Mitsuko R. Ito

Urocortin and corticotropin-releasing factor receptor expression in the human colonic mucosa

Urocortin is a newly identified member of the CRF neuropeptide family. Urocortin has been found to bind with high affinity to CRF receptors. The present study investigated urocortin and CRF receptor expression in human colonic mucosa. Non-pathologic sections of adult colorectal tissues were obtained from patients with colorectal cancer at surgery. Urocortin expression was examined using immunohistochemistry and messenger (m) RNA in situ hybridization. Isolated lamina propria mononuclear cells (LPMC) and epithelial cells were also analyzed by flow cytometry for the characterization of urocortin-positive cells, and by RT-PCR for detection of urocortin, CRF, and CRF receptor mRNA. Urocortin peptide distribution at various stages of human development (n = 35, from 11 weeks of gestation to 6 years of age) was examined by immunohistochemistry using surgical and autopsy specimens. Immunoreactive urocortin and urocortin mRNA were predominantly detected in lamina propria macrophages. Urocortin peptide expression was detected from as early as three months of age, but not before birth or in neonates. Urocortin, CRF receptor type 1 and type 2 alpha mRNA were detected in LPMC. CRF receptor type 2 beta mRNA, a minor isoform in human tissues, was also detected in LPMC, but at lower levels. Urocortin is locally synthesized in lamina propria macrophages and may act on lamina propria inflammatory cells as an autocrine/paracrine regulator of the mucosal immune system. The appearance of urocortin after birth indicates that the exposure to dietary intake and/or luminal bacteria after birth may contribute to the initiation of urocortin expression in human gastrointestinal tract mucosa.

Severe hypercholesterolemia, impaired fat tolerance, and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E.

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had ∼60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were ∼3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced arthrosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.

A Signal Adaptor SLAM-Associated Protein Regulates Spontaneous Autoimmunity and Fas-Dependent Lymphoproliferation in MRL-Faslpr Lupus Mice

Autoantibody production and lymphadenopathy are common features of systemic autoimmune disease. Targeted or spontaneous mutations in the mouse germline have generated many autoimmune models with these features. Importantly, the models have provided evidence for the gene function in prevention of autoimmunity, suggesting an indispensable role for the gene in normal immune response and homeostasis. We describe here pathological and genetic characterizations of a new mutant strain of mice, the mutation of which spontaneously occurred in the Fas-deficient strain, MRL/Mp.Faslpr (MRL/lpr). MRL/lpr is known to stably exhibit systemic lupus erythematosus-like diseases. However, the mutant mice barely displayed autoimmune phenotypes, though the original defect in Fas expression was unchanged. Linkage analysis using (mutant MRL/lpr × C3H/lpr)F2 mice demonstrated a nucleotide insertion that caused loss of expression of small adaptor protein, signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). SAP is known to be a downstream molecule of SLAM family receptors and to mediate the activation signal for tyrosine kinase Fyn. Recent studies have shown pleiotropic roles of SAP in T, B, and NK cell activations and NKT cell development. The present study will provide evidence for an essential role for SAP in the development of autoimmune diseases, autoantibodies, and lymphadenopathy in MRL/lpr lupus mice.

Antagonist of Secondary Lymphoid-Tissue Chemokine (CCR Ligand 21) Prevents the Development of Chronic Graft-Versus-Host Disease in Mice

The use of receptor antagonists for chemokines is an alternative approach to blocking chemokine actions and has the potential to provide novel therapeutics. We determined the receptor antagonist properties of murine N-terminally truncated secondary lymphoid tissue chemokine (SLC)/6Ckine/CCR ligand 21 analogs and evaluated the preventive effects of SLC antagonists on chronic graft-vs-host disease (GVHD) in a murine model by blocking the homing of donor CCR7-expressing T cells into the recipient’s lymphoid organs. SLC analogs truncated >4 aa residues from the N terminus showed a loss of chemotaxis and Ca2+ influx of CCR7-expressing cells and also inhibited SLC-stimulated chemotaxis and SLC-induced Ca2+ influx completely. To determine whether SLC antagonist inhibits the development of chronic GVHD, chronic GVHD was induced by injecting DBA/2 spleen cells into (C57BL/6 × DBA/2) F1 mice. Total numbers of spleen cells and host B cells, serum levels of IgE, and of total IgG and IgG1 of anti-DNA Abs in SLC antagonist-treated GVHD mice were significantly lower than those in control PBS-treated GVHD mice. This was due to a reduction in the levels of activated donor CD4+ T cells and a decrease in IL-4 production, resulting in a reduction in the numbers of activated host B cells. Therefore, our results suggest that SLC antagonist has beneficial effects for the prevention of chronic GVHD.

