Julie M. Rumble

Neutrophil-related factors as biomarkers in EAE and MS

Using a mouse model of multiple sclerosis (MS), the authors show that neutrophils expand in the bone marrow and accumulate in the circulation before clinical onset of disease. Early in disease development, neutrophils infiltrate the CNS, which is suppressed by G-CSF receptor deficiency and blockade of CXCL1 to ameliorate disease. In patients with MS, systemic expression of neutrophil-related mediators correlates with new lesion formation, lesion burden, and clinical disability.

CD30 activates both the canonical and alternative NF-κB pathways in anaplastic large cell lymphoma cells

CD30 is a member of the tumor necrosis factor receptor superfamily whose expression is up-regulated on anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) cells. Many different outcomes of CD30 stimulation have been reported, including cell cycle arrest, apoptosis, and activation of the prosurvival transcription factor, NF-κB, although this last activity is much less well defined in ALCL cells. In order to better understand the signaling properties of CD30 in cancer, we established a system for the stimulation of CD30 with its physiological ligand. Using this system, CD30 was stimulated on ALCL and HL cells, and the subsequent CD30 signaling properties were characterized. We show that a fraction of ALCL cells rapidly underwent apoptosis following CD30 stimulation, whereas HL cells were unaffected. The surviving ALCL cells exhibited robust activation of both the canonical and alternative NF-κB pathways as measured by nuclear translocation of RelA, p50, RelB, and p52, and this culminated in the transactivation of classical NF-κB-responsive genes. With prolonged CD30 stimulation, ALCL cells underwent cell cycle arrest that correlated with expression of the cell cycle inhibitor p21waf1. Furthermore, p21waf1 expression and cell cycle arrest were found to depend predominantly on the canonical NF-κB pathway, since it was reversed by RNA interference-mediated suppression of RelA. In contrast, suppression of the p100/p52 NF-κB subunit had little effect on p21waf1. These data reveal that in ALCL cells, in contrast to other cell types, CD30 stimulation elicits p21waf1-mediated arrest through the canonical but not the alternative NF-κB pathway.

The lymphoid chemokine, CXCL13, is dispensable for the initial recruitment of B cells to the acutely inflamed central nervous system

Abstract Cases of progressive multifocal leukoencephalopathy can occur in patients treated with the B cell depleting anti-CD20 antibody, rituximab, highlighting the importance of B cell surveillance of the central nervous system (CNS). The lymphoid chemokine, CXCL13, is critical for B cell recruitment and functional organization of peripheral lymphoid tissues, and CXCL13 levels are often elevated in the inflamed CNS. To more directly investigate the role of CXCL13 in CNS B cell migration, its role in animal models of infectious and inflammatory demyelinating disease was examined. During acute alphavirus encephalitis where viral clearance depends on the local actions of anti-viral antibodies, CXCL13 levels and B cell numbers increased in brain tissue over time. Surprisingly, however, CXCL13-deficient animals showed normal CNS B cell recruitment, unaltered CNS virus replication and clearance, and intact peripheral anti-viral antibody responses. During experimental autoimmune encephalomyelitis (EAE), CNS levels of CXCL13 increased as symptoms emerged and equivalent numbers of B cells were identified among the CNS infiltrates of CXCL13-deficient mice compared to control animals. However, CXCL13-deficient mice did not sustain pathogenic anti-myelin T cell responses, consistent with their known propensity to develop more self-limited EAE. These data show that CXCL13 is dispensable for CNS B cell recruitment in both models. The disease course is unaffected by CXCL13 in a CNS infection paradigm that depends on a pathogen-specific B cell response, while it is heightened and prolonged by CXCL13 when myelin-specific CD4+ T cells drive CNS pathology. Thus, CXCL13 could be a therapeutic target in certain neuroinflammatory diseases, but not by blocking B cell recruitment to the CNS.

Cytoprotective effects of IAPs revealed by a small molecule antagonist.

