Autoimmune Mouse Strains Research Articles

Differences in T regulatory cells between mouse strains frequently used in immunological research: Treg cell quantities and subpopulations in NOD, B6 and BALB/c mice

Foxp3+ Regulatory T cells (Tregs) are essential for the maintenance of tolerance to self. Therefore, it is expected that lower numbers and/or less than optimal function could impact on the functioning of the immune system, and thereby contributing to the development of autoimmune diseases. In the present report, by comparing Tregs from most frequently used mouse strains in immunological research (C57BL/6 (B6), BALB/c and NOD), we provide evidence showing that the NOD mouse strain, highly predisposed to develop autoimmune responses, exhibit a generalized decreased in Tregs counts with enhanced proportions of CD44hiCD62Llow Tregs when compared with BALB/c mice. No major differences were observed in Helios+ or Helios- Tregs between strains. The expression of CXCR3, CCR5 and CCR6 on Tregs from all strains showed minor proportions of CXCR3+ and CCR5+ cells in NOD Tregs. Naïve CD4+CD25- T cells from NOD mice also showed decreased capacity to induce in vitro iTregs when compared with B6 and BALB/c mice. Lower expression of molecules involved in Treg suppressor mechanisms such as CD25, LAP-1, CD39 and PD-1 was observed both in NOD iTregs and Tregs from lymph nodes of NOD mice. Moreover, in vitro assays showed that Tregs from NOD mice exhibited reduced ability to suppress proliferation of CD4+CD25- responder T cells when compared with B6 and BALB/c mice. Major differences were consistently observed between NOD and BALB/c mice, whereas no major differences were found for many of the analyzed parameters between the NOD and B6 mice, suggesting that highly and mildly autoimmune prone mouse strains may share some Tregs features. On the contrary, BALB/c Tregs were in major quantities, expressed higher levels of Foxp3 and exhibited more potent ability to inhibit effector T cell proliferation, data that could be related to its natural resistance to the induction of different experimental autoimmune conditions. Altogether our results demonstrate a generalized Treg cell dysfunction in NOD mice, a strain characterized by its high predisposition to develop spontaneous and induced autoimmune diseases.

MRL Strains Have a BAFFR Mutation without Functional Consequence.

It has been shown that B cell activating factor receptor (BAFFR) is critical for B cell development and survival. In this study, we sought to evaluate the expression and function of BAFFR across multiple stains of mice that vary in their potential to develop systemic autoimmune disease. The inability of a commercial antibody to bind to BAFFR in the autoimmune prone mouse strains, MRL and MRL/Lpr led to the discovery of a mutation in TNFRSF13C gene (encoding BAFFR) that resulted in a Pro44Ser substitution in the N-terminus near the BAFF binding site in these strains. To define the biological consequences of mutant BAFFR, we compared the expression and activity of BAFFR in MRL and MRL/Lpr mice to BALB/c, which express the consensus version of TNFRSF13C. B cells from MRL and MRL/Lpr mice expressed mutant BAFFR on surface and were capable of responding to BAFF as exhibited by BAFF-mediated reduction in apoptosis and NF-κB2 activation. Signaling through MAPK ERK1/2 was not significantly induced by BAFF in MRL/Lpr mice; however, MAPK ERK1/2 signaling was intact in MRL mice. The inability of MRL/Lpr B cells to significantly activate ERK1/2 in response to BAFF was due to the high basal activity of the signaling pathway in these cells. In fact, basal activity of ERK1/2 in B cells correlated with the degree of autoimmune susceptibility exhibited by each strain. In addition, aged MRL/Lpr mice with severe autoimmune disease had high BAFF levels, low surface BAFFR, and high basal NF-κB2 activation, a pattern which is attributed to the high frequency of antibody secreting cells. We conclude that P44S BAFFR mutation does not hinder BAFFR function or enhance B cell activity in MRL/Lpr and MRL mice and that other susceptibility loci on the MRL background contributed to the hyperactivity of these cells.

Mechanisms of Coagulation-Complement pathway interplay in ischemia/reperfusion induced tissue injury.