Genetic dissection of the complex pathological manifestations of collagen disease in MRL/lpr mice

An MRL strain of mice bearing a Fas‐deletion mutant gene, lpr, MRL/MpJ‐lpr/lpr (MRL/lpr) develops collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren’s syndrome, respectively. Development of such lesions seems dependent on host genetic background since the congenic C3H/HeJ‐lpr/lpr (C3H/lpr) mice rarely develop them. To identify the gene loci affecting each lesion, a genetic dissection of these complex pathological manifestations was carried out. First, histopathological features in MRL/lpr, C3H/lpr, (MRL/lpr × C3H/lpr) F1 intercross, and MRL/lpr × (MRL/lpr × C3H/lpr) F1 backcross mice were analyzed. Genomic DNA of the backcross mice were subjected to association studies by Chi‐squared analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. As a result, gene loci recessively associated with each lesion were mapped on different chromosomal positions. We concluded that each of these lesions in MRL/lpr mice is under the control of a different set of genes, suggesting that the complex pathological manifestations of collagen disease result from polygenic inheritance.

Role of macrophages in the development of arteritis in MRL strains of mice with a deficit in Fas‐mediated apoptosis

The lpr and gld genes have been shown to encode the Fas antigen deletion mutant and the Fas ligand (FasL) mutant, respectively. An MRL strain of mice bearing the gld gene was observed to spontaneously develop granulomatous arteritis, similar to that in mice bearing the lpr gene, indicating that arteritis in this strain is due to an inefficient Fas–FasL interaction resulting in an incapacity for Fas‐mediated apoptosis. The arterial lesions in both strains were characterized by a remarkable perivascular accumulation of activated macrophages bearing Mac‐2 antigen, following the infiltration of CD4+ cells, and this resulted in the destruction of the arterial wall. Almost all of these infiltrating cells were Fas‐positive, as determined in MRL/gld mice. Macrophage colony‐stimulating factor (M‐CSF), which is present at increased levels in MRL/lpr mice, but not in MRL/Mp‐ +/+ (MRL/+) mice, induced the expression of Mac‐2 antigen and Fas antigen on spleen adherent cells of MRL/+ mice. Moreover, continuous infusion of M‐CSF into the peritoneal cavity or subcutis of MRL/+ mice induced the release of oxygen radicals of peritoneal macrophages or granuloma formation associated with the massive accumulation of Mac‐2+ cells, respectively. These findings suggest that macrophages in these mice, which may be activated by M‐CSF and may avoid Fas‐mediated apoptosis, play a critical role as effector cells in the destruction of arterial wall.

Bone marrow cell transfer of autoimmune diseases in a MRL strain of mice with a deficit in functional Fas ligand: Dissociation of arteritis from glomerulonephritis

MRL/MpTn‐gld/gld (MRL/gld) mice, which are deficient in a functional Fas ligand (FasL), spontaneously develop autoimmune diseases involving both lethal glomerulonephritis and systemic arteritis, while MRL/Mp‐+/+ (MRL/+) and C3H/HeJ‐gld/gld (C3H/gld) do not. To determine the cells responsible for the development of glomerulonephritis and arteritis, we transferred bone marrow cells from MRL/gld mice to undiseased MHC‐compatible gld/gld or +/+ mice. In bone marrow irradiation chimeras, MRL/gld bone marrow cells were transferred to lethally irradiated MRL/+ or C3H/HeJ‐+/+ (C3H/+) mice, and both recipients developed glomerulonephritis associated with hypergammaglobulinemia without causing graft‐versus‐host (GVH)‐like diseases. However, a striking difference between them was that MRL/+ recipients developed arteritis, but C3H/+ recipients did not. In bone marrow mixed chimeras formed by transferring MRL/gld bone marrow cells to unirradiated mice, the MRL/gld bone marrow cells induced glomerulonephritis in C3H/gld mice, but not in C3H/+ and MRL/+ mice. These results indicate that bone marrow cells from MRL/gld mice can cause glomerulonephritis in mice, even in those with a C3H background, possibly if they survive longer by escaping from Fas‐mediated apoptosis, while the development of arteritis requires the MRL genetic background in the re‐cipients. This is the first report of the transfer of arteritis in lupus mice to undiseased recipients.

