Daniele Fanoni

Role of inflammatory cells, cytokines and matrix metalloproteinases in neutrophil-mediated skin diseases

Pyoderma gangrenosum (PG) is a rare, immune‐mediated inflammatory skin disease presenting with painful ulcers having undermined edges. Less commonly, bullous and vegetative variants exist. Histology consists of a neutrophil‐rich dermal infiltrate. We characterized immunohistochemically the infiltrate in different variants of PG and in another neutrophilic dermatosis as Sweets syndrome. We studied 21 patients with PG, eight with Sweets syndrome and 20 controls, evaluating skin immunoreactivity for inflammatory cell markers (CD3, CD163 and myeloperoxidase), cytokines [tumour necrosis factor (TNF)‐α, interleukin (IL)‐8 and IL‐17], metalloproteinases (MMP‐2 and MMP‐9) and vascular endothelial growth factor (VEGF). Immunoreactivities of CD3, CD163, myeloperoxidase, TNF‐α, IL‐8, IL‐17, MMP‐2, MMP‐9 and VEGF were significantly higher in both PG and Sweets syndrome than in controls (P = 0·0001). Myeloperoxidase (neutrophil marker), IL‐8 (cytokine chemotactic for neutrophils) and MMP‐9 (proteinase‐mediating tissue damage) were expressed more significantly in both ulcerative and bullous PG than in vegetative PG as well as in Sweets syndrome (P = 0·008–P = 0·0001). In ulcerative PG, the expression of CD3 (panT cell marker) and CD163 (macrophage marker) were significantly higher in wound edge than wound bed (P = 0·0001). In contrast, the neutrophil marker myeloperoxidase was expressed more significantly in wound bed than wound edge (P = 0·0001). Our study identifies PG as a paradigm of neutrophil‐mediated inflammation, with proinflammatory cytokines/chemokines and MMPs acting as important effectors for the tissue damage, particularly in ulcerative and bullous PG where damage is stronger. In ulcerative PG, the wound bed is the site of neutrophil‐recruitment, whereas in the wound edge activated T lymphocytes and macrophages pave the way to ulcer formation.

Expression of tissue factor by eosinophils in patients with chronic urticaria.

Background: Although several cases of chronic urticaria (CU) are currently regarded as autoimmune in origin, associated with histamine-releasing autoantibodies, an activation of blood coagulation via tissue factor (TF) and a strong expression of TF in lesional skin have been described. Eosinophils, which are involved in CU skin lesions, have recently been demonstrated as the major source of TF in human blood. We assessed whether eosinophils are the cellular source of TF in CU skin lesions. Methods: Twenty patients with severe CU were studied. Skin biopsy specimens were taken from wheals. The control group consisted of specimens of perilesional normal skin from different types of skin tumours (10) and various skin disorders with non-eosinophilic infiltrates, including leukocytoclastic vasculitis (7), lichen planus (8) and mastocytosis (3). TF expression was evaluated by immunohistochemical methods using an anti-TF monoclonal antibody. Co-localization of TF and eosinophil cationic protein, a classic cell marker of eosinophils, was investigated by double-staining studies using 2 specific monoclonal antibodies in the 4 specimens showing the highest TF reactivity scores. Results: All specimens from patients with CU clearly showed TF expression that was absent in all normal control specimens (p = 0.0001) and in the skin disorders with non-eosinophilic infiltrates (p = 0.001–0.0001). The double-staining experiments for TF and eosinophil cationic protein clearly showed that the TF-positive cells were eosinophils. Conclusions: Eosinophils are the main source of TF in CU lesional skin. This finding highlights the role of these cells in the pathophysiology of CU and might pave the way for new therapeutic strategies.

New monoclonal antibodies against B-cell antigens: Possible new strategies for diagnosis of primary cutaneous B-cell lymphomas

Reactivities of the monoclonal antibodies (mAbs) of the 9th Human Leukocyte Differentiation Antigen Workshop, in order to define specific antigenic expression of the primary cutaneous B-cell lymphomas (PC-BCL), were analyzed by immunohistology on human tonsil and on PC-BCL, such as follicular centre B-cell lymphomas (FCL), marginal zone lymphomas (MZL) and diffuse large B-cells lymphomas leg-type (DLBL-LT). We identified some subgroups of mAbs that were exclusively or preferentially positive in one lymphoma cell type: the PC-FCL subgroup of mAbs includes PD1/CD279, GCET-1, hFCRL1/CD307a, FCRL2/CD307b, CXCR5/CD185, B7-DC/CD273, MRC/CD200, CD130, CXCR4/CD184, Siglec-5/14, CD150, on the other hand subgroup of mAbs in PC-MZL includes BTLA/CD272, BLIMP-1, hCD38. No specific subgroup of mAbs was found to label PC-DLBCL. This study may be useful to better define specific antigen profile of different PC-BCL entities leading to a correct diagnosis.

Association of Pyoderma Gangrenosum, Acne, and Suppurative Hidradenitis (PASH) Shares Genetic and Cytokine Profiles With Other Autoinflammatory Diseases

AbstractThe association of pyoderma gangrenosum, acne, and suppurative hidradenitis (PASH) has recently been described and suggested to be a new entity within the spectrum of autoinflammatory syndromes, which are characterized by recurrent episodes of sterile inflammation, without circulating autoantibodies and autoreactive T-cells. We conducted an observational study on 5 patients with PASH syndrome, analyzing their clinical features, genetic profile of 10 genes already known to be involved in autoinflammatory diseases (AIDs), and cytokine expression pattern both in lesional skin and serum. In tissue skin samples, the expressions of interleukin (IL)-1&bgr; and its receptors I and II were significantly higher in PASH (P = 0.028, 0.047, and 0.050, respectively) than in controls. In PASH patients, chemokines such as IL-8 (P = 0.004), C-X-C motif ligand (CXCL) 1/2/3 (P = 0.028), CXCL 16 (P = 0.008), and regulated on activation, normal T cell expressed and secreted (RANTES) (P = 0.005) were overexpressed. Fas/Fas ligand and cluster of differentiation (CD)40/CD40 ligand systems were also overexpressed (P = 0.016 for Fas, P = 0.006 for Fas ligand, P = 0.005 for CD40, and P = 0.004 for CD40 ligand), contributing to tissue damage and inflammation. In peripheral blood, serum levels of the main proinflammatory cytokines, that is, IL-1&bgr;, tumor necrosis factor-&agr;, and IL-17, were within the normal range, suggesting that in PASH syndrome, the inflammatory process is mainly localized into the skin. Four out of our 5 PASH patients presented genetic alterations typical of well-known AIDs, including inflammatory bowel diseases, and the only patient lacking genetic changes had clinically evident Crohn disease. In conclusion, overexpression of cytokines/chemokines and molecules amplifying the inflammatory network, along with the genetic changes, supports the view that PASH syndrome is autoinflammatory in origin.

Activation of blood coagulation in bullous pemphigoid: role of eosinophils, and local and systemic implications.

Background  Bullous pemphigoid (BP) is a blistering skin disease caused by autoantibodies to hemidesmosomal proteins, with eosinophils participating in blister formation. Eosinophils are a source of tissue factor (TF), an initiator of blood coagulation.

Treatment of Refractory Pemphigus with the Anti-CD20 Monoclonal Antibody (Rituximab)

Background: Pemphigus is a severe blistering disorder caused by autoantibodies to desmogleins 1 and 3. Because some patients with pemphigus never enter into remission, new immunosuppressants are warranted. Rituximab is a chimeric monoclonal antibody binding to the CD20 antigen on B cells, which proved to be effective in recalcitrant pemphigus. Objectives: To evaluate the efficacy and safety of rituximab in refractory pemphigus and to investigate its effects on the autoantibody profile. Patients and Methods: Six patients with recalcitrant pemphigus were treated. Rituximab was administered intravenously at a dosage of 375 mg/m2 body surface once weekly for 4 weeks. Results: Three pemphigus foliaceus patients and 1 with mucocutaneous pemphigus vulgaris (PV) showed complete response over a follow-up period of up to 18 months. In one oral PV, control of the disease was achieved using pulse therapy with cyclophosphamide following rituximab withdrawal. In one PV with vegetating features, good improvement was obtained after 6 rituximab infusions. All patients tolerated the treatment well. Anti-desmoglein autoantibodies significantly decreased only in pemphigus foliaceus. Conclusions: This study highlights that rituximab is a valuable drug for refractory pemphigus, although the response of mucous membranes and cutaneous folds may be delayed.