Abstract Impaired fibrinolysis in systemic lupus erythematosus (SLE) is a contributing factor in disease complications including hyper-coagulation, tissue ischemia/reperfusion (IR), and increased risk of thrombosis: including stroke and deep vein thrombosis. Aberrant complement activation has been identified as a major factor in tissue injury in SLE. Previous work in the laboratory focused on mechanisms of complement activation in IR induced tissue injury in B6.MRL/lpr mice, an autoimmune prone mouse strain with a propensity to develop lymphadenopathy, glomerulonephritis, and polyarthritis. This work identified a C3-independent mechanism of downstream complement C5a generation contributing to tissue injury. Evidence of interplay between the coagulation and complement pathways was identified. Current work elucidates the mechanism by which the two pathways interact. Using a superior mesenteric artery IR model, we utilized complement and specific extrinsic or intrinsic coagulation pathway inhibitors to evaluate and compare resulting tissue pathology, generation of secondary inflammatory mediators, extent of innate immune activation, and generation of breakdown products involved in regulating feedback regulatory loops after ischemic/reperfusion injury. Inhibition of the extrinsic pathway had a greater impact on downstream complement activation, tissue pathology, vascular integrity, and recruitment of innate immune components versus the inhibition of the extrinsic pathway. Understanding the mechanisms of interplay between the pathways in the autoimmune mouse has significant clinical implications – especially in autoimmune diseases in which inflammation and hypercoagulability are known manifestations.

Correcting the NLRP3 inflammasome deficiency in macrophages from autoimmune NZB mice with exon skipping antisense oligonucleotides.

Inflammasomes are molecular complexes activated by infection and cellular stress, leading to caspase-1 activation and subsequent interleukin-1β (IL-1β) processing and cell death. The autoimmune NZB mouse strain does not express NLRP3, a key inflammasome initiator mediating responses to a wide variety of stimuli including endogenous danger signals, environmental irritants and a range of bacterial, fungal and viral pathogens. We have previously identified an intronic point mutation in the Nlrp3 gene from NZB mice that generates a splice acceptor site. This leads to inclusion of a pseudoexon that introduces an early termination codon and is proposed to be the cause of NLRP3 inflammasome deficiency in NZB cells. Here we have used exon skipping antisense oligonucleotides (AONs) to prevent aberrant splicing of Nlrp3 in NZB macrophages, and this restored both NLRP3 protein expression and NLRP3 inflammasome activity. Thus, the single point mutation leading to aberrant splicing is the sole cause of NLRP3 inflammasome deficiency in NZB macrophages. The NZB mouse provides a model for addressing a splicing defect in macrophages and could be used to further investigate AON design and delivery of AONs to macrophages in vivo.

The complete mitochondrial genome for an autoimmune thyroiditis mouse strain NOD.H-2h4

Autoimmune thyroiditis is a chronic organ-specific autoimmune disease characterized by mononuclear cell infiltration of the thyroid gland and destruction of thyroid follicles by the infiltrating inflammatory cells. We sequenced the complete mitochondrial genome sequencing of an autoimmune thyroiditis mouse strain NOD.H-2h4 for the first time. The total length of the mitogenome was 16,310 bp and contains 159 SNPs compared with the house mouse reference sequence.

Analysis of the misregulation of RhoA and RhoH in autoimmune mice. (HUM7P.302)

