Andreas Kerstan

cFLIPL Inhibits Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated NF-κB Activation at the Death-inducing Signaling Complex in Human Keratinocytes

Human keratinocytes undergo apoptosis following treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via surface-expressed TRAIL receptors 1 and 2. In addition, TRAIL triggers nonapoptotic signaling pathways including activation of the transcription factor NF-κB, in particular when TRAIL-induced apoptosis is blocked. The intracellular protein cFLIPL interferes with TRAIL-induced apoptosis at the death-inducing signaling complex (DISC) in many cell types. To study the role of cFLIPL in TRAIL signaling, we established stable HaCaT keratinocyte cell lines expressing varying levels of cFLIPL. Functional analysis revealed that relative cFLIPL levels correlated with apoptosis resistance to TRAIL. Surprisingly, cFLIPL specifically blocked TRAIL-induced NF-κB activation and TRAIL-dependent induction of the proinflammatory target gene interleukin-8. Biochemical characterization of the signaling pathways involved showed that apoptosis signaling was inhibited at the DISC in cFLIPL-overexpressing keratinocytes, although cFLIPL did not significantly impair enzymatic activity of the receptor complex. In contrast, recruitment and modification of receptor-interacting protein was blocked in cFLIPL-overexpressing cells. Taken together, our data demonstrate that cFLIPL is not only a central antiapoptotic modulator of TRAIL-mediated apoptosis but also an inhibitor of TRAIL-induced NF-κB activation and subsequent proinflammatory target gene expression. Hence, cFLIPL modulation in keratinocytes may not only influence apoptosis sensitivity but may also lead to altered death receptor-dependent skin inflammation.

cFLIP Regulates Skin Homeostasis and Protects against TNF-Induced Keratinocyte Apoptosis

FADD, caspase-8, and cFLIP regulate the outcome of cell death signaling. Mice that constitutively lack these molecules die at an early embryonic age, whereas tissue-specific constitutive deletion of FADD or caspase-8 results in inflammatory skin disease caused by increased necroptosis. The function of cFLIP in the skin in vivo is unknown. In contrast to tissue-specific caspase-8 knockout, we show that mice constitutively lacking cFLIP in the epidermis die around embryonic days 10 and 11. When cFLIP expression was abrogated in adult skin of cFLIPfl/fl-K14CreERtam mice, severe inflammation of the skin with concomitant caspase activation and apoptotic, but not necroptotic, cell death developed. Apoptosis was dependent of autocrine tumor necrosis factor production triggered by loss of cFLIP. In addition, epidermal cFLIP protein was lost in patients with severe drug reactions associated with epidermal apoptosis. Our data demonstrate the importance of cFLIP for the integrity of the epidermis and for silencing of spontaneous skin inflammation.

Cutting Edge: Distinct TCR- and CD28-Derived Signals Regulate CD95L, Bcl-xL, and the Survival of Primary T Cells

TCR-driven clonal expansion of T cells is limited by activation-induced cell death through CD95/CD95L interactions. This cell-contact dependent mechanism is attenuated by costimulation through CD28. Here, we show that primary rat lymph node T cells activated by “superagonistic” CD28-specific mAb, which do not require TCR-engagement for full T cell activation, do not up-regulate CD95L. CD28 superagonist activated T cells are highly resistant to artificial CD95 cross-linking, and display a marked up-regulation of the survival factor Bcl-xL. Consistently, NF-κB factors, known to promote Bcl-xL transcription, are strongly activated by superagonistic CD28 mAb stimulation. In contrast, a weaker induction of NFAT, which positively regulates the CD95L gene, in CD28 activated cells as compared with TCR- or TCR/CD28-stimulated cells was observed. Thus, by recruiting the mitogenic activity of CD28 in the absence of TCR engagement, the anti-apoptotic signals provided by costimulation are revealed without interfering proapoptotic effects induced by TCR stimulation.

