Inhibition of FAK prevents blister formation in the neonatal mouse model of pemphigus vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis and by autoantibodies against desmoglein 3 localized on desmosomes. In addition, caspases also seem to participate in this blistering disease. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in cytoskeleton remodelling and formation and disassembly of cell adhesion structures. We have previously demonstrated that HER (human epidermal growth factor receptor related) isoforms, Src (Rous sarcoma) and mammalian target of rapamycin (mTOR), three molecules implicated in signalling processes, take part in suprabasal acantholysis and apoptosis induced by PV-IgG in a mouse model. Our aim was to investigate whether upregulation of FAK is implicated in the development of PV lesions. Herein, using a mouse model, PV-IgG administration showed an increased level of FAK phosphorylated on 397 and 925 tyrosine residues in the basal layer of epidermis. When mice were pretreated with a FAK inhibitor (FI), the acantholysis of the basal layer of epidermis was absent. More interestingly, we observed that phosphorylated FAK (Y397/925) decreased when HER isoforms, Src, mTOR and pan-caspases inhibitors were employed before PV-IgG administration. In addition, pretreatment with the FI before PV-IgG injection prevented the changes in both Bax and Bcl-2 expression and caspase-9 and caspase-3 activities induced by PV-IgG. Finally, FI reduced the expression of phosphorylated Src and mTOR in the basal cells of epidermis. In conclusion, our data reveal a novel role of phosphorylated FAK (Y397/925) in PV development involving HER isoforms, Src and mTOR kinase

The role of nitric oxide synthases in pemphigus vulgaris in a mouse model

Pemphigus vulgaris (PV) is a blistering autoimmune disease characterized by IgG autoantibodies against desmoglein 3. Nitric oxide synthases (NOS) may contribute to the increase of inflammation in tissues by the generation of nitrotyrosine residues (NTR). OBJECTIVES: To investigate whether the production of NTR mediated by NOS may participate in the development of inflammation and acantholysis in PV. METHODS: Mice were pretreated or not with NOS, tyrosine-kinase (TK) or nuclear factor (NF)-kappaB inhibitors, and then injected with PV-IgG. PV manifestations were examined in all mice. The expression of NTR, constitutive NOS (cNOS) [endothelial NOS (eNOS) and neuronal NOS (nNOS)], inducible NOS (iNOS) and NF-kappaB factor were studied in epidermis of mice using immunohistochemical techniques. RESULTS: After PV-IgG injection, expressions of NTR, iNOS, eNOS and nNOS increased in acantholytic cells, as did nuclear translocation of NF-kappaB in the basal cells of the epidermis. Pretreatment of mice with inhibitors of TK, nNOS and nonselective NOS, completely prevented NTR expression and the clinical and histological findings of PV in mice. TK inhibitor genistein inhibited both nNOS and iNOS expression on the membrane of basal keratinocytes, and nuclear translocation of NF-kappaB. CONCLUSIONS: Upregulation of cNOS and iNOS, NTR generation and nuclear translocation of NF-kappaB may contribute to increased inflammation and tissue damage in PV lesions. The absence of the clinical and histological findings of PV and NTR expression in mice injected with PV-IgG, through pretreatment with TK and nNOS inhibitors, provides compelling evidence that these signalling molecules should be considered as potential therapeutic targets in PV

Immunomodulation induced by synthetic peptides derived from Staphylococcus aureus protein A

Peptides from 10 to 22 amino acids containing sequences encompassed by Staphylococcus aureus protein A were synthesized. Some of these peptides, when present in cultures of lymphomononuclear cells from healthy donors or from cancer patients (melanoma, breast carcinoma, non-Hodgkin lymphoma and renal cell carcinoma) promoted: (i) changes in the phenotype of the lymphomononuclear population, (ii) stimulation of monocytes (release of IL-1 and TNF-alpha), and (iii) an increase in cytotoxicity against K562, Daudi and HT-29 cells. Isolated monocytes responded also to those peptides with a release of IL-1 and TNF alpha and an increase of cytotoxicity against HT-29 cells. It was found that the active peptides had the following structural pattern: a length of at least 15 amino-acid residues with a proline at position 6, valine, leucine, isoleucine, glycine, alanine or lysine at position 2, and glutamic or aspartic acid at position 11. Replacement of Pro at position 6 with any other residue turned the peptide inactive. Replacement of residues at positions 2 and 11 with amino-acid residues other than those required for activity resulted in compounds with a marked decrease in the immunomodulating properties described, or lacking these properties altogether

