Isabelle Daigle

T helper (Th) 2 predominance in atopic diseases is due to preferential apoptosis of circulating memory/effector Th1 cells

T cells constitute a large population of cellular infiltrate in atopic/allergic inflammation and a dysregulated, Th2‐biased peripheral immune response appears to be an important pathogenetic factor. In atopic dermatitis, circulating cutaneous lymphocyte‐associated antigen‐bearing (CLA+) CD45RO+ T cells with skin‐specific homing property represent an activated memory/effector T cell subset. They express high levels of Fas and Fas ligand and undergo activation‐induced apoptosis. The freshly purified CLA+ CD45RO+ T cells of atopic individuals display distinct features of in vivo‐triggered apoptosis such as pro‐caspase degradation and active caspase‐8 formation. In particular, the Th1 compartment of activated memory/effector T cells selectively undergoes activation‐induced cell death, skewing the immune response toward surviving Th2 cells in atopic dermatitis patients. The apoptosis of circulating memory/effector T cells was confined to atopic individuals whereas non‐atopic patients such as psoriasis, intrinsic‐type asthma, contact dermatitis, intrinsic type of atopic dermatitis, bee venom allergic patients, and healthy controls showed no evidence for enhanced T cell apoptosis in vivo. These results define a novel mechanism for peripheral Th2 response in atopic diseases. —Akdis, M., Trautmann, A., Klunker, S., Daigle, I., Küçüksezer, U. C., Deglmann, W., Disch, R., Blaser, K., Akdis, C. A. T helper (Th) 2 predominance in atopic dermatitis is due to preferential apoptosis of circulating memory/effector Th1 cells. FASEB J. 17, 1026–1035 (2003)

Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils

Death domain–containing receptors of the tumor necrosis factor (TNF)/nerve growth factor (NGF) family can induce apoptosis upon activation in many cellular systems. We show here that a conserved phosphotyrosine-containing motif within the death domain of these receptors can mediate inhibitory functions. The Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), SHP-2 and SH2-containing inositol phosphatase (SHIP) bound to this motif in a caspase-independent but cell-dependent manner. We also found that stimulation of death receptors disrupted anti-apoptosis pathways initiated (at least under certain conditions) by survival factors in neutrophils. In these cells, activation of the tyrosine kinase Lyn, an important anti-apoptotic event, was prevented as a consequence of death-receptor stimulation, most likely through association of the receptor with activated SHP-1. Thus, we provide molecular and functional evidence for negative signaling by death receptors.

Critical role for caspases 3 and 8 in neutrophil but not eosinophil apoptosis.

Background: Apoptosis is a necessary process to control cell numbers in multicellular organisms. In many chronic inflammatory diseases, reduced cell death of different types of granulocytes is one important mechanism for cell accumulation. Here, we studied the role of caspases in neutrophil and eosinophil apoptosis in the presence or absence of granulocyte-macrophage stimulating factor and anti-CD95 monoclonal antibodies, respectively. Methods: Granulocytes were isolated from human blood using standard protocols. Immunoblot and functional studies with cell-permeable specific peptide inhibitors were performed to analyze caspase involvement. Fas receptor and Fas ligand expression was analyzed by RT-PCR, flow cytometry, and immunoblotting. Cell death was analyzed by ethidium bromide exclusion test. Results: Caspases 3 and 8 are critically involved in the regulation of neutrophil apoptosis in vitro. In contrast, these two caspases did not appear to play a major role in the regulation of eosinophil apoptosis. However, the broad-range caspase inhibitor VAD prevented eosinophil death, indicating that caspases are also involved within the apoptotic machinery of eosinophils. Functional inhibitor studies suggested that caspase 9 is crucial for both caspase 3 and 8 activation, at least in neutrophils. In contrast, spontaneous apoptosis of neutrophils or eosinophils is unlikely to be the consequence of Fas ligand/Fas receptor molecular interactions. Conclusion: The data of this study indicate differences in the usage of caspases between neutrophils and eosinophils.

CD137 activation abrogates granulocyte-macrophage colony-stimulating factor-mediated anti-apoptosis in neutrophils.

CD137 (ILA / 4‐1BB) is a member of the TNF / NGF receptor family, and has previously been suggested to be involved in T cell activation and differentiation. Here, we demonstrate that blood neutrophils from control individuals and patients with cystic fibrosis express CD137 mRNA and surface protein. In contrast, lung neutrophils derived from patients with cystic fibrosis did not express detectable CD137 levels. Such CD137‐deficient neutrophils could also be generated from normal neutrophils by TNF‐α stimulation in vitro. TNF‐α was found to be highly expressed in epithelial cells from cystic fibrosis but not normal lungs, suggesting that TNF‐α might account for reduced neutrophil CD137 levels under inflammatory conditions in vivo. To investigate whether CD137 is involved in the regulation of apoptosis, neutrophils were activated with functional anti‐CD137 antibody in the presence or absence of different neutrophil survival factors in vitro. Activation of CD137 abrogated GM‐CSF‐mediated anti‐apoptosis in normal but not in CD137‐deficient neutrophils. Moreover, G‐CSF‐ and IFN‐γ‐mediated neutrophil anti‐apoptosis was not affected by anti‐CD137 antibody treatment. In conclusion, these data suggest that CD137 activation may limit GM‐CSF‐mediated anti‐apoptosis of neutrophils. The absence of this anti‐inflammatory mechanism in inflammatory responses might be associated with massive neutrophil accumulation and consequent tissue damage.