cFLIPL inhibits TNF-related apoptosis-inducing ligand-mediated NF-κB activation at the death inducing signalling complex (DISC) in human keratinocytes

Human keratinocytes undergo apoptosis following treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via surface-expressed TRAIL receptors 1 and 2. In addition, TRAIL triggers nonapoptotic signaling pathways including activation of the transcription factor NF-κB, in particular when TRAIL-induced apoptosis is blocked. The intracellular protein cFLIPL interferes with TRAIL-induced apoptosis at the death-inducing signaling complex (DISC) in many cell types. To study the role of cFLIPL in TRAIL signaling, we established stable HaCaT keratinocyte cell lines expressing varying levels of cFLIPL. Functional analysis revealed that relative cFLIPL levels correlated with apoptosis resistance to TRAIL. Surprisingly, cFLIPL specifically blocked TRAIL-induced NF-κB activation and TRAIL-dependent induction of the proinflammatory target gene interleukin-8. Biochemical characterization of the signaling pathways involved showed that apoptosis signaling was inhibited at the DISC in cFLIPL-overexpressing keratinocytes, although cFLIPL did not significantly impair enzymatic activity of the receptor complex. In contrast, recruitment and modification of receptor-interacting protein was blocked in cFLIPL-overexpressing cells. Taken together, our data demonstrate that cFLIPL is not only a central antiapoptotic modulator of TRAIL-mediated apoptosis but also an inhibitor of TRAIL-induced NF-κB activation and subsequent proinflammatory target gene expression. Hence, cFLIPL modulation in keratinocytes may not only influence apoptosis sensitivity but may also lead to altered death receptor-dependent skin inflammation.

Wasp venom immunotherapy induces activation and homing of CD4+CD25+ forkhead box protein 3–positive regulatory T cells controlling TH1 responses

BACKGROUND Despite a growing interest in CD4(+)CD25(+) forkhead box protein 3 (Foxp3)-positive regulatory T (Treg) cells, the fundamental parameters of the activation and homing of these cells during wasp venom immunotherapy (VIT) are largely unknown. OBJECTIVE We investigated longitudinally the phenotype and function of Treg cells in a well-characterized homogeneous group of patients with wasp venom allergy during VIT. METHODS In 30 patients peripheral Treg cells were ex vivo monitored for their activation status and homing capacities by means of flow cytometric analysis before and after 1 and 6 months of VIT. In addition, the in vitro suppressive activity of Treg cells, as well as cytokine secretion, in response to wasp venom was analyzed. RESULTS One month after initiating VIT, the proportion of both CD4(+)CD25(+)Foxp3(+) and CD4(+)Foxp3(+) Treg cells significantly decreased in peripheral blood. Coexpression of the lymph node homing receptors CCR7/CD62L were induced in CD4(+)Foxp3(+)CD45RO(+) Treg cells, indicating recirculation of VIT-activated Treg cells in secondary lymphoid organs. In vivo imaging by means of color duplex ultrasonography of the axillary draining lymph nodes demonstrated a VIT-induced 4-fold augmentation in afferent arterial blood flow. Furthermore, increased activation markers (CD45RO and HLA-DR) of Treg cells correlated with effective in vitro suppression of wasp venom-driven T-cell proliferation. After 1 month of VIT, Treg cell depletion in vitro greatly enhanced wasp venom-induced IFN-γ secretion. CONCLUSIONS Allergen exposure during VIT simultaneously induces the activation and selective homing of circulating Treg cells. Functionally, on the one hand, Treg cells balance the immune reaction toward tolerance, and on the other hand, they are involved in controlling overwhelming T(H)1 responses.