An imbalance in Akt/mTOR is involved in the apoptotic and acantholytic processes in a mouse model of pemphigus vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by the presence of IgG autoantibodies against Dsg3. Our aim was to investigate the molecular events implicated in the development and localization of apoptosis and acantholysis in PV. We used a passive transfer mouse model together with immunohistochemical (IHC) techniques and the TUNEL assay, with quantification analysis in the basal layer of the epidermis. The activated signalling molecules analysed and apoptotic cells detected showed an identical localization. Herein, we found for the first time in vivo an increased expression of activated HER receptor isoforms in the basal layer in PV lesions. Besides, we observed the almost total lack of activated Akt compared with a higher level of activated mTOR within the basal cells of the epidermis. Our observations strongly support that the restriction of acantholysis to the basal layer may be due, at least in part, to the selective and increased presence of activated HER receptor isoforms in these cells. After phosphorylation of HER receptor isoforms, intracellular signalling pathways are activated in the basal layer. In addition, the imbalance in Akt/mTOR that takes place in the basal cells may provide intracellular signals necessary for the development of apoptosis and acantholysi

Inducible nitric oxide synthase in human lymphomononuclear cells activated by synthetic peptides derived from extracellular matrix proteins.

Synthetic peptides with sequences present in extracellular matrix proteins are capable of causing the expression of the inducible form of nitric oxide synthase (iNOS), detected by immunocytochemistry, and the release of NO by human lymphomononuclear cells incubated in their presence. Active peptides are 15-mers containing a characteristic 2-6-11 motif in which the amino acid residue at position 2 is Leu, Ile, Val, Gly, Ala or Lys; the residue at position 6 is always Pro; and residue 11 is Glu or Asp. The induction of iNOS in human monocytes and macrophages could be involved in the cytotoxicity against tumor cell lines also elicited by these peptides

Cutaneous Biology: In vivo blockade of pemphigus vulgaris acantholysis by inhibition of intracellular signal transduction cascades

Pemphigus vulgaris (PV) is an autoimmune disease characterized by mucocutaneous intraepithelial blisters and pathogenic autoantibodies against desmoglein 3. The mechanism of blister formation in pemphigus has not been defined; however, in vitro data suggest a role for activation of intracellular signalling cascades. OBJECTIVES: To investigate the contribution of these signalling pathways to the mechanism of PV IgG-induced acantholysis in vivo. METHODS: We used the passive transfer mouse model. Mice were injected with IgG fractions of sera from a patient with PV, with or without pretreatment with inhibitors of proteins that mediate intracellular signalling cascades. RESULTS: Inhibitors of tyrosine kinases, phospholipase C, calmodulin and the serine/threonine kinase protein kinase C prevented PV IgG-induced acantholysis in vivo. CONCLUSIONS: These observations strongly support the role of intracellular signalling cascades in the molecular mechanism of PV IgG-induced acantholysi

Papel de las isoformas HER y la mTOR en la acantólisis del pénfigo vulgar en un modelo murino

El pénfigo vulgar (PV) es una enfermedad ampollosa autoinmune que afecta a piel y mucosas, caracterizada por la presencia de autoanticuerpos IgG frente a la desmogleína 3 (Dsg3) que provocan una ruptura intraepitelial de la epidermis llamada acantólisis. El mecanismo por el cual se produce la acantólisis no es del todo conocido. Nuestro objetivo en este trabajo fue investigar los eventos moleculares implicados en el desarrollo y la localización de la apoptosis y la acantólisis en el PV. Parra ello empleamos el modelo murino de tranferencia pasiva con estudios de inmunohistoquímica con un análisis cuantitativo y la técnica TUNEL para el estudio de la apoptosis. Tanto las moléculas de señalización activadas analizadas como las células apoptóticas detectadas mostraron la misma localización. Hemos observado por primera vez in vivo un aumento de la expresión de las isoformas activadas de los receptores HER en la capa basal de la epidermis en las lesiones de PV. La importancia de este hallazgo se ve reforzada por el hecho de que el pretratamiento de los ratones con erlotinib (inhibidor de HER1, HER2 Y HER3) inhibió tanto la enfermedad clínica como histológica. Además, hemos hallado una casi nula expresión de la Akt activada comparada con un nivel más elevado de la mTOR activada dentro de las células basales de la epidermis. Tras el pretratamiento de los animales con rapamicina (inhibidor de mTRO) pudimos observar cómo se anularon las manifestaciones clínicas e histológicas del PV. Nuestras observaciones sugieren que la acntólisis podría estar restringida a la capa basal, al menos en parte, por el aumento selectivo de las isoformas activadas de los receptores HER que ocurre en estas células. Después de la forsforilación de las isoformas de los receptores HER, se activarían vías de señalización intracelular en la capa basal epidérmica. Además, el desequilibrio de Akt/mTOR que tiene lugar en las células de la capa basal podría aportar las señales intracelulares necesarias para el desarrollo de la apoptosis y la acantólisis

