Association between E-selectin expression and histopathological modification of glomerular lesions by non-nephritogenic IgM antibodies in experimental lupus nephritis.

OBJECTIVE: To examine the role played by E-selectin in bystander IgM-mediated modification of glomerular lesions in experimental lupus nephritis. METHODS: Experimental lupus SCID mice were induced by an intraperitoneal injection of clone 7B6.8, which was derived from a MRL/lpr mouse and shown to induce wire-loop type glomerular lesions. Mice were subsequently administered clone Sp6, a non-nephritogenic IgM antibody- producing hybridoma. E-selectin expression was then evaluated in glomeruli showing histopathological conversion of lesions from wire-loop-like to a cell-proliferative form. We also investigated the effects of a circulating soluble form of E-selectin (sE-selectin) on the modification of glomerular lesions in this lupus model. RESULTS: In experimental lupus mice, glomerular E-selectin expression significantly increased during the conversion from wire-loop-like glomerular lesions to a cell-proliferative type mediated by a non-nephritogenic bystander IgM antibody in presence of a nephritogenic antibody. Intraglomerular infiltration of CD68 + macrophages correlated significantly with the glomerular level of E-selectin expression. In addition, overexpression of circulating sE-selectin significantly suppressed conversion to cell-proliferative glomerular lesions and glomerular macrophage infiltration in these lupus model mice. CONCLUSIONS: The histopathological modification of lupus nephritis by non-nephritogenic bystander IgM antibodies is associated in part with glomerular E-selectin expression.

Evidence for reactivation of human herpesvirus 6 in generalized lymphadenopathy in a patient with drug-induced hypersensitivity syndrome.

The present case provides direct evidence of human herpesvirus 6 reactivation in resected lymph node tissue in a patient with drug-induced hypersensitivity syndrome. This case clearly demonstrates that appropriate pathological evaluation of lymphadenopathy for drug-induced hypersensitivity syndrome, which mimics malignant lymphoma in clinical, radiological, and pathological findings, is required.

Enhanced expression of the soluble form of E-selectin attenuates progression of lupus nephritis and vasculitis in MRL/lpr mice.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that causes inflammatory tissue damage, including lupus nephritis and vasculitis. Local generation of adhesion molecules and expression of their ligands on inflammatory cells appears to contribute to the progression of SLE. We found significantly increased E-selectin expression in the glomeruli and renal interstitial microvasculature of MRL/MpJ-lpr/lpr (MRL/lpr) lupus model mice. This was accompanied with infiltration of inflammatory cells, especially macrophages and CD8(+) T cells. Similarly, in 21 patients with proliferative lupus nephritis, there was a significant correlation between renal E-selectin levels and macrophage and CD8(+) T cell infiltration in the affected kidneys. By contrast, in transgenic MRL/lpr mice exhibiting elevated levels of circulating soluble E-selectin (sE-selectin) protein, which competitively inhibits E- and P-selectin-mediated extravasation of inflammatory cells, the progression of lupus nephritis and vasculitis was significantly suppressed and survival was significantly prolonged. This improvement was accompanied by significant reductions in renal infiltration by macrophages and CD8(+) T cells. These results suggest that E-selectin plays a crucial role in lupus nephritis and vasculitis by mediating renal infiltration of inflammatory cells, and that because it inhibits this process, sE-selectin could potentially serve as an effective treatment for lupus nephritis and vasculitis.

Fractalkine expression and CD16+ monocyte accumulation in glomerular lesions: association with their severity and diversity in lupus models.

Fractalkine (Fkn) is expressed on injured endothelial cells and is a membrane-bound chemokine that attracts cells expressing its receptor, CX3CR1, including CD16(+) monocytes (CD16(+) Mos). To clarify the role played by Fkn in the development of glomerular lesions in lupus nephritis, we examined Fkn expression and CD16(+) Mo accumulation induced in experimental C.B-17/Inc-scid/scid (SCID) lupus model mice by injection of IgG(3)-producing hybridoma clones obtained from MRL/lpr mice. Glomerular Fkn expression and accumulation of CD16(+) Mos were semiquantitatively evaluated using laser capture microdissection and real-time PCR. Injection of the 2B11.3 and 7B6.8 clones induced formation of glomerular proliferative and wire-loop lesions, respectively. Immunohistological analysis of the localization of Fkn and CD16(+) Mos revealed that Fkn expression and CD16(+) Mo accumulation were markedly elevated in glomerular lesions induced by 2B11.3, whereas no elevation was detected in those induced by 7B6.8. In addition, to examine the contribution of glomerular Fkn to the development of proliferative lesions, L cells producing an Fkn antagonist (Fkn-AT) were transplanted into SCID mice exhibiting proliferative lupus nephritