Paola Toto

Immune Modulation in Pemphigus Vulgaris: Role of CD28 and IL-10

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by Abs to the desmosomal cadherin desmoglein-3. Although the autoantibodies have been shown to be pathogenic, the role of the cellular immune system in the pathology of pemphigus-induced acantholysis is unclear. To further delineate the potential role of T cell-signaling pathways in the pathogenesis of PV, we performed passive transfer experiments with PV IgG in gene-targeted mutant mice. Our results demonstrated that CD28-deficient mice (lacking a costimulatory signal for T cell activation) are 5-fold more sensitive to the development of PV than wild-type mice. To evaluate whether the higher incidence of disease was due to an impairment in intercellular adhesion of keratinocytes, we performed an in vitro acantholysis, using CD28−/− mice keratinocytes. No alteration in in vitro adhesion was detected in CD28−/−-type keratinocytes. Because the CD28 molecule plays a pivotal role in the induction of Th2 cytokines, we examined the levels of a prototypic Th2 cytokine (IL-10) in CD28−/− mice. Lower levels of IL-10 mRNA were found in lesions from CD28−/− mice. To determine whether pemphigus susceptibility in CD28−/− was related to IL-10 deficiency, we performed passive transfer experiments in IL-10−/− mice that demonstrated increased blisters compared with controls. To confirm that IL-10 is involved in the pathogenesis, rIL-10 was given with PV IgG. IL-10 significantly suppressed the disease activity. These data suggest a potential role of IL-10 in PV.

Rethinking the role of tumour necrosis factor-alpha in ultraviolet (UV) B-induced immunosuppression: altered immune response in UV-irradiated TNFR1R2 gene-targeted mutant mice.

Background Ultraviolet (UV) B‐induced immunosuppression, implicated in the pathogenesis of skin cancers, is postulated to be mediated in part by cis‐urocanic acid (cis‐UCA) via tumour necrosis factor (TNF)‐α. TNF‐α produces morphological changes in Langerhans cells indistinguishable from those induced by UVB exposure and antibodies against TNF‐α have been demonstrated to inhibit UVB‐induced immunosuppression in vivo.

Role of Th2 cytokines, RANTES and eotaxin in AIDS-associated eosinophilic folliculitis.

The pathogenesis of AIDS-associated eosinophilic folliculitis is still unknown. The expression of chemokines and Th2-type cytokines is increased in other conditions associated with tissue eosinophilia and in allergic reactions. We evaluated the mRNA expression by reverse transcriptase polymerase chain reaction of two Th2 cytokines (interleukin-4 and interleukin-5) and of two chemokines (RANTES and eotaxin) in the skin of 6 patients with AIDS-associated eosinophilic folliculitis; the tissue localization of eotaxin was shown by immunohistochemistry. We demonstrated the increased expression of interleukin-4, interleukin-5, RANTES and eotaxin in lesional skin of the patients compared to normal skin of HIV+ individuals. We concluded that a Th2 pattern is present in AIDS-associated eosinophilic folliculitis. The cytokine milieu in this disease may favour a Th2 immune response to an unknown antigen, whereby RANTES and eotaxin act in synergy with interleukin-4 and interleukin-5 to mediate tissue inflammation.

In vitro C3 mRNA expression in pemphigus vulgaris : Complement activation is increased by IL-1α and TNF-α

Background: Pemphigus vulgaris (PV) is a potentially life-threatening disease, characterized immunohistologically by IgG deposits and complement activation on the surface of keratinocytes. Complement activation has been implicated in the pathogenesis with C3 deposits in about 90% of patients. Objective: In order to further elucidate the role of complement in PV and to define which cytokines play a role in C3 mRNA expression, we performed an in vitro study in human keratinocytes. Methods: Normal human epidermal keratinocytes (NHuK) were incubated with PV serum and C3 mRNA was measured. We previously had shown that IL-1α and TNF-α are expressed in PV in vivo and in vitro. Since cytokines are able to modulate complement activation, mRNA expression was evaluated in a similar experiment after pretreatment using antibodies against IL-1α and TNF-α. Results: Incubation of NHuK with PV sera caused their detachment from the plates after 20–30 minutes with a complete acantholysis within 12 hours. An early C3 mRNA expression was seen after 30 minutes with a peak level after 1 hour. Blocking studies, using antibodies against human IL-1α and TNF-α in NHuK together with PV-IgG, showed reduction of in vitro induced acantholysis and inhibition of C3 mRNA expression. Conclusions: This study supports the hypothesis that complement C3 is important in PV acantholysis and that complement activation is increased by IL-1α and TNF-α.

Lupus vulgaris developing at the site of misdiagnosed scrofuloderma.

Cutaneous tuberculosis is a rare form of extrapulmonary tuberculosis primarily occurring in developing countries. The recent increase in the incidence of tuberculosis, especially due to human immunodeficiency virus (HIV) infections, has led to a resurgence of extrapulmonary forms of this disease. We describe a case of lupus vulgaris in a 33‐year‐old woman who had a 5‐year history of a slowly growing plaque on her neck. The lesion was located at the site of surgery repairing the scar resulting from the incision of a subcutaneous abscess during childhood. This lesion was misdiagnosed as bacterial abscess. Histopathologic examination of the plaque revealed non‐caseating tuberculoid granulomas consisting of lymphocytes, epithelioid and giant cells. Staining for acid‐fast bacilli and culture from biopsied tissue was negative. Polymerase chain reaction (PCR) for detection of Mycobacterium tuberculosis DNA, performed on a skin biopsy specimen, was positive. A diagnosis of lupus vulgaris developing at the site of a previous misdiagnosed scrofuloderma was made. Conventional antitubercular therapy with rifampicin, isoniazid and ethambutol was administered for 6 months, resulting in resolution of the lesion.

