X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis.

X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.

Clinical and immunological overlap between autoimmune lymphoproliferative syndrome and common variable immunodeficiency.

Autoimmune lymphoproliferative syndrome (ALPS) is mainly caused by defects in the CD95 pathway. Raised CD3+TCRαß+CD4-CD8- double negative T cells and impaired T cell apoptosis are hallmarks of the disease. In contrast, the B cell compartment has been less well studied. We found an altered distribution of B cell subsets with raised transitional B cells and reduced marginal zone B cells, switched memory B cells and plasma blasts in most of 22 analyzed ALPS patients. Moreover, 5 out of 66 ALPS patients presented with low IgG and susceptibility to infection revealing a significant overlap between ALPS and common variable immunodeficiency (CVID). In patients presenting with lymphoproliferation, cytopenia, hypogammaglobulinemia and impaired B cell differentiation, serum biomarkers were helpful in addition to apoptosis tests for the identification of ALPS patients. Our observations may indicate a role for apoptosis defects in some diseases currently classified as CVID.

Reduced memory B cells in patients with hyper IgE syndrome.

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection.

Fas/APO-1 gene transfer for human malignant glioma.

Human malignant glioma cells are susceptible to apoptosis induced by antibodies to Fas/APO-1, a cytokine receptor protein of the nerve growth factor/tumor necrosis factor receptor superfamily. Here we show that a critical level of cell surface expression of Fas/APO-1 is a prerequisite for induction of glioma cell apoptosis via Fas/APO-1. Although Fas/APO-1 mRNA was expressed in three Fas/APO-1 antibody-resistant glioma cell lines, these cells expressed either little Fas/APO-1 protein (LN-319 and LN-405) or an abnormal Fas/APO-1 protein that was not translocated to the cell membrane and therefore functionally inactive (LN-308). Although all glioma cell lines expressed mRNA for Fas/APO-1-delta TM, a soluble form of Fas/APO-1 lacking the transmembrane domain, none of the cell lines released detectable amounts of soluble Fas/APO-1, a potential endogenous antagonist of Fas/APO-1-mediated glioma cell apoptosis. Stable transfection of three resistant glioma cell lines with a human Fas/APO-1 cDNA expression vector dramatically enhanced cell surface expression of Fas/APO-1 and induced susceptibility to Fas/APO-1 antibody-mediated apoptosis. These data indicate that malignant glioma cells, unlike other tumor cells, uniformly harbor the intracellular cascade required for Fas/APO-1-mediated apoptosis. Low level of Fas/APO-1 expression results from inefficient transcription and translation of the Fas/APO-1 gene or the synthesis of mutant Fas/APO-1 proteins. gamma-Interferon, tumor necrosis factor-alpha, and interleukin 1 beta augmented Fas/APO-1-mediated apoptosis of Fas/APO-1-transfected glioma cells by acting on the subcellular suicidal cascade triggered by Fas/APO-1 activation. Dexamethasone attenuated Fas/APO-1 antibody-induced apoptosis, not only of constitutively Fas/APO-1-positive glioma cells, but also of Fas/APO-1-transfected glioma cells. The antiapoptotic effect of dexamethasone could be overcome by preexposure of the glioma cells to gamma-interferon or by coexposure to Fas/APO-1 antibodies and cycloheximide. Thus, Fas/APO-1 gene transfer and combined immunotherapy using Fas/APO-1 antibodies and cytokines may overcome Fas/APO-1 antibody resistance of Fas/APO-1-negative human malignant glioma cells, which may represent subpopulations within single gliomas or form a separate subgroup of human malignant gliomas.

Fas/Apo-1 activates nuclear factor kappa B and induces interleukin-6 production.

Fas antigen/Apo-1 (Fas) and the p55 tumor necrosis factor receptor (TNF-R) are two related cell surface molecules that induce apoptosis in susceptible cells. With regard to their cytoplasmic homology region, we investigated whether Fas like the TNF-R activates nuclear factor kappa B (NF-kappa B), using human SV80 fibroblasts transfected with the cDNA encoding human Fas. In this cell line Fas mobilizes the p50/p65 heterodimeric form of NF-kappa B and induces interleukin-6 (IL-6) production. Compared to NF-kappa B activation via the TNF-R differences in kinetics and signal intensity were observed. Peak activation occurred 2 hr after Fas compared to 1 hr after TNF-R stimulation. Furthermore, when equitoxic concentrations of anti-Fas antibody and TNF were applied, TNF triggered a stronger NF-kappa B response. Studies using inhibitors of signal transduction suggest that both receptors mediate NF-kappa B activation via similar routes: D609, an inhibitor of the phospatidylcholine-specific phospholipase C, had an inhibitory effect, while the protein kinase C inhibitor staurosporine had an enhancing effect on both Fas and TNF-R induced NF-kappa B mobilization. Interestingly, D609 had no influence on Fas and TNF-R mediated cytotoxicity arguing against an involvement of NF-kappa B in the cell death pathway triggered by these receptors. This is the first indication that Fas may activate genes via NF-kappa B and may thus in addition to its role as a cell death inducing receptor serve a much broader range of biological functions.