Abstract When cultured in the presence of apoptotic cells, cell bodies or lipids, RhoA is misregulated in macrophages from all 6 autoimmune mouse strains but none of the 7 normal strains tested. Misregulation of RhoA results in defective morphology, altered cytokine production, and increased adhesion. The same macrophages cultured without apoptotic cell lipids had properly regulated RhoA and behaved normally. Normal mouse macrophages cultured with the Rho inhibitor C3 toxin, had defective macrophage morphology and behavior. Therefore, we proposed that autoimmunity is due to an apoptotic cell recognition signaling defect and that normal macrophage function may be restored by a chemical reversal of the defect. Our new data demonstrate that lupus strain, MRL, macrophages cultured in the presence of C3 no longer had defects in morphology and function. The fact that a RhoA inhibitor restored RhoA function indicates that the misregulation in autoimmune macrophages involves a RhoA regulator also affected by C3 and not RhoA itself. RhoH is a known inhibitor of RhoA and it is only expressed in hematopoietic cells. Experiments comparing RhoH expression in BALB and MRL macrophages suggest at least four different sizes of RhoH protein are expressed in normal macrophages in response to lipid exposure and that lupus macrophages do not express the multiple forms of RhoH even in the presence of lipids. A lack of proper RhoH expression could be behind the autoimmune macrophage defect.

Contribution of VH Replacement Products in Mouse Antibody Repertoire

VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

Analysis of Hematopoietic Stem Cell populations in control and autoimmune mouse strains. (111.48)

Abstract Hematopoietic stem cells (HSC) are cells capable of self renewal and multilineage differentiation. Flow cytometry is often used to isolate very pure populations of HSC. To successfully isolate live HSC using FACS, cells are typically identified using a combination of cell surface markers, a dye or both. Several methods have successfully been used for the isolation of these cells in mice, including KLS (c-kit/Sca-1/Lineage negative), side population (Hoechst Red vs. Hoechst Blue), and more recently the SLAM panel (CD150+/CD48-/CD41-). It has been demonstrated that transplantation of all these cell populations results in bone marrow reconstitution in lethally irradiated mice. Using combinations of these signatures, we examined various mouse strains to determine whether there were strain-specific differences in the HSC populations. Specifically, we isolated HSCs by FACS from two control (C57BL/6 and Balb/C) and two autoimmune (SJL and NZB) strains. The data suggest that certain signatures are more suitable for the isolation of prospective HSC populations from certain strains of mice and that the HSC compartment is variable is size from strain to strain. In addition we will discuss interesting findings with respect to expression of these markers in the spleens of the different strains.

CD40 glycoforms and TNF-receptors 1 and 2 in the formation of CD40 receptor(s) in autoimmunity

The CD40–CD154 dyad is an intensely studied field as is glycosylation status and both impact immunological functions and autoimmune conditions. CD40 has several isoforms, is modified by glycosylation, and trimerizes to form the functional receptor. We described a CD4+CD40+ T cell (Th40) subset which is expanded in autoimmunity and is necessary and sufficient in transferring type 1 diabetes. Glycosylation impacts immunological events and T cells from autoimmune mouse strains express 30–40% less GlcNAc-branched N-glycans than T cells from non-autoimmune strains, a decrease known to activate T cells. Here we demonstrate that several CD40 receptor constellations exist on CD4 T cells. However, rather than containing different isoforms of CD40 they contain different glycoforms of isoform I. The glycoform profile is dependent on availability of CD154 and autoimmune NOD mice express a high level of a less glycosylated form. Interestingly, CD40 stimulation induces some CD40 receptor constellations that contain TNF-receptors 1 and 2 and targeting of those alters CD40 signaling outcomes in NOD Th40 cells. CD40-stimulation in vivo of non-autoimmune BALB/c mice expands the Th40 population and alters the CD40 glycoform profile of those cells to appear more like that of autoimmune prone NOD mice.Further understanding the dynamics and composition of the different CD40 receptor constellations will provide important insights into treatment options in autoimmunity.

Vκ polymorphisms in NOD mice are spread throughout the entire immunoglobulin kappa locus and are shared by other autoimmune strains