Deregulated expression of members of the IAP (inhibitor of apoptosis) family has been identified in a wide variety of neoplastic cells, and synthetic IAP antagonists represent a promising novel class of chemotherapeutic agents. Early work focused on the ability of these compounds to block the caspase-inhibitory function of XIAP (X-linked IAP). However, recent studies have shown that IAP antagonists, although primarily designed to target XIAP, trigger ubiquitin-mediated degradation of two related proteins, c-IAP (cellular IAP) 1 and c-IAP2, and through this process potentiates the death of tumour cells via autocrine cellular-signalling pathways. In this context, the relative contribution of XIAP as a target of this class of compounds is unclear. In the present study, we examine the involvement of XIAP using a recently described synthetic IAP antagonist, AEG40730, and through comparison of a human XIAP-depleted tumour cell line with its isogenic wild-type control line. Treatment with nanomolar concentrations of AEG40730 resulted in the loss of both XIAP and c-IAP1 proteins, albeit with different kinetics. Although XIAP-deficient HCT116 cells retained some sensitivity to external apoptotic stimuli, the results suggest that IAP antagonists, such as AEG40730, exert their apoptosis-enhancing effects through XIAP in addition to the c-IAPs. These results indicate that IAP antagonists can target multiple IAPs to augment distinct pro-apoptotic signalling pathways, thereby revealing the potential for these compounds in cancer therapy and underscoring the promise of IAP-targeted therapies.

VIAF, a Conserved Inhibitor of Apoptosis (IAP)-interacting Factor That Modulates Caspase Activation

Inhibitor of apoptosis (IAP) proteins are involved in the suppression of apoptosis, signal transduction, cell cycle control and gene regulation. Here we describe the cloning and characterization of viral IAP-associated factor (VIAF), a highly conserved, ubiquitously expressed phosphoprotein with limited homology to members of the phosducin family that associates with baculovirus Op-IAP. VIAF bound Op-IAP both in vitro and in intact cells, with each protein displaying a predominantly cytoplasmic localization. VIAF lacks a consensus IAP binding motif, and overexpression of VIAF failed to prevent Op-IAP from protecting human cells from a variety of apoptotic stimuli, suggesting that VIAF does not function as an IAP antagonist. VIAF was unable to directly inhibit caspase activation in vitro and a reduction of VIAF protein levels by RNA interference led to a decrease in Bax-mediated caspase activation, suggesting that VIAF functions to co-regulate the apoptotic cascade. Finally, VIAF is a substrate for ubiquitination mediated by Op-IAP. Thus, VIAF is a novel IAP-interacting factor that functions in caspase activation during apoptosis.

PHENOTYPIC DIFFERENCES BETWEEN MICE DEFICIENT IN XIAP AND SAP, TWO FACTORS TARGETED IN X-LINKED LYMPHOPROLIFERATIVE SYNDROME (XLP)

Mutations in the X-linked inhibitor of apoptosis (XIAP) have recently been identified in patients with the rare genetic disease, X-linked lymphoproliferative syndrome (XLP), which was previously thought to be solely attributable to mutations in a distinct gene, SAP. To further understand the roles of these two factors in the pathogenesis of XLP, we have compared mice deficient in Xiap with known phenotypes of Sap-null mice. We show here that in contrast to Sap-deficient mice, animals lacking Xiap have apparently normal NKT cell development and no apparent defect in humoral responses to T cell-dependent antigens. However, Xiap-deficient cells were more susceptible to death upon infection with the murine herpesvirus MHV-68 and gave rise to more infectious virus. These differences could be rescued by restoration of XIAP. These data provide insight into the differing roles of XIAP and SAP in the pathogenesis of XLP.

Diverse functions within the IAP family

Inhibitors of apoptosis (IAPs) are a diverse group of signaling molecules that function in a wide range of cellular roles, from the inhibition of caspases to the promotion of cell-cycle progression. These diverse functions may well be linked to the many different domains that are contained within

APOPTOTIC SENSITIVITY OF MURINE IAP-DEFICIENT CELLS

Although numerous studies have implicated the IAPs (inhibitor of apoptosis proteins) in the control of apoptotic cell death, analyses of murine Iap-targeted cells have not revealed significant differences in their susceptibility to apoptosis. In the present study, we show that, under defined conditions, murine cells lacking XIAP (X-linked inhibitor of apoptosis) and c-IAP (cellular IAP) 2, but not c-IAP1, exhibit heightened apoptotic sensitivity to both intrinsic and extrinsic apoptotic stimuli.

In Vitro Polarization of T-Helper Cells

In vitro polarization of CD4(+) T cells along distinct T-helper (Th) lineages is critical for defining the factors and properties that determine the differentiation, stability, and effector functions of each Th subset. Furthermore, polarized cells can be transferred into naïve syngeneic mice to investigate their trafficking patterns and pathological or therapeutic roles in the setting of infection, autoimmunity, and neoplasia. In this chapter, we describe methods for generating and characterizing a spectrum of CD4(+) Th cell lines in vitro. Protocols are provided that use naïve wild-type or T cell receptor (TCR) transgenic CD4+ T cells, or a polyclonal population of primed CD4+ T cells from immunized mice.