If Pemphigus Vulgaris IgG are the Cause of Acantholysis, New IgG-Independent Mechanisms are the Concause NICOLA CIRILLO, 1,2,3 * MICHELE LANZA, 4 FELICE FEMIANO, 2 GIOVANNI MARIA GAETA, 1 ALFREDO DE ROSA, 2 FERNANDO GOMBOS, 1,2 AND ALESSANDRO LANZA 1,2 1 Regional Center on Craniofacial Malformations-MRI, 1st School of Medicine and Surgery, II University of Naples, Naples, Italy 2 Department of Odontostomatology, 1st School of Medicine and Surgery, II University of Naples, Naples, Italy 3 Department of Experimental Medicine, 1st School of Medicine and Surgery, II University of Naples, Naples, Italy 4 Centro Grandi Apparecchiature, 1st School of Medicine and Surgery, II University of Naples, Naples, Italy Pemphigus vulgaris (PV) is a disease of epidermal adhesion. Its pathogenesis is currently traced back to the action of autoantibodies against antigens located within the intercellular substance of keratinocytes, such as desmogleins and acetylcholine receptors. In the present paper, we sought to elucidate the non-IgG-mediated effects of PV sera on keratinocytes. Results showed that PV sera depleted of IgG were able to induce well-defined changes on keratinocyte morphology and metabolic activity. Indeed, PV IgG-free sera determined marked alterations on cell shape, accompanied by partial loss of keratinocyte–keratinocyte interactions within 48 h after treatment. Furthermore, PV IgG-depleted sera caused a sharp reduction of cell viability along with a less sustained weakening of intercellular adhesion strength. In light of the above findings, loss of cell–cell adhesion in PV occurs as a result of the cooperating action of both IgG and non-IgG-mediated mechanisms. These data have remarkable consequences on experimental models of PV and might open new ‘‘biological’’ approaches to its therapy. Thus, researchers are well advised that PV pathophysiology cannot be faithfully reproduced by leaving non-IgG serum factors out of consideration. J. Cell. Physiol. 212: 563–567, 2007. ß 2007 Wiley-Liss, Inc. Pemphigus vulgaris (PV) is a chronic autoimmune disease targeting skin and mucous membranes. It is characterized clinically by aphlegmasic flaccid blisters and erosions (Lanza et al., 2006a). PV lesions develop as a consequence of the loss of cell–cell adhesion among keratinocytes leading to the formation of intraepithelial cleft, or acantholysis. Although a critical role for autoantibodies in PV seems undeniable, however, in recent years PV pathogenesis has been revisiting, mostly because it has become clear that induction of cell–cell detachment is an active process that appears to be more complex than the simple interaction of antibodies with adhesion molecules (Caldelari et al., 2001; Waschke et al., 2005). Furthermore, autoimmunity in pemphigus seems to be not just restricted to desmogleins; indeed, compelling evidence now attests to the role of IgG against keratinocyte cholinergic receptors in the disruption of cell–cell contacts leading keratinocytes to separate from one another (Nguyen et al., 2000a,b). Recent studies have provided evidence that pemphigus acantholysis is related to a series of cytokines, such as IL-1a, TNF-a (Feliciani et al., 2000), IL-6 (Lopez-Robles et al., 2001), IL-10 (Bhol et al., 2000), FasL (Puviani et al., 2003). Although it is not clear whether cytokines play a pathogenic action, yet they could be critical in regulating the delicate equilibrium ensuring the maintenance of cell adhesion. To investigate the role of serum factors other than IgG in PV pathogenesis, we examined the effects of IgG-depleted sera on keratinocyte monolayers. The results obtained in the present study raised intriguing perspectives, as they represent the first demonstration that PV sera can exert relevant effects on keratinocyte shape and metabolic activity in absence of autoantibodies. Materials and Methods Serum collection, preparation of IgG-free sera and IgG purification Serum samples were collected from four patients with active PV (named PV1-4) and three healthy donors (named C1-3), following well-established diagnostic criteria (Lanza et al., 2006b). Serum samples were incubated with appropriate amounts of Protein A-Sepharose microbeads (Sigma, St. Louis, MO) for 2 h, followed by centrifugation at 10,000g. The supernatant was transferred to a new tube and stored at 808C as IgG-free serum. Bound IgG were released from the beads with glycine 0.1 M and pH was rapidly adjusted by adding large volumes of TBS. IgG were then lyophilized and stored at 808C. Alternatively, sera were filtered with 50-kDa cut-off filters (Biomax-50, Millipore Corporation, Billerica, MA). IgG-free sera obtained with the two methods were tested separately in pilot essays and found to exert similar effects. Experiments shown in the present paper were realized by using 50 kDa-filtered PV1 serum. Results were confirmed by probing all sera in at least two independent experiments. *Correspondence to: Nicola Cirillo, Department of Odontostomatology, Second University of Naples, Via Luigi de Crecchio, 7, 80138 Naples, Italy. E-mail: cirillo.sun@libero.it Received 9 February 2007; Accepted 19 March 2007 DOI: 10.1002/jcp.21111 RAPID COMMUNICATION 563 Journal of Journal of Cellular Physiology Cellular Physiology ß 2007 WILEY-LISS, INC.