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Pathogenicity of autoantibodies in anti-p200 pemphigoid.

Vafia K, Groth S, Beckmann T, Hirose M, Dworschak J, Recke A, Ludwig RJ, Hashimoto T, Zillikens D, Schmidt E - PLoS ONE (2012)

Bottom Line: In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG.Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions.In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ.

View Article: PubMed Central - PubMed

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

ABSTRACT
Recently, the C-terminus of laminin γ1 has been identified as target antigen in anti-p200 pemphigoid and the disease was renamed as anti-laminin γ1 pemphigoid. However, the pathogenic relevance of these autoantibodies has not yet been demonstrated. Therefore, we employed an ex vivo model of autoantibody-mediated leukocyte-dependent neutrophil activation and dermal-epidermal separation (DES) using cryosections of human skin. We showed that anti-p200 pemphigoid sera (n = 7) induced DES in a time-dependent manner, in contrast to sera from healthy controls. Furthermore, laminin γ1-specific IgG and serum depleted from anti-laminin γ1 reactivity were generated using the recombinant C-terminus of laminin γ1 (LAMC1-term; amino acids 1364 to 1609). Interestingly, both fractions labeled the dermal-epidermal-junction (DEJ) by indirect immunofluorescence microscopy on human foreskin and recognized a 200 kDa protein by immunoblotting with dermal extract. Human and rabbit IgG against LAMC1-cterm failed to attract neutrophils at the DEJ and to induce DES. In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG. Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions. In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ. We conclude that autoantibodies in anti-p200 pemphigoid sera are pathogenic while pathogenicity is not mediated by autoantibodies against laminin γ1. Further studies are needed to identify the pathogenically relevant autoantigen in anti-p200 pemphigoid.

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Passive transfer of rabbit anti-mLAMC1-cterm IgG into adult mice is not pathogenic.Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into adult C57BL/6 and BALB/c mice. Injection of 15 mg rabbit anti-mLAMC1-cterm IgG every second day for 10 days did not result in clinical or histopathological (a, e) lesions on day 12. Linear deposition of rabbit IgG at the dermal-epidermal junction (DEJ) was only observed in 2 of 5 C57BL/6 (b) and one of 5 BALB/c mice (f), while staining of murine C3 was negative in all mice (c, g). At day 12, in sera of all 10 mice, rabbit IgG labeled the basal keratinocytes at the DEJ of normal mouse skin (d, h) and reacted with recombinant mLAMC1-cterm by ELISA (i) and immunoblotting (j, C57BL/6, lanes 1; BALB/c, lane 2) and the 200 kDa p200 protein in extract of murine dermis (k, C57BL/6 lane 1; BALB/c, lane 2). Normal mouse sera (j and k, C57BL/6, lane 3; BALB/c, lane 4) were used as controls.
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pone-0041769-g006: Passive transfer of rabbit anti-mLAMC1-cterm IgG into adult mice is not pathogenic.Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into adult C57BL/6 and BALB/c mice. Injection of 15 mg rabbit anti-mLAMC1-cterm IgG every second day for 10 days did not result in clinical or histopathological (a, e) lesions on day 12. Linear deposition of rabbit IgG at the dermal-epidermal junction (DEJ) was only observed in 2 of 5 C57BL/6 (b) and one of 5 BALB/c mice (f), while staining of murine C3 was negative in all mice (c, g). At day 12, in sera of all 10 mice, rabbit IgG labeled the basal keratinocytes at the DEJ of normal mouse skin (d, h) and reacted with recombinant mLAMC1-cterm by ELISA (i) and immunoblotting (j, C57BL/6, lanes 1; BALB/c, lane 2) and the 200 kDa p200 protein in extract of murine dermis (k, C57BL/6 lane 1; BALB/c, lane 2). Normal mouse sera (j and k, C57BL/6, lane 3; BALB/c, lane 4) were used as controls.

Mentions: C57BL/6 (n = 5) and BALB/c (n = 5) adult mice were injected, every second day, for 12 days, with 15 mg of rabbit anti-mLAMC1-cterm IgG. None of the mice treated showed blisters clinically or histopathologically (Fig. 6a, e). IF microscopy of perilesional mouse skin revealed weak, linear, deposits of rabbit IgG in 2 C57BL/6 and one BALB/c mice (Fig. 6b, f), while murine complement C3 at the DEJ was negative in all cases (Fig. 6c, g). All mice showed serum autoantibodies against the DEJ with titers ranging from 1∶30 to 1.40 by indirect IF microscopy (Fig. 6d, h), to p200 by immunoblotting (titre 1∶105) (Fig. 6k), and to mLAMC1-cterm (titre 1∶105) by ELISA and immunoblotting (Fig. 6 i, j).