Design, engineering and in vitro evaluation of MHC class-II targeting allergy vaccines

Background:  The worldwide increasing incidence of allergic diseases requires the development of new, efficient vaccination strategies, the only curative treatment with a long‐lasting effect. Current allergen‐specific immunotherapy protocols suffer from limited efficacy and a long treatment time.

Human Mast Cells Undergo TRAIL-Induced Apoptosis

Mast cells (MC), supposedly long-lived cells, play a key role in allergy and are important contributors to other inflammatory conditions in which they undergo hyperplasia. In humans, stem cell factor (SCF) is the main regulator of MC growth, differentiation, and survival. Although human MC numbers may also be regulated by apoptotic cell death, there have been no reports concerning the role of the extrinsic apoptotic pathway mediated by death receptors in these cells. We examined expression and function of death receptors for Fas ligand and TRAIL in human MC. Although the MC leukemia cell line HMC-1 and human lung-derived MC expressed both Fas and TRAIL-R, MC lines derived from cord blood (CBMC) expressed only TRAIL-R. Activation of TRAIL-R resulted in caspase 3-dependent apoptosis of CBMC and HMC-1. IgE-dependent activation of CBMC increased their susceptibility to TRAIL-mediated apoptosis. Results suggest that TRAIL-mediated apoptosis may be a mechanism of regulating MC survival in vivo and, potentially, for down-regulating MC hyperplasia in pathologic conditions.

IL-10 controls Aspergillus fumigatus- and Pseudomonas aeruginosa-specific T-cell response in cystic fibrosis.

Up to 90% of patients with cystic fibrosis (CF) are chronically colonized with Pseudomonas aeruginosa, and 10% to 50% of CF patients are colonized with Aspergillus fumigatus. Despite an extensive inflammatory reaction, patients cannot eliminate the microorganisms. The present study demonstrates that an IL-10 mediated T-cell tolerance to major infectious agents A. fumigatus and P. aeruginosa plays an important role in the control of T-cell-mediated inflammatory responses in CF. Peripheral blood mononuclear cells of CF patients secreted significantly higher amounts of IL-10. T-cell response against recombinant A. fumigatus antigens rAsp f 3, rAsp f 4, rAsp f 6, and heatinactivated P. aeruginosa was controlled by IL-10. Proliferation and interferon-γ production was significantly increased when endogenous IL-10 was blocked in aspergillus and pseudomonas antigen-stimulated cells of CF patients. The role of IL-10 was further documented by increased spontaneous proliferation of peripheral blood mononuclear cells of CF patients after preincubation with antisense oligonucleotides blocking the synthesis of IL-10 receptor-associated kinases janus tyrosine kinase 1 and tyrosine kinase 2. Together, these data demonstrate an important role of IL-10-mediated peripheral T-cell tolerance to P. aeruginosa and A. fumigatus in the control of the intensity of the inflammatory T-cell response in CF.

Induction of the IL-10 gene via the Fas receptor in monocytes - an anti-inflammatory mechanism in the absence of apoptosis

Death receptors play an important role in controlling cell numbers and immune responses. In contrast to TNF receptors, little is known about non‐apoptosis functions of the Fas receptor (CD95, APO‐1). Here we demonstrate that Fas receptor engagement results in the induction of the IL‐10 gene in monocytes, but not in lymphocytes or dendritic cells. In contrast, TNF‐α stimulated IL‐10 production in dendritic cells but not monocytes. Fas receptor‐mediated transcriptional activation of the IL‐10 gene was followed by the release of large amounts of the cytokine in cell cultures and occurred in the absence of apoptosis induction. Since caspase activation did not occur in monocytes following Fas receptor engagement, it is unlikely that caspases are involved in IL‐10 gene activation. Monocyte‐derived IL‐10 suppressed T cell proliferation induced by anti‐CD3 monoclonal antibody without affecting CD3‐mediated transmembrane signal transduction. In conclusion, we report about a novel pathway initiated via the Fas receptor leading to transcriptional activation of at least one cytokine gene. Fas ligand‐induced IL‐10 production in monocytes might represent an important anti‐inflammatory mechanism in secondary immune responses.

Auto-reactive IgE responses to acidic ribosomal P2 protein in systemic lupus erythematosus

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Fas Ligand Reduces Viability in Primary Melanoma Short-Term Cell Cultures More than in Metastatic Melanoma Short-Term Cell Cultures

Background: Apoptotic pathway aberrations are reported as important tumor progression factors in melanoma. Objective: Effect of soluble Fas ligand (sFasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on short-term cultured melanoma cell viability from different stages of melanoma. Results: Recombinant human FasL reduced viability after 18 h in a dose-dependent manner in 4 of 5 cell cultures from primary tumors and 1 of 9 cell cultures from metastatic melanoma (67.5 vs. 96.4%, p = 0.007). DNA fragmentation on flow cytometry confirmed apoptosis. Incubation with TRAIL had no effect on melanoma cell viability. Immunohistochemistry showed Fas in 3 of 4 primary and in 6 of 7 metastatic lesions, no FasL in primary lesions, and FasL in 5 of 7 metastatic lesions. Conclusion: Melanoma short-term cell cultures from primary tumors show decreased viability under FasL, but not TRAIL stimulation rather than short-term cell cultures derived from metastases.