The diversity of immunoglobulin (Ig) and T cell receptor (TCR) genes available to form the lymphocyte repertoire has the capacity to produce a broad array of both protective and harmful specificities. In type 1 diabetes (T1D), the presence of antibodies to insulin and other islet antigens predicts disease development in both mice and humans, and demonstrate that immune tolerance is lost early in the disease process. Anti-insulin T cells isolated from T1D-prone non-obese diabetic (NOD) mice use polymorphic TCRalpha chains, suggesting that the available T cell repertoire is altered in these autoimmune mice. To probe whether insulin-binding B cells also possess polymorphic V genes, Ig light chains were isolated and sequenced from NOD mice that harbor an Ig heavy chain transgene. Three insulin-binding Vkappa genes were identified, all of which were polymorphic to the closest germline sequence matches present in the GenBank database. Additional analysis of over 300 light chain sequences from multiple sources, including germline DNA, shows that polymorphisms are spread throughout the entire NOD Igkappa locus, as these polymorphic sequences represent 43 distinct Vkappa genes which belong to 14 Vkappa families. Database searches reveal that a majority of polymorphic Vkappa genes identified in NOD are identical to Vkappa genes isolated from SLE-prone NZBxNZW F1 or MRL strains of mice, suggesting that a shared Igkappa haplotype may be present. Predicted amino acid changes preferentially occur in CDR, and thus could alter antigen recognition by the germline B cell repertoire of autoimmune versus non-autoimmune mouse strains.

Endogenous humoral autoreactive immune responses to apoptotic cells: Effects on phagocytic uptake, chemotactic migration and antigenic spread

Enhanced cell death and deficient clearance of cellular debris are thought to contribute to increased self-antigen exposure in systemic autoimmune disease. To investigate the characteristics of early humoral autoimmune responses, six monoclonal antibodies were generated from two autoimmune prone strains of mice. All antibodies specifically bound the surface of late-stage apoptotic cells. Similar antibody reactivities were present in the sera of patients with systemic lupus erythematosus. While IgM antibodies significantly reduced the phagocytic uptake of apoptotic thymocytes, IgG antibodies enhanced uptake. Poly-reactivity was demonstrated in the recognition of ribonucleoproteins and lipids. An antibody reactive towards lysophosphatidylcholine reversed lysophosphatidylcholine-mediated inhibition of LPS-induced TNF-alpha production and adversely affected the transmigration of phagocytes towards an apoptotic stimulus. In several instances, CDR were characterized by the accumulation of somatic mutations. Anti-idiotypic antibodies generated upon immunization bound distinct cellular moieties and self-antigens. Poly-specific, apoptotic cell-reactive autoantibodies can therefore directly impact upon the course of disease by influencing phagocytic uptake of apoptotic cells, by inducing a pro-inflammatory environment through neutralization of bioactive lipids, by blinding phagocytes to the presence of dying cells through the negation of lipidic chemotactic signals, and by mediating diversification of the humoral autoimmune response via the idiotypic network.

Expansion and Hyperactivity of CD1d-Restricted NKT Cells during the Progression of Systemic Lupus Erythematosus in (New Zealand Black × New Zealand White)F1 Mice

CD1d-restricted NKT cells expressing invariant TCR alpha-chain rearrangements (iNKT cells) have been reported to be deficient in humans with a variety of autoimmune syndromes and in certain strains of autoimmune mice. In addition, injection of mice with alpha-galactosylceramide, a specific glycolipid agonist of iNKT cells, activates these T cells and ameliorates autoimmunity in several different disease models. Thus, deficiency and reduced function in iNKT cells are considered to be risk factors for the development of such diseases. In this study we report that the development of systemic lupus erythematosus in (New Zealand Black (NZB) x New Zealand White (NZW))F(1) mice was paradoxically associated with an expansion and activation of iNKT cells. Although young (NZB x NZW)F(1) mice had normal levels of iNKT cells, these expanded with age and became phenotypically and functionally hyperactive. Activation of iNKT cells in (NZB x NZW)F(1) mice in vivo or in vitro with alpha-galactosylceramide indicated that the immunoregulatory role of iNKT cells varied over time, revealing a marked increase in their potential to contribute to production of IFN-gamma with advancing age and disease progression. This evolution of iNKT cell function during the progression of autoimmunity may have important implications for the mechanism of disease in this model of systemic lupus erythematosus and for the development of therapies using iNKT cell agonists.

Impaired migration of NOD mouse thymocytes: a fibronectin receptor-related defect.

