Expansion of CD22lo B cells in the spleen of autoimmune-prone flaky skin mice

Similar to murine models with compromised CD22/SHP-1 function, flaky skin (fsn) mutant mice exhibit lymphocyte hyperactivation and an autoimmune phenotype characterized by circulating autoantibodies to dsDNA and glomerulonephritis. Immunophenotyping of fsn/fsn splenic B cells was performed to determine if abnormalities in CD22 expression contributed to the phenotype. We identified an expansion of an IgM(bright) CD22lo population consistent with immature B-lymphocytes. While normal B-lymphocytes require IL-4 to achieve down-modulation of CD22 expression in response to BCR cross-linking, culture with anti-IgM alone led to reduced CD22 expression in fsn/fsn mice. Furthermore, when IL-4 was added to fsn/fsn cultures, no further reduction in CD22 expression was observed. This suggested that fsn/fsn B cells were pre-activated in vivo by chronic IL-4 exposure. A portion of these CD22lo cells expressed the B-1 surface marker CD11b. We contend that decreased activation thresholds among CD22lo B-lymphocytes contributes to the expansion of immature and B-1 B cell populations and to the development of autoimmune pathology in fsn/fsn mice.     

Characterization of early endosome antigen 1 in neural tissues

The finding that patients and mice bearing autoantibodies directed against early endosome antigen 1 (EEA1) develop neurological signs and deficits prompted an investigation of EEA1 distribution, localization, and interaction with synaptic proteins found in neural tissues. We detected EEA1 in a variety of neural tissues and in cells of neural origin where it co-localized with SNAP-25. The interaction between EEA1 and SNAP-25 was dependent on the leucine zipper and a newly identified methyl-accepting domain of EEA1. The C-terminal zinc-binding FYVE finger motif (EEA1(1271-1411)) of EEA1 also interacted with native SNAP-25 but only in the presence of 100microM Ca(2+). In contrast, EEA1 did not bind to cysteine string protein or synapsin in these binding assays. These results suggest that EEA1 is involved in neuronal synaptic vesicle function and axonal transport and growth. EEA1 may undergo calcium-dependent conformational changes that are required for binding to SNAP-25.     

Combined treatment with lisofylline and exendin-4 reverses autoimmune diabetes

Type 1 diabetes mellitus (T1DM) is an autoimmune disease leading to near complete pancreatic beta-cell destruction. New evidence suggests that beta-cell regeneration is possible, but ongoing autoimmune damage prevents restoration of beta-cell mass. We tested the hypothesis that simultaneously blocking autoimmune cytokine damage and supplying a growth-promoting stimulus for beta-cells would provide a novel approach to reverse T1DM. Therefore, in this study we combined lisofylline to suppress autoimmunity and exendin-4 to enhance beta-cell proliferation for treating autoimmune-mediated diabetes in the non-obese diabetic (NOD) mouse model. We found that this combined therapy effectively reversed new-onset diabetes within a week of therapy, and even maintained euglycemia up to 145 days after treatment withdrawal. The therapeutic effect of this regimen was associated with improved beta-cell metabolism and insulin secretion, while reducing beta-cell apoptosis. It is possible that such combined therapy could become a new strategy to defeat T1DM in humans.     

Mouse stefins A1 and A2 (Stfa1 and Stfa2) differentiate between papain-like endo- and exopeptidases

Stefin A (Stfa) acts as a competitive inhibitor of intracellular papain-like cysteine proteases which play important roles in normal cellular functions such as general protein turnover, antigen processing and ovarian follicular growth and maturation. In the mouse there are at least three different variants of Stfa (Stfa1, Stfa2 and Stfa3). Recent genetic studies identified structural polymorphisms in Stfa1 and Stfa2 as candidates for Aod1b, a locus controlling susceptibility to day three thymectomy (D3Tx)-induced autoimmune ovarian disease (AOD). To evaluate the functional significance of these polymorphisms, recombinant allelic proteins were expressed in Escherichia coli, purified and characterized. The polymorphisms do not markedly alter the folding characteristics of the two proteins. Stfa1 and Stfa2 both act as fast and tight binding inhibitors of endopeptidases papain and cathepsins L and S, however their interaction with exopeptidases cathepsins B, C and H was several orders of magnitude weaker compared to human, porcine and bovine Stfa. Notwithstanding, the K(i) values for the interactions of Stfa1-b from AOD resistant C57BL/6J mice was 10-fold higher than that of the Stfa1-a allele from susceptible A/J mice for papain, cathepsins B, C and H but not L and S. In contrast, the inhibitory activities of Stfa2-a and Stfa2-b were found to be roughly equivalent for all targets peptidases.     

