The Pathophysiological Significance of Nondesmoglein Targets of Pemphigus Autoimmunity

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115

A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on keratinocytes (KCs) activating the signaling kinases and executioner caspases that damage KCs, causing their shrinkage, detachment from neighboring cells, and rounding up (apoptolysis). In this study, we found that PV antibody binding leads to activation of epidermal growth factor receptor kinase, Src, p38 MAPK, and JNK in KCs with time pattern variations from patient to patient. Both extrinsic and intrinsic apoptotic pathways were also activated. Although Fas ligand neutralizing antibody could inhibit the former pathway, the mechanism of activation of the latter remained unknown. PV antibodies increased cytochrome c release, suggesting damage to mitochondria. The immunoblotting experiments revealed penetration of PVIgG into the subcellular mitochondrial fraction. The antimitochondrial antibodies from different PV patients recognized distinct combinations of antigens with apparent molecular sizes of 25, 30, 35, 57, 60, and 100 kDa. Antimitochondrial antibodies were pathogenic because their absorption abolished the ability of PVIgG to cause keratinocyte detachment both in vitro and in vivo. The downstream signaling of antimitochondrial antibodies involved JNK and late p38 MAPK activation, whereas the signaling of anti-desmoglein 3 (Dsg3) antibody involved JNK and biphasic p38 MAPK activation. Using KCs grown from Dsg3؊/؊ mice, we determined that Dsg3 did not serve as a surrogate antigen allowing antimitochondrial antibodies to enter KCs. The PVIgG-induced activation of epidermal growth factor receptor and Src was affected neither in Dsg3KCs nor due to absorption of antimitochondrial antibodies. These results demonstrated that apoptolysis in PV is a complex process initiated by at least three classes of autoantibodies directed against desmosomal, mitochondrial, and other keratinocyte self-antigens. These autoantibodies synergize with the proapoptotic serum and tissue factors to trigger both extrinsic and intrinsic pathways of cell death and break the epidermal cohesion, leading to blisters. Further elucidation of the primary signaling events downstream of PV autoantigens will be crucial for the development of a more successful therapy for PV patients.Pemphigus vulgaris (PV) 3 is an IgG autoantibody-mediated disease of skin and mucosa caused by a loss of epidermal cohesion and manifested by progressive blistering and non-healing erosions. The mechanism of detachment of keratinocytes (KCs) in PV, termed acantholysis, is a subject of intensive research. Elucidation of the pathophysiologic mechanism of acantholysis should facilitate development of novel pharmacologic approaches to prevent and treat blistering without systemic corticosteroids, a mainstay of treatment of PV patients. Acantholysis can be blocked both by inhibitors of signaling kinases, such as p38 MAPK (3), mammalian target of rapamycin (4), Src, and epidermal growth factor receptor (EGFR) kinase (2, 4 -7) and by other tyrosine kinases, phospholipase C, calmodulin...

Section: Discussionsupporting

confidence: 92%

Antimitochondrial Autoantibodies in Pemphigus Vulgaris

Marchenko

Chernyavsky

Arredondo

et al. 2010

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115

“…MP-Passive transfer of PV IgG to neonatal Balb/c mice caused gross and microscopic changes consistent with experimental pemphigus in vivo, as reported elsewhere (3,57,58). We first determined the dose of PV IgG appropriate for testing the clinical efficacy of MP.…”

Section: Development Of In Vivo Model For Testing Clinical Efficacy Ofmentioning

confidence: 76%

“…All IgG fractions were isolated using 40% ammonium sulfate followed by dialysis against phosphate-buffered saline (PBS; Sigma), lyophilized, weighed, and reconstituted in PBS, as detailed elsewhere (57,58). This study had been approved by the University of California, Davis, Human Subjects Review Committee.…”

Section: Pemphigus and Control Igg Fractions-mentioning

confidence: 99%

“…Semi-quantitative IF Assays-The PV IgG deposits in the epidermis of experimental and control mice were visualized by incubating cryostat section of mouse skin for 1 h at room temperature with fluorescein isothiocyanate-conjugated (FITC) donkey anti-human IgG antibody (Pierce), as detailed elsewhere (58). To visualize adhesion molecules in KC treated with MP, the second passage normal human foreskin KC were grown to confluence in 4-chamber microscope slides (Lab-Tek Chamber Slide System, Nalge Nunc International Corp., Naperville, IL) in KGM containing 0.09 mM Ca 2ϩ (Invitrogen), fed with fresh medium (control) or medium containing 0.25 mM MP (experiment), and incubated in a humid 5% CO 2 incubator overnight.…”

Section: Pemphigus and Control Igg Fractions-mentioning

confidence: 99%

See 1 more Smart Citation

Pemphigus Vulgaris IgG and Methylprednisolone Exhibit Reciprocal Effects on Keratinocytes

Nguyen

Arredondo

Chernyavsky

et al. 2004

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“…The pathophysiology of PV includes an array of IgG autoantibodies reacting with keratinocyte self-antigens with the apparent molecular mass ranging from 12 to 190 kDa (reviewed in Ref. 5), including a 130-kDa keratinocyte polypeptide (6,7). The notion that autoantibodies are the main cause of PV stems from the fact that passive transfer of pemphigus, but not normal, IgGs to neonatal mice can induce skin lesions characteristic of PV (8).…”

mentioning

confidence: 99%

Pemphigus Vulgaris Antibody Identifies Pemphaxin

Nguyen

Ndoye

Grando

2000

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119

Because pemphigus vulgaris (PV) IgGs adsorbed on the rDsg3-Ig-His baculoprotein induced blisters in neonatal mice, it was proposed that anti-desmoglein 3 (Dsg 3) autoantibody causes PV. However, we found that rDsg3-Ig-His absorbs autoantibodies to different antigens, including a non-Dsg 3 keratinocyte protein of 130 kDa. This prompted our search for novel targets of PV autoimmunity. The PV IgG eluted from a 75-kDa keratinocyte protein band both stained epidermis in a pemphigus-like pattern and induced acantholysis in keratinocyte monolayers. Screening of a keratinocyte gt11 cDNA library with this antibody identified clones carrying cDNA inserts encoding a novel molecule exhibiting ϳ40% similarity with annexin-2, named pemphaxin (PX). Recombinant PX (rPX-His) was produced in Escherichia coli M15 cells, and, because annexins can act as cholinergic receptors, its conformation was tested in a cholinergic radioligand binding assay. rPX-His specifically bound [3 H]acetylcholine, suggesting that PX is one of the keratinocyte cholinergic receptors known to be targeted by disease-causing PV antibodies. Preabsorption of PV sera with rPX-His eliminated acantholytic activity, and eluted antibody immunoprecipitated native PX. This antibody alone did not cause skin blisters in vivo, but its addition to the preabsorbed PV IgG fraction restored acantholytic activity, indicating that acantholysis in PV results from synergistic action of antibodies to different keratinocyte self-antigens, including both acetylcholine receptors and desmosomal cadherins.