We previously showed intrathymic alterations in non-obese diabetic (NOD) mice, including the appearance of giant perivascular spaces, filled with mature thymocytes, intermingled with an extracellular matrix network. This raised the hypothesis of a defect in thymocyte migration with partial arrest of exiting thymocytes in the perivascular spaces. Herein, we investigated the expression of receptors for fibronectin [very late antigen (VLA)-4 and VLA-5] and laminin (VLA-6), known to play a role in thymocyte migration. When compared with two normal and one other autoimmune mouse strains, a decrease of VLA-5 expression in NOD thymocytes was noticed, being firstly observed in late CD4/CD8 double-negative cells, and more pronounced in mature CD4(+) and CD8(+) thymocytes. Functionally, thymocyte exit from the lymphoepithelial complexes, the thymic nurse cells, was reduced. Moreover, NOD thymocyte adhesion to thymic epithelial cells as well as to fibronectin was diminished, and so was the migration of NOD thymocytes through fibronectin-containing transwell chambers. In situ, intra-perivascular space thymocytes were VLA-5-negative, suggesting a correlation between the thymocyte arrest within these structures and loss of VLA-5 expression. Overall, our data reveal impairment in NOD thymocyte migration, and correspond to the first demonstration of a functional fibronectin receptor defect in the immune system.

Surprisingly minor influence of TRAV11 (Valpha14) polymorphism on NK T-receptor mCD1/alpha-galactosylceramide binding kinetics.

Defects in natural killer T (NK T) cell function and of interleukin-4 -production in SJL and NOD mice have been linked to susceptibility to autoimmune disease. As SJL and NOD mice both carry the T-cell receptor (TCR) alpha-chain locus "c" (Tcra(c)) haplotype, found in few other strains, we have attempted to determine the influence of Tcra polymorphism on NK T-cell recognition of ligand, selection, and immune responses. The majority of NK T cells use an "invariant" TRAV11J15 (previously called AV14J18 or Valpha14 Jalpha281) alpha- chain paired with either TRBV13-2, BV29, or BV1 to recognize ligands presented by mCD1 molecules, including the glycolipid alpha-galactosylceramide (alpha-GalCer). Sequencing of TRAV11 from the mouse strains B10.A (encoding the Tcra(b) haplotype), B10.A- Tcra(c), and NOD (Tcra(c)) shows that Tcra(c) has a single TRAV11 gene (TRAV11*01) and that Tcra(b) has a single expressed gene (TRAV11*02), plus a closely related pseudogene. There is no apparent difference in alpha-chain J-region usage or in the CDR3alpha sequence at the TRAV11-J15 junction between the haplotypes in TRAV11-bearing NK T cells. Using Biacore and tetramer-binding and decay assays, we have determined that the interaction between Tcra(c) TRAV11*01 NK T TCR and the mCD1/alpha-GalCer complex is slightly weaker than that of Tcra(b) (i.e., TRAV11*02) NK T TCR. These differences are minor compared with differences between agonist and antagonist ligands in other TCR systems, suggesting that it is unlikely that TCR polymorphism explains the defect in NK T cells in the autoimmune mouse strains.

Structure of complement receptor (CR) 2 and CR2-C3d complexes.

Using X-ray crystallography, we have determined the structure of the first two short consensus repeats (SCRs) of human complement receptor (CR) 2 in complex with C3d. These studies revealed: (i) a primary site of interaction for C3d within SCR2 of CR2, (ii) a hydrophobic patch holding SCR1 to SCR2 in a rigid V-shape, (iii) a dimer formed by interactions between SCR1 of each molecule, (iv) several non-linear sequences on C3d that interact with CR2 and (v) mutations of C3d amino acids within the co-crystal interface that resulted in decreased binding. In addition, a polymorphism that results in decreased C3d binding and introduces a new glycosylation site predicted to disrupt the dimer interface was found in the New Zealand White autoimmune mouse strain. Although the co-crystal complex results are in agreement with a subset of prior studies, our additional findings, which demonstrate an extended SCR1-SCR2 structure in solution and differences in the kinetics of ligand-receptor interactions with longer forms of CR2, have suggested a more complex receptor-ligand interaction. To characterize this interaction further, several approaches directed at the determination of solution phase interactions as well as the analysis of the three-dimensional structure of CR2 alone and key CR2 mutants will be necessary.