Complement-dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein assembly in orthogonal arrays

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which binding of pathogenic autoantibodies (NMO-IgG) to astrocyte aquaporin-4 (AQP4) causes complement-dependent cytotoxicity (CDC) and inflammation. We previously reported a wide range of binding affinities of NMO-IgGs to AQP4 in separate tetramers versus intramembrane aggregates (orthogonal arrays of particles, OAPs). We report here a second, independent mechanism by which CDC is affected by AQP4 assembly. Utilizing lactate dehydrogenase release and live/dead cell cytotoxicity assays, we found in different cell lines, and with different monoclonal and patient-derived NMO-IgGs, that CDC was greatly (>100-fold) reduced in cells expressing M1- versus M23-AQP4. Studies using a M23-AQP4 mutant containing an OAP-disrupting mutation, and in cells expressing AQP4 in different M1/M23 ratios, indicated that NMO-IgG-dependent CDC requires AQP4 OAP assembly. In contrast, antibody-dependent cell-mediated cytotoxicity produced by natural killer cells did not depend on AQP4 OAP assembly. Measurements of C1q binding and complement attack complex (C9neo) supported the conclusion that the greatly enhanced CDC by OAPs is due to efficient, multivalent binding of C1q to clustered NMO-IgG on OAPs. We conclude that AQP4 assembly in OAPs is required for CDC in NMO, establishing a new mechanism of OAP-dependent NMO pathogenesis. Disruption of AQP4 OAPs may greatly reduce NMO-IgG dependent CDC and NMO pathology.     

Immunological functions of leptin and adiponectin

Recent years have seen several advances in our understanding of the functions of adipose tissue regarding not only the energy storage, but also the regulation of complex metabolic and endocrine functions. In this context, leptin and adiponectin, the two most abundant adipocyte products, represent one of the best example of adipocytokines involved in the control of energy expenditure, lipid and carbohydrate metabolism as well as in the regulation of immune responses. Leptin and adiponectin secretion is counter-regulated in vivo, in relation to degree of adiposity, since plasma leptin concentrations are significantly elevated in obese subjects in proportion to body mass index while adiponectin secretion decreases in relation to the amount of adipose tissue. In this review we focus on the main biological activities of leptin and adiponectin on the lipid and carbohydrate metabolism and on their contribute in regulation of innate and adaptive immune responses.     

Target specificity of an autoreactive pathogenic human γδ-T cell receptor in myositis

In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the αβ-T cell receptor (αβ-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the γδ-TCR (γδ-T cell-mediated myositis). We investigated the antigen specificity of a human γδ-TCR previously identified in an autoimmune tissue lesion of γδ-T cell-mediated myositis. We show that this Vγ1.3Vδ2-TCR, termed M88, recognizes various proteins from different species. Several of these proteins belong to the translational apparatus, including some bacterial and human aminoacyl-tRNA synthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from Escherichia coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short α-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both γδ-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized γδ-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a γδ-T cell and by autoantibodies reveals an unexpected link between T cell and antibody responses in autoimmune myositis.     

Roles of Hopx in the differentiation and functions of immune cells

Homeodomain only protein (Hopx, HOPX) is a highly evolutionarily conserved, homeodomain-containing, small protein expressed in multiple tissues and cell types, including those of hematopoietic origin. The quasi-ubiquitous presence of Hopx contrasts with its specialized and context-dependent roles in various cell lineages. Recently, versatile functions of Hopx have been revealed in immune cells, including T lymphocytes with effector and regulatory roles. The induction of Hopx expression can indicate early developmental and differentiation pathways, and early Hopx expression characterizes the recently identified pre-effector T cells that become destined for subsequent effector differentiation. Further, specific molecular mechanisms of Hopx are indispensable for the functional homeostasis of peripherally induced regulatory T cells (pTreg cells). Here we offer a perspective on these diverse roles of Hopx in immune cells and discuss the recent advances that helped to clarify the relevant functions and mechanisms of Hopx.