Tannic acid induces in vitro acantholysis of keratinocytes via IL-1alpha and TNF-alpha.

The mechanism of acantholysis in pemphigus vulgaris (PV) is an intriguing argument since several chemical mediators are implicated. We previously reported a central role for IL-1α and TNF-α, both able to regulate complement activation and plasminogen activators. Very little is known about what triggers the disease (drugs, viruses or food). In this study, we evaluate the molecular role of tannins in acantholysis. By HPLC chromatography we measured tannic acid (TA) and gallic acid (GA) in blister fluid of 4 groups of patients divided according to their dietary habits, including a regular diet, a diet rich in tannins, a diet free of tannins, and a group of pemphigus patients. Blister fluid was obtained from patients using a suction blister apparatus. We show that people with a diet rich in tannins have increased tannin metabolites (TA and GA) in the skin in respect to controls (tannin-rich diet: GA = 194.52±2.39 nmol/ml; TA = 348.28±1.4 nmol/ml versus tannin-Mediterranean diet: GA = 15.28±1.63 nmol/ml; TA = 22.81±1.68 nmol/ml). PV patients showed similar values to the Mediterranean diet population (PV patients: GA = 95.8±1.97 nmol/ml; TA = 199.09±4.15 nmol/ml versus Mediterranean diet: GA = 83.53±2.35 nmol/ml; TA = 195.1±2.50 nmol/ml). In an in vitro acantholysis system using TA and PV-IgG we show that TA 0.1 mM in NHEK culture is able to induce acantholysis. This effect was able to amplify the acantholytic action of PV-IgG in vitro. A blocking study using anti IL-1α and anti TNF-α antibodies showed a reduction in TA-induced acantholysis. Taken together, these results suggest that a diet rich in tannins could be a trigger in genetically predisposed patients. If these data are confirmed, a complementary diet poor in tannins may be useful in patients affected by PV.

A Th2-like cytokine response is involved in bullous pemphigoid. the role of IL-4 and IL-5 in the pathogenesis of the disease.

Bullous Pemphigoid is an autoimmune bullous disorder characterized by production of IgG against an hemidesmosomal antigen (230 kDa, 180 kDa) responsible for blistering of the skin. In the past several mediators have been implicated in the pathogenesis of the disease such as proteases and collagenases secreted by local inflammatory cells. In order to investigate the role of cytokines in BP, the cytokine pattern was evaluated by an immunohistochemical analysis and by reverse transcriptase polymerase chain reaction procedure in 13 BP patients. Cytokines examined were interleukin (IL)-2, IL-4, IL-5, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. The T cell inflammatory infiltrate was also characterized by monoclonal antibodies showing CD3+, CD4+ T cells with a perivascular and scattered distribution in lesional skin. IL-4 and IL-5 were detected in a similar distribution to the inflammatory infiltrate. IL-4 and IL-5 mRNA levels were also revealed by RT-PCR. Proinflammatory cytokines such as TNF-alpha, IL-6 and Th1-like cytokines (IL-2 and INF-gamma) were not detected neither as proteins nor as mRNA. Since IL-4 and IL-5 are important in eosinophil chemoattraction, maturation and functional activity, the presence of IL-4 and IL-5 in BP suggest that these cytokines could be important in the pathogenesis of the disease.

Coexisting pemphigus vulgaris and pemphigus foliaceus in the same patient

A 79-year-old man was admitted to our clinic with a 3-month history of diffuse scaly and bullous lesions. Skin examination revealed multiple blisters with an annular shape on the lower dorsum and abdominal areas (Fig. 1), while on the scalp and in the retroauricolar area lesions appeared as erythematous scales and crusts (Fig. 2). Symptoms referred were burning and itching. Past medical history of the patient revealed that he was affected by prostate cancer, treated with ormonal therapy. Chest X-ray and routine laboratory investigations were within normal limits. A TC scan and total body scintigraphy excluded distant metastasis. A paraneoplastic pemphigus was suspected and skin samples from scaly eythematous and bullous lesions were taken for histopathological examination and direct immunofluorescence. Indirect immunofluorescence on

Skin Toxicity from Glyphosate‐Surfactant Formulation

Glyphosate (N‐[phosphonomethyl]glycine) is a nonselective herbicide used in agriculture as a foliage spray for the control and the destruction of herbaceous plants. Adverse skin reactions due to contact with this compound have been rarely described. We report a case of a 78‐year‐old woman presenting with extensive chemical burns on her trunk and legs caused by accidental contact with a glyphosate‐surfactant formulation. The lesions healed in four weeks without scarring.

'Suction split' as a routine method to differentiate epidermolysis bullosa acquisita from bullous pemphigoid

Epidermolysis bullosa acquisita (EBA) and bullous pemphigoid (BP) are diseases with similar clinical, histological, and immunofluorescent findings. Diagnosis requires the use of immunoelectron microscopy, immuno‐precipitation or immunoblotting, but in recent years the differential diagnosis has been based on a cheaper technique named salt split skin. This study demonstrates that with a suction blister the fracture is at the same level as that obtained with the sodium split method and that it is also faster and cheaper. Suction blisters on normal skin and autoimmune perilesional bullous lesions, obtained with a hand vacuum pump, were studied by direct immunofluorescence and electron microscopy to evaluate the level of the split on normal suction split skin. Normal human split skin was also used as a substrate for an indirect immunofluorescent study using sera of patients with BP (68 sera), EBA (10 sera) and cicatricial pemphigoid (CP) (16 sera). Direct immunofluorescent examination was also done on perilesional skin after artificial separation obtained with a hand‐vacuum pump in patients with the same diseases listed above (32 BP, 11 CP, 6 EBA).