Experimental lupus nephritis in severe combined immunodeficient (SCID) mice : remodelling of the glomerular lesions by bystander IgM antibodies

MRL/Mp‐lpr/lpr (MRL/lpr) mice develop glomerular lesions with regular variations in their histopathological manifestations, similar to those in lupus nephritis. These lesions are mainly either cell‐proliferative or wire loop‐like and are associated with glomerular deposits of immunoglobulins, most frequently IgG and IgM. We previously established a nephritogenic IgG3‐producing hybridoma clone, B1, from an MRL/lpr mouse, which induces only a ‘wire loop‐like’ type of glomerular lesion when injected into SCID mice. Injection of SCID mice with an anti‐trinitrophenyl IgM antibody‐producing hybridoma clone, Sp6, following injection of the B1 clone, however, resulted in the development of a ‘cell‐proliferative’ type of glomerular lesion, associated with an accumulation of both antibodies in glomeruli. This accumulation occurred even though Sp6 IgM antibodies did not react with B1 IgG3 antibodies and vice versa. A mutant clone of Sp6, T/13μE/3.1, which produces antibodies deficient in C1q binding, produced a similar effect as that of the Sp6 clone, i.e. ‘cell‐proliferative’ lesions. Again the B1 antibodies did not react with T/13μE/3.1‐IgM antibodies and vice versa. We therefore conclude that bystander IgM antibodies contribute to the remodelling of glomerular lesions in situ, following glomerular injury by the nephritogenic antibodies.

Vascular lesions in mice with a deficit in Fas-mediated apoptosis and their transfer

The lpr and gld genes are thought to result in an incapacity for Fas-mediated apoptosis of T and B cells and the development of subsequent autoimmune disease. A newly established gld-congenic strain of mice, MRL/MpTn-gld/gld (MRL/gld), was found to develop vascular lesions involving arteritis and glomerulonephritis (GN), which were similar to those observed in the MRL/Mp-lpr/lpr (MRL/lpr) strain. However, comparative studies with a C3H/HeJ strain bearing lpr or gld revealed that these lesions developed only in mice with an MRL background. We were successful in transferring GN to normal MHC-compatible gld/gld and irradiated +/+ mice by bone marrow cells of MRL/gld mice, but were unsuccessful using those of C3H/gld mice. Transfer of arteritis, however, was only successful in mice with an MRL background. Nephritogenic monoclonal antibodies obtained from an MRL/lpr and an MRL/gld mouse were shown to be bone marrow-derived and rich in clonal diversity, and at least two of these were capable of causing glomerular injury by different mechanisms. Development of GN and systemic arteritis in MRL/lpr and MRL/gld mice will be dependent not only on their incapacity for Fas-mediated apoptosis but also on bone marrow cells and peripheral cells with intrinsic defects.

Suppression of experimental lupus nephritis by aberrant expression of the soluble E-selectin gene

Circulating leukocytes, particularly neutrophils and monocytes, are important effector cells in the induction of many forms of glomerulonephritis. Adhesion molecules, especially selectins, are also thought to be critical for the development of this disease. We examined the possible suppressive effect of soluble E‐selectin on the development of experimental lupus nephritis induced by the injection of a hybridoma clone (2B11.3) derived from an MRL/MpJ‐lpr/lpr lupus mouse. This clone produces IgG3 antibodies that induce severe proliferative glomerulonephritis resembling lupus nephritis when injected into normal mice. Transgenic mice with a soluble E‐selectin gene were injected intraperitoneally with the hybridoma cells and histopathologically examined on day 15. As a result, the development of glomerulonephritis was significantly suppressed. This suppression was characterized by fewer inflammatory cell infiltrates, compared with non‐transgenic litter mates, despite the fact that there were no remarkable differences in immunoglobulin deposits or expression of E‐selectin between the two groups. These findings suggest that by controlling inflammatory cell infiltration, soluble E‐selectin plays a preventative role in the development of a particular type of lupus nephritis.