies induce dermal-epidermal separation when co-incubated with cryosections of human skin and leucocytes from healthy volunteers. IgG autoantibodies trigger complement and/or leucocyte activation resulting in specific pathology in several autoimmune conditions. In these diseases, IgG1 and IgG3 isotypes, but not the IgG4 subclass, are thought to trigger inflammatory pathways resulting in tissue damage. The capacity of IgG4 autoantibodies to mediate tissue damage has not yet been demonstrated. In this study, we isolated IgG1 and IgG4 autoantibodies from bullous pemhigoid patients'serum and analysed their blister-inducing potential in our cryosection assay. As expected, complement-fixing IgG1 autoantibodies induced sub-epidermal splits in this experimental model. Purified IgG4 did not fix complement, but, interestingly, like IgG1, activated leucocytes and induced dermal-epidermal separation. The potential of IgG4 autoantibodies to induce Fc-dependent dermal-epidermal separation was significantly lower compared to IgG1. Our results demonstrate that IgG4 autoantibodies are able to activate leucocytes and point to a hitherto less recognized function of IgG4. Moreover, for the first time, we clearly demonstrate that BP IgG4 autoantibodies have the capacity to induce leucocyte-dependent tissue damage.

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A blocking monoclonal antibody against CD16 significantly inhibits the autoantibody-induced dermal–epidermal separation. Cryo-sections of human skin were incubated with IgG, IgG1 and IgG4 purified from BP patients 1 and 2 (n = 4 sections/antibody preparation). Subsequently, granulocytes were treated for 10 min at room temperature with 3G8 or a control antibody prior to incubation with skin sections. Dermal–epidermal separation is represented as means ± SEM of the percent of the total length of the dermal–epidermal junction (DEJ) for each section.
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fig05: A blocking monoclonal antibody against CD16 significantly inhibits the autoantibody-induced dermal–epidermal separation. Cryo-sections of human skin were incubated with IgG, IgG1 and IgG4 purified from BP patients 1 and 2 (n = 4 sections/antibody preparation). Subsequently, granulocytes were treated for 10 min at room temperature with 3G8 or a control antibody prior to incubation with skin sections. Dermal–epidermal separation is represented as means ± SEM of the percent of the total length of the dermal–epidermal junction (DEJ) for each section.

Mentions: When incubated with the cryosections of human skin in the presence of leucocytes purified from healthy donors, both serum (n = 6) (Fig. 4A) and IgG1 autoantibodies (n = 3) (Fig. 4B) from BP patients induced dermal–epidermal separation. Importantly, subsequent addition of leucocytes to cryosections previously treated with non-complement fixing IgG4 autoantibodies (n = 5) also resulted in dermal–epidermal separation (Fig. 4C). Sub-epidermal splits were not observed in frozen skin sections incubated with serum samples from healthy controls (Fig. 4D). Fc RIII (CD16) is constitutively expressed in a high-copy number on neutrophils and was shown to mainly mediate both antibody-induced blistering in a mouse model of BP [46] and autoantibody-induced leucocyte attachment to the basement membrane in the cryosection model [47]. Therefore, to investigate the Fc-dependent leucocyte activation in our model, we blocked the binding of IgG autoantibodies to CD16 on granulocytes using the mAb 3G8. Cryosections of human skin were incubated with IgG, IgG1 and IgG4 purified from two BP patients (n = 4 sections/antibody preparation). Subsequently, granulocytes were treated for 10 min at room temperature with 3G8 or a control antibody prior to incubation with skin sections. The mAb 3G8, but not the control antibody, caused a 86.6, 85 and 85.4% inhibition of dermal–epidermal separation induced by IgG, IgG1 and IgG4, respectively (Fig. 5).


IgG4 autoantibodies induce dermal-epidermal separation.