Histopathology of anti-laminin 5 mucous membrane pemphigoid

BACKGROUND Anti-laminin 5 mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by autoantibodies against the major basement membrane component laminin 5 (laminin 332, epiligrin). OBJECTIVE AND METHODS We reviewed 17 biopsy specimens from 9 patients with anti-laminin 5 MMP in an attempt to define typical histopathologic features of the disease. RESULTS Fifteen specimens showed subepidermal blister formation, while two biopsy specimens revealed an epithelial ulcer. In 11 biopsies a sparse to moderate inflammatory infiltrate composed of lymphocytes and neutrophils with some eosinophils was observed. Four biopsies showed a dense infiltrate dominated by neutrophils in two cases and by eosinophils in one case. The remaining biopsy revealed a dense lymphoplasmacellular infiltrate without granulocytes. Scarring of the upper dermis was present only in 5 specimens. Immunohistochemical analysis localized type IV collagen to the dermal side of the blister, suggesting that split formation occurred within the lamina lucida of the cutaneous basement membrane. LIMITATIONS The number of patients studied was relatively small. CONCLUSIONS Histopathology of anti-laminin 5 MMP is characterized by subepidermal blistering and a sparse to moderate superficial lymphohistiocytic infiltrate with neutrophils and/or eosinophils. Both infiltrate density and composition may vary, making anti-laminin 5 MMP indistinguishable from other autoimmune subepidermal blistering diseases by histopathology alone. Scarring is present only in a minority of cases and is not a sensitive clue to the diagnosis of anti-laminin 5 MMP.

Effector pathways during eczematous dermatitis: where inflammation meets cell death.

Abstract:  During eczematous skin inflammation, the main constituents of the skin, keratinocytes (KC), play an important role in inducing and shaping the immunological response to environmental stimuli. This review focuses on the epidermal inflammation caused by keratinocyte‐T cell interactions arising from a disturbed barrier function of the skin. In eczematous dermatitis, activated dermis‐ and epidermis‐infiltrating T cells target KC for apoptosis. In turn, damaged KC respond by secreting inflammatory mediators, thus effecting further recruitment of immunocytes to inflamed skin. Further advances will come from identification of the immunoregulatory mechanisms involved in the pathogenesis of eczematous dermatitis. Potential therapeutic interventions are discussed.

Aminopenicillin‐associated exanthem: lymphocyte transformation testing revisited

The lymphocyte transformation test (LTT) has been promoted as in‐vitro test for diagnosis of drug hypersensitivity. For determination of statistical LTT sensitivity, series of patients with clinically uniform reactions followed by complete drug hypersensitivity work‐up are mandatory. Assessment of LTT specificity requires control patients who tolerated exposure to the drug studied.

Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGFβ1 transgenic mice

Please cite this paper as: Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGFβ1 transgenic mice. Experimental Dermatology 2009; 19: 406–415.

High-Risk Human Papillomavirus Infection in Bowen’s Disease of the Nail Unit: Report of Three Cases and Review of the Literature

Background: Bowen’s disease (BD) of the nail unit is associated with human papillomavirus (HPV) infection. Objective: This study aimed to investigate the frequency of high-risk HPV infection, gender, age and digital distribution in this condition. Methods: Biopsy specimens of 3 consecutive cases with periungual BD were investigated for the presence of HPV DNA by in situ hybridization and by polymerase chain reaction (PCR). Furthermore, 74 cases of ungual BD conducted with HPV genotyping as reported in the literature were reviewed. Results: PCR of biopsy specimens revealed in 2 cases infection with HPV-16 and in 1 case with HPV-73. Additionally, in 1 HPV-16-positive case HPV-31/33 was detected by in situ hybridization. In line, review of the literature demonstrated a clear association of HPV-positive BD with high-risk HPV types. Interestingly, age at diagnosis was significantly lower in women. Whereas in both genders the second to fourth fingers on both hands were commonly diseased, only in men the thumbs were also prominently affected. Conclusions: Infection with high-risk HPV types is common in BD of the nail unit suggesting the aetiological cause. Therefore, patients and partners should be closely followed up for digital and genital HPV-associated lesions.