Evolution of catalytic antibody repertoire in autoimmune mice

We have attempted to efficiently obtain catalytic antibodies (catAbs) with amidase/esterase activity in the expanded sequence space of the antibody repertoire. In doing so, we used an autoimmune mouse strain, MRL/ lpr, that is known to produce enhanced levels of autoantibodies. We applied different types of haptens, such as alkylphosphonate ( 1) , alkylphosphonamidate ( 2) , alkylphosphonamidate (OMe) ( 3) , and difluoroketone ( 4) , that are supposed to mimic the transition state of the substrate in the ester/amide hydrolysis. Among them, hapten ( 2 ) could not be used, as it was readily broken down after synthesis. Upon immunization with hapten ( 1 ), catAbs preferentially evolved in MRL/ lpr mice, but this did not happen upon immunization with haptens ( 3 ) and ( 4 ). Independently, immunization to MRL/ lpr mice with vitamin B 6 phosphonate hapten ( 5) successfully elicited the catAbs with the ability to activate vitamin B 6 prodrugs. The common observation seen in these two cases is that most of the catAbs derived from MRL/ lpr mice by hapten ( 1 ) and half of them by hapten ( 5 ) had a Lys at H95, which is at the junctional N region between the V H and J H gene segments. Despite the conservation of Lys (H95), analyses of the N-region and utilization of the D gene segment in the heavy chain gene showed that these catAbs were from several independent clones of the same family. Studies of site-directed mutagenesis suggest that, in the catAbs elicited from hapten ( 1 ), a Lys (H95) and a His (L91) are involved in the catalytic function. Both residues are known to interact with the phosphonate moiety of hapten ( 1 ). Such studies also suggest that, in the catAbs elicited from hapten ( 5 ), a Lys (H95) and a His (H35) are involved in the catalytic function. These basic amino acids seem to be important for binding to the phosphonate hapten, as they were not changed even after extensive evolution following multiple mutations. By contrast, in normal BALB/c mice, immunization of hapten ( 1 ) resulted in eliciting catAbs in lower yield and the majority were the non-catAbs, whose sequences were quite different from those of the catAbs from MRL/ lpr mice. They were clonally related to one another and most of them originated from a single clone. The positions of the interacting key residues in the CDRs that interact with the phosphorus moiety strongly differ between our catAbs and other reported catAbs with esterase/amidase activity, which were elicited by the phosphonate/phosphonamidate haptens from normal mice. Further comparison of antibodies elicited by the phosphorus haptens, such as DNA, RNA, phosphocholine, and phosphotyrosine, indicated that none of them had sequence similarity in the basic amino acids and their positions in the CDRs, except for one example, which is anti-DNA antibody elicited from C3H- lpr mice. Analysis based on the classification of canonical structures of the antibodies again suggested that our catAbs derived from MRL/ lpr mice belong to an unusual class that is not listed in the literature. Taken together, the above evidence suggests that the unique catalytic subsets that existed in the initial repertoire in the MRL/ lpr mice could effectively be captured by the phosphonate haptens through the interaction with the Lys at H95. In the BALB/c mice, however, another noncatalytic subset with an ability to bind only to a moiety other than the phosphonate moiety alternatively evolved, because of the lowest abundance or elimination of the catalytic subsets.

Cevimeline hydrochloride hydrate (Saligren capsule 30 mg): a review of its pharmacological profiles and clinical potential in xerostomia

Cevimeline hydrochloride hydrate is a muscarinic receptor agonist with a chemical structure of a quinuclidine. Intraduodenal administration of cevimeline hydrochloride hydrate dose-dependently increased salivary secretion in normal mice and rats, two strains of autoimmune disease mice, and X-irradiated rats. The clinical efficacy of the cevimeline hydrochlide hydrate at 30 mg t.i.d. during 4 weeks has been demonstrated in double blind comparative study with placebo. In addition, its treatments in 52 weeks have increased salivary flow and improved subjective and objective symptoms of patients with xerostomia in Sjögren's syndrome.