El papel de la FAK en la acantólisis del Pénfigo vulgar en un modelo murino

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis, and autoantibodies against desmoglein 3 localized on desmosomes. In addition, caspases also seem to participate in this blistering disease. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in cytoskeleton remodelling and formation and disassembly of cell adhesion structures. We have previously demonstrated that HER (human epidermal growth factor receptor related) isoforms, Src (Rous sarcoma) and mTOR (mammalian target of rapamycin), three molecules implicated in signalling processes, take part in suprabasal acantholysis and apoptosis induced by PV-IgG in a mouse model. Our aim was to investigate if upregulation of FAK is implicated in the development of PV lesions. Herein, using a mouse model, PV-IgG administration showed an increased level of FAK phosphorylated on 397 and 925 tyrosine residues in the basal layer of epidermis. When mice were pretreated with a FAK inhibitor the acantholysis of the basal layer of epidermis was absent. More interestingly, we observed that phosphorylated FAK (Y397/925) decreased when HER isoforms, Src, mTOR, and pan-caspases inhibitors were employed before PV-IgG administration. In addition, pretreatment with the FAK inhibitor before PV-IgG injection avoided the changes of both Bax and Bcl-2 expression and caspases-9 and –3 activities induced by PV-IgG. Finally, FAK inhibitor reduced expression of phosphorylated Src and mTOR in the basal cells of epidermis. In conclusion, our data reveal a novel biochemical mechanism for phosphorylated FAK (Y397/925) in PV development involving HER isoforms, Src and mTOR kinases

El papel de la FAK en la acantólisis del Pénfigo vulgar en un modelo murino

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis, and autoantibodies against desmoglein 3 localized on desmosomes. In addition, caspases also seem to participate in this blistering disease. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in cytoskeleton remodelling and formation and disassembly of cell adhesion structures. We have previously demonstrated that HER (human epidermal growth factor receptor related) isoforms, Src (Rous sarcoma) and mTOR (mammalian target of rapamycin), three molecules implicated in signalling processes, take part in suprabasal acantholysis and apoptosis induced by PV-IgG in a mouse model. Our aim was to investigate if upregulation of FAK is implicated in the development of PV lesions. Herein, using a mouse model, PV-IgG administration showed an increased level of FAK phosphorylated on 397 and 925 tyrosine residues in the basal layer of epidermis. When mice were pretreated with a FAK inhibitor the acantholysis of the basal layer of epidermis was absent. More interestingly, we observed that phosphorylated FAK (Y397/925) decreased when HER isoforms, Src, mTOR, and pan-caspases inhibitors were employed before PV-IgG administration. In addition, pretreatment with the FAK inhibitor before PV-IgG injection avoided the changes of both Bax and Bcl-2 expression and caspases-9 and –3 activities induced by PV-IgG. Finally, FAK inhibitor reduced expression of phosphorylated Src and mTOR in the basal cells of epidermis. In conclusion, our data reveal a novel biochemical mechanism for phosphorylated FAK (Y397/925) in PV development involving HER isoforms, Src and mTOR kinases

Nitric oxide activates granule-associated DNase in human monocytes

Activated and differentiated human monocytes with a CD14+CD16+ phenotype were found to contain a DNase activity associated with secretion granules. Activated cells were obtained from patients with autoimmune diseases. Activation and differentiation of monocytes were also achieved after incubation of PBMC from healthy subjects with protein A (SpA) or immunopotentiating peptides. DNase activity corresponded to a 66-kDa protein, similar to that described in granules from T lymphocytes, active preferentially on double-strand DNA. DNA fragmentation activity increased when NO donors were present; the activity was higher in the presence of Ca2+, and at low pH values. The Ca2+-dependent activity was inhibited by Zn2+. NO-dependent activity was additive with that of Ca2+-dependent and it was not inhibited by Zn2+. Dithiothreitol did not modify the effect of NO on DNase activity. Incubation of PBMC in the presence of NMLA, an inhibitor of NO synthases, decreased this DNase activity. Data reported clearly suggest a regulatory role of NO in granule-associated DNase activit