Pathogenicity of autoantibodies in anti-p200 pemphigoid.

Vafia K, Groth S, Beckmann T, Hirose M, Dworschak J, Recke A, Ludwig RJ, Hashimoto T, Zillikens D, Schmidt E - PLoS ONE (2012)

Passive transfer of rabbit anti-mLAMC1-cterm IgG into adult mice is not pathogenic.Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into adult C57BL/6 and BALB/c mice. Injection of 15 mg rabbit anti-mLAMC1-cterm IgG every second day for 10 days did not result in clinical or histopathological (a, e) lesions on day 12. Linear deposition of rabbit IgG at the dermal-epidermal junction (DEJ) was only observed in 2 of 5 C57BL/6 (b) and one of 5 BALB/c mice (f), while staining of murine C3 was negative in all mice (c, g). At day 12, in sera of all 10 mice, rabbit IgG labeled the basal keratinocytes at the DEJ of normal mouse skin (d, h) and reacted with recombinant mLAMC1-cterm by ELISA (i) and immunoblotting (j, C57BL/6, lanes 1; BALB/c, lane 2) and the 200 kDa p200 protein in extract of murine dermis (k, C57BL/6 lane 1; BALB/c, lane 2). Normal mouse sera (j and k, C57BL/6, lane 3; BALB/c, lane 4) were used as controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404064&req=5

pone-0041769-g006: Passive transfer of rabbit anti-mLAMC1-cterm IgG into adult mice is not pathogenic.Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into adult C57BL/6 and BALB/c mice. Injection of 15 mg rabbit anti-mLAMC1-cterm IgG every second day for 10 days did not result in clinical or histopathological (a, e) lesions on day 12. Linear deposition of rabbit IgG at the dermal-epidermal junction (DEJ) was only observed in 2 of 5 C57BL/6 (b) and one of 5 BALB/c mice (f), while staining of murine C3 was negative in all mice (c, g). At day 12, in sera of all 10 mice, rabbit IgG labeled the basal keratinocytes at the DEJ of normal mouse skin (d, h) and reacted with recombinant mLAMC1-cterm by ELISA (i) and immunoblotting (j, C57BL/6, lanes 1; BALB/c, lane 2) and the 200 kDa p200 protein in extract of murine dermis (k, C57BL/6 lane 1; BALB/c, lane 2). Normal mouse sera (j and k, C57BL/6, lane 3; BALB/c, lane 4) were used as controls.
Mentions: C57BL/6 (n = 5) and BALB/c (n = 5) adult mice were injected, every second day, for 12 days, with 15 mg of rabbit anti-mLAMC1-cterm IgG. None of the mice treated showed blisters clinically or histopathologically (Fig. 6a, e). IF microscopy of perilesional mouse skin revealed weak, linear, deposits of rabbit IgG in 2 C57BL/6 and one BALB/c mice (Fig. 6b, f), while murine complement C3 at the DEJ was negative in all cases (Fig. 6c, g). All mice showed serum autoantibodies against the DEJ with titers ranging from 1∶30 to 1.40 by indirect IF microscopy (Fig. 6d, h), to p200 by immunoblotting (titre 1∶105) (Fig. 6k), and to mLAMC1-cterm (titre 1∶105) by ELISA and immunoblotting (Fig. 6 i, j).

Bottom Line: In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG.Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions.In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ.

View Article: PubMed Central - PubMed

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

ABSTRACT
Recently, the C-terminus of laminin γ1 has been identified as target antigen in anti-p200 pemphigoid and the disease was renamed as anti-laminin γ1 pemphigoid. However, the pathogenic relevance of these autoantibodies has not yet been demonstrated. Therefore, we employed an ex vivo model of autoantibody-mediated leukocyte-dependent neutrophil activation and dermal-epidermal separation (DES) using cryosections of human skin. We showed that anti-p200 pemphigoid sera (n = 7) induced DES in a time-dependent manner, in contrast to sera from healthy controls. Furthermore, laminin γ1-specific IgG and serum depleted from anti-laminin γ1 reactivity were generated using the recombinant C-terminus of laminin γ1 (LAMC1-term; amino acids 1364 to 1609). Interestingly, both fractions labeled the dermal-epidermal-junction (DEJ) by indirect immunofluorescence microscopy on human foreskin and recognized a 200 kDa protein by immunoblotting with dermal extract. Human and rabbit IgG against LAMC1-cterm failed to attract neutrophils at the DEJ and to induce DES. In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG. Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions. In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ. We conclude that autoantibodies in anti-p200 pemphigoid sera are pathogenic while pathogenicity is not mediated by autoantibodies against laminin γ1. Further studies are needed to identify the pathogenically relevant autoantigen in anti-p200 pemphigoid.

Show MeSH
Related in: MedlinePlus