Spontaneous formation of germinal centers in autoimmune mice

The mechanisms of autoantibody production are not well understood. Germinal centers (GC) may be important sites of immune disregulation in autoimmune diseases. In this study, we document the presence of spontaneous GC formation in the spleens of several autoimmune mouse strains that spontaneously develop autoimmune Type I diabetes and a lupus-like disease. In contrast, mouse strains that do not develop lupus did not exhibit spontaneous formation of GC. In all of the autoimmune strains studied, GC were present at 1-2 months of age, a time that closely parallels the appearance of autoantibodies. Like the GC that develop after purposeful immunization, GC in autoimmune mice contained B220(+), PNA(+), and GL-7(+) B cells, and FDC-M1(+) follicular dendritic cells. In addition, spontaneously formed GC in autoimmunity and those caused by immunization were abrogated in a similar way by a short-term treatment with anti-CD40 ligand antibody. These data indicate that spontaneously forming GC in autoimmunity are similar to those appearing after purposeful immunization.

Altered Circadian Rhythms of the Stress Hormone and Melatonin Response in Lupus-prone MRL/MP- fas Ipr Mice

The immune system interacts with the hypothalamo-pituitary-adrenal axis via so-called glucocorticoid increasing factors, which are produced by the immune system during immune reactions, causing an elevation of systemic glucocorticoid levels that contribute to preservation of the immune reactions specificities. Previous results from our laboratory had already shown an altered immuno-neuroendocrine dialogue via the hypothalamo-pituitary-adrenal axis in autoimmune disease-prone chicken and mouse strains. In the present study, we further investigated the altered glucocorticoid response via the hypothalamo-pituitary-adrenal axis in murine lupus. We established the circadian rhythms of corticosterone, dehydroepiandrosterone-sulfate, adrenocorticotropic hormone and melatonin, as well as the time response curves after injection of interleukin-1 of the first three parameters in normal SWISS and lupus-prone MRL/MP- fas Ipr mice. The results show that lupus-prone MRL/MP- fas Ipr mice do not react appropriately to changes of the light/dark cycle, circadian melatonin rhythms seem to uncouple from the light/dark cycle, and plasma corticosterone levels are elevated during the resting phase. Diurnal changes of dehydroepiandrosterone-sulfate and adrenocorticotropic hormone were normal compared to healthy controls. These data indicate that MRL/ MP- fas Ipr mice not only show an altered glucocorticoid response mediated via the hypothalamo pituitary adrenal axis to IL-1, but are also affected by disturbances of corticosterone and melatonin circadian rhythms. Our findings may have implications for intrathymic T cell development and the emergence of autoimmune disease.

Abnormal cochlear connective tissue mineralization in the Palmerston North autoimmune mouse

Inner ear fibrosis and osteogenesis are common features of human autoimmune disease, although the cellular mechanisms are unknown. The Palmerston North (PN) autoimmune strain mouse has been shown to develop modiolar sclerotic lesions with progression of its systemic disease. Therefore, lesion development was studied in the cochleas of PN mice to gain insight into potential autoimmune osteogenic processes in the human ear. Cochleas from PN mice were examined with electron microscopy to characterize the cellular and extracellular matrix changes that lead to abnormal mineralization. Initially, activated fibroblasts produced extracellular matrix fibers, ranging in size from fine fibrils to larger collagen-like fibers. These proliferating fibers appeared to ‘seed’ the mineralizing lesions by serving as the framework for mineral deposition. As mineralization continued, the foci grew in size and fused to form large sclerotic masses within the connective tissue. However, the lesions never invaded nor degraded the normal modiolar bone. These observations of abnormal mineralization of cochlear connective tissue fibers show some parallels with human cochlear autoimmune osteogenesis, suggesting similar molecular processes may be involved.