Mihai S, Chiriac MT, Herrero-González JE, Goodall M, Jefferis R, Savage CO, Zillikens D, Sitaru C - J. Cell. Mol. Med. (2007 Sep-Oct)

A blocking monoclonal antibody against CD16 significantly inhibits the autoantibody-induced dermal–epidermal separation. Cryo-sections of human skin were incubated with IgG, IgG1 and IgG4 purified from BP patients 1 and 2 (n = 4 sections/antibody preparation). Subsequently, granulocytes were treated for 10 min at room temperature with 3G8 or a control antibody prior to incubation with skin sections. Dermal–epidermal separation is represented as means ± SEM of the percent of the total length of the dermal–epidermal junction (DEJ) for each section.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401274&req=5

fig05: A blocking monoclonal antibody against CD16 significantly inhibits the autoantibody-induced dermal–epidermal separation. Cryo-sections of human skin were incubated with IgG, IgG1 and IgG4 purified from BP patients 1 and 2 (n = 4 sections/antibody preparation). Subsequently, granulocytes were treated for 10 min at room temperature with 3G8 or a control antibody prior to incubation with skin sections. Dermal–epidermal separation is represented as means ± SEM of the percent of the total length of the dermal–epidermal junction (DEJ) for each section.
Mentions: When incubated with the cryosections of human skin in the presence of leucocytes purified from healthy donors, both serum (n = 6) (Fig. 4A) and IgG1 autoantibodies (n = 3) (Fig. 4B) from BP patients induced dermal–epidermal separation. Importantly, subsequent addition of leucocytes to cryosections previously treated with non-complement fixing IgG4 autoantibodies (n = 5) also resulted in dermal–epidermal separation (Fig. 4C). Sub-epidermal splits were not observed in frozen skin sections incubated with serum samples from healthy controls (Fig. 4D). Fc RIII (CD16) is constitutively expressed in a high-copy number on neutrophils and was shown to mainly mediate both antibody-induced blistering in a mouse model of BP [46] and autoantibody-induced leucocyte attachment to the basement membrane in the cryosection model [47]. Therefore, to investigate the Fc-dependent leucocyte activation in our model, we blocked the binding of IgG autoantibodies to CD16 on granulocytes using the mAb 3G8. Cryosections of human skin were incubated with IgG, IgG1 and IgG4 purified from two BP patients (n = 4 sections/antibody preparation). Subsequently, granulocytes were treated for 10 min at room temperature with 3G8 or a control antibody prior to incubation with skin sections. The mAb 3G8, but not the control antibody, caused a 86.6, 85 and 85.4% inhibition of dermal–epidermal separation induced by IgG, IgG1 and IgG4, respectively (Fig. 5).

Bottom Line: In these diseases, IgG1 and IgG3 isotypes, but not the IgG4 subclass, are thought to trigger inflammatory pathways resulting in tissue damage.Purified IgG4 did not fix complement, but, interestingly, like IgG1, activated leucocytes and induced dermal-epidermal separation.The potential of IgG4 autoantibodies to induce Fc-dependent dermal-epidermal separation was significantly lower compared to IgG1.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Lübeck, Lübeck, Germany.

ABSTRACT
Bullous pemphigoid (BP) is a sub-epidermal autoimmune blistering disease associated with autoantibodies to the dermal-epidermal junction (DEJ). Patients' autoantibodies induce dermal-epidermal separation when co-incubated with cryosections of human skin and leucocytes from healthy volunteers. IgG autoantibodies trigger complement and/or leucocyte activation resulting in specific pathology in several autoimmune conditions. In these diseases, IgG1 and IgG3 isotypes, but not the IgG4 subclass, are thought to trigger inflammatory pathways resulting in tissue damage. The capacity of IgG4 autoantibodies to mediate tissue damage has not yet been demonstrated. In this study, we isolated IgG1 and IgG4 autoantibodies from bullous pemhigoid patients'serum and analysed their blister-inducing potential in our cryosection assay. As expected, complement-fixing IgG1 autoantibodies induced sub-epidermal splits in this experimental model. Purified IgG4 did not fix complement, but, interestingly, like IgG1, activated leucocytes and induced dermal-epidermal separation. The potential of IgG4 autoantibodies to induce Fc-dependent dermal-epidermal separation was significantly lower compared to IgG1. Our results demonstrate that IgG4 autoantibodies are able to activate leucocytes and point to a hitherto less recognized function of IgG4. Moreover, for the first time, we clearly demonstrate that BP IgG4 autoantibodies have the capacity to induce leucocyte-dependent tissue damage.

Show MeSH
Related in: MedlinePlus