Neurons induced to express major histocompatibility complex class I antigen are killed via the perforin and not the Fas (APO-1/CD95) pathway.

Cytotoxic T lymphocytes (CTL) kill target cells by perforin-mediated pore formation, induction of apoptosis by the Fas ligand, or both. It has been demonstrated that depolarized neurons can be induced to express major histocompatibility complex (MHC) class I antigens by interferon-gamma. Evidence for antigen-dependent CTL-mediated killing was obtained by transfecting neurons with MHC class I cDNA. The present study was designed to investigate the mechanisms of killing of cerebellar granule neurons depolarized by high K+ concentrations and thereby inducible for MHC class I antigen expression. We found that neurons express only low levels of Fas (APO-1/CD95) and are resistant to Fas ligand-mediated killing even when pretreated with cytokines. However, granules extracted from CTL as well as purified perforin induce almost complete lysis of neurons. These data suggest that CTL-mediated elimination of neurons involves the perforin, but not the Fas pathway of target cell killing.

CD40 function in nonhematopoietic cells. Nuclear factor kappa B mobilization and induction of IL-6 production.

CD40 is a member of the TNF receptor family that was first characterized as an important T-B cell interaction molecule. This receptor is also expressed on many other cell types, including normal basal epithelium, carcinomas, and transformed cell lines. The functions of CD40 in non-B cells are largely unknown. Our studies demonstrate that CD40 mediates nuclear factor kappa B (NF-kappa B) mobilization and IL-6 production in nonhematopoietic cells. Stimulation of the transformed fibroblast cell line SV80 with CD40 ligand (CD40L) or anti-CD40 Ab resulted in the production of IL-6; this could be increased by IFN-gamma pretreatment, which is known to up-regulate CD40 expression. Studies with transfectants overexpressing CD40 demonstrated that activation of CD40 alone is sufficient to induce IL-6 production. The transcription factor NF-kappa B appears to play a central role in CD40-mediated activation of the IL-6 gene; NF-kappa B mobilization directly preceded CD40-mediated IL-6 production, and suppression of NF-kappa B mobilization with the metabolic inhibitor D609 also suppressed the IL-6 response. A striking similarity to the requirements for TNF-induced IL-6 production, which is mediated by the p55TNF receptor in SV80 cells, was observed. In view of the intracellular homologies between CD40 and the p55TNF receptor, it should be considered that the two receptors share common components in their signaling pathways that lead to IL-6 production.

Puromycin-sensitive aminopeptidase. Sequence analysis, expression, and functional characterization.

Among the molecular mechanisms that control the cell division cycle, proteolysis has emerged as a key regulatory process enabling cells to pass critical check points. Such proteolysis involves a cascade of enzymes including a multisubunit complex termed 26S proteasome. Here we report on the analysis of a novel mouse cDNA encoding the puromycin-sensitive aminopeptidase (PSA) and on its expression in COS cells and 3T3 fibroblasts. PSA is 27-40% homologous to several known Zn(2+)-binding aminopeptidases including aminopeptidase N. Immunohistochemical analysis revealed that PSA is localized to the cytoplasm and to the nucleus and associates with microtubules of the spindle apparatus during mitosis. Furthermore, puromycin and bestatin both arrested the cell cycle, leading to an accumulation of cells in G2/M phase, and ultimately induced cells to undergo apoptosis at concentrations that inhibit PSA. Control experiments including cycloheximide further suggested that the induction of apoptosis by puromycin was not attributable to inhibition of protein synthesis. Taken together, these data favor the novel idea that PSA participates in proteolytic events essential for cell growth and viability.

Local Fas/APO-1 (CD95) ligand-mediated tumor cell killing in vivo.

Fas/APO-1 (CD95) is a cell surface receptor which mediates apoptosis when ligated by specific antibodies or by its recently cloned natural ligand, FasL. We have studied the cytotoxic potential of FasL in vivo using Fas/APO-1-expressing Yac-1 cells as targets. Supernatant harvested from Neuro-2a cells transfected with the murine FasL cDNA contains FasL and transduces a potent apoptotic signal to Yac-1 cells in vitro. Specificity of FasL-mediated cytotoxicity was confirmed by competition assays using soluble Fas or anti-Fas/APO-1 F(ab')2 fragments which specifically interfere with FasL-Fas/APO-1 interactions. Intraperitoneal injection of FasL-containing supernatant efficiently killed Yac-1 target cells which had been implanted in capsules into the peritoneal cavity of mice. Analysis of the target cells revealed DNA fragmentation and nuclear changes typical of apoptosis. As previously shown, intraperitoneal injection of anti-Fas/APO-1 antibodies caused liver failure (Ogasawara, J., Watanabe, F.R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S., Nature 1993. 364:806) and was observed at doses which did not reduce Yac-1 cell viability. In contrast, FasL did not induce histopathology in the liver when applied intraperitoneally at doses cytotoxic for Yac-1 cells. However, intravenous administration of FasL induced lethal liver hemorrhages and hepatocyte apoptosis. Thus, locally applied FasL kills tumor cells very efficiently without systemic toxicity and may therefore represent a candidate for local tumor treatment.