Cutaneous extranodal NK/T-cell lymphoma: a clinicopathologic study of 5 patients with array-based comparative genomic hybridization

Extranodal natural killer/T-cell (ENK/T) lymphoma is a rare neoplasm, subcategorized into ENK/T-nasal (ENK/T-N) and ENK/T-nasal type (ENK/T-NT) lymphomas. ENK/T-NT lymphoma with initial presentation in the skin is known as primary cutaneous ENK/T-NT (PC-ENK/T-NT) lymphoma. The aim of this study was to investigate pathogenesis, genomic alterations, and prognosis of cutaneous ENK/T lymphomas to provide further insights into clinicopathologic features and genetic mechanism of lymphomagenesis. A retrospective case study of 5 white patients affected by ENK/T lymphoma (4 PC-ENK/T-NT and 1 ENK/T-N with cutaneous involvement) was performed. Most of the cases presented with multiple nodules and ulcerations localized on the extremities. A considerable percentage had disease in advanced stage with a 12-month survival rate of 40%. Genomic alterations were detected by array-based comparative genomic hybridization that showed gains of 1q, 7q and loss of 17p in the cases of PC-ENK/T-NT lymphomas and gain of 7q and loss of 9p, 12p, 12q in the case of ENK/T-N lymphoma. In conclusion, ENK/T lymphoma is a very aggressive entity, and, in our cases, the exclusively cutaneous presentation was not associated with a better prognosis. The results of our array comparative genomic hybridization analysis could be useful to better define the different ENK/T lymphoma subgroups with cutaneous involvement.

Inflammatory cells, cytokines and matrix metalloproteinases in amicrobial pustulosis of the folds and other neutrophilic dermatoses.

Amicrobial pustulosis of the folds (APF) is a rare cutaneous disease characterized by relapsing sterile pustules frequently associated with autoimmune disorders. Although APF pathophysiology is still undefined, scattered reports suggest involvement of neutrophils. The aim of the present study is to evaluate the role of the skin inflammatory infiltrate, selected multifunctional cytokines and effectors of tissue damage in APF and other neutrophilic dermatoses. We studied, by immunohistochemical methods, inflammatory cell markers (CD3, CD163, myeloperoxidase), cytokines (TNF-α, IL-8, IL-17), metalloproteinases (MMP-2, MMP-9) and vascular-endothelial-growth-factor (VEGF) in lesional skin from six patients with APF, 11 with pyoderma gangrenosum (PG), 7 with Sweets syndrome, and in 20 normal skin samples. Immunoreactivities of CD3, CD163, myeloperoxidase, TNF-α, IL-8, IL-17, MMP-2, MMP-9 and VEGF were significantly higher in APF, PG and Sweets syndrome than in controls (p=0.0001). IL-8 was more expressed in PG than in APF (P=0.002) and Sweets syndrome (p=0.001). In APF, MMP-9 reactivity was higher than in Sweets syndrome, (p=0.035), but less intense than in PG (p=0.020). Our study supports the role of proinflammatory cytokines/chemokines and MMPs as important effectors for the tissue damage in APF similarly to classic neutrophilic dermatoses.

Diagnosis and disease severity assessment of epidermolysis bullosa acquisita by ELISA for anti-type VII collagen autoantibodies: an Italian multicentre study

Background  Epidermolysis bullosa acquisita (EBA) is a rare autoimmune mucocutaneous bullous disease caused by autoantibodies against type VII collagen, a component of anchoring fibrils that stabilizes dermoepidermal adherence. Type VII collagen is composed of a collagenous domain linked by the noncollagenous (NC)1 and NC2 domains.

Coagulation activation in autoimmune bullous diseases

The main autoimmune blistering skin disorders are pemphigus vulgaris (PV) and bullous pemphigoid (BP). They differ in the inflammatory infiltrate, which is more intense in BP. Inflammation is known to activate coagulation in several disorders. Local and systemic activation of coagulation was evaluated in BP and PV. We studied 20 BP patients (10 active and 10 remittent), 23 PV patients (13 active and 10 remittent) and 10 healthy subjects. The coagulation markers prothrombin fragment F1+2 and D‐dimer were measured by enzyme‐immunoassays in plasma. The presence of tissue factor (TF), the main initiator of blood coagulation, was evaluated immunohistochemically in skin specimens from 10 patients with active PV, 10 patients with active BP and 10 controls. Plasma F1+2 and D‐dimer levels were significantly high in active BP (P = 0·001), whereas in active PV the levels were normal. During remission, F1+2 and D‐dimer plasma levels were normal in both BP and PV. TF immunoreactivity was found in active BP but neither in active PV nor in normal skin. TF reactivity scores were higher in active BP than in controls or active PV (P = 0·0001). No difference in TF scores was found between active PV and controls. BP is associated with coagulation activation, which is lacking in PV. This suggests that BP but not PV patients have an increased thrombotic risk. The observation that thrombotic complications occur more frequently in BP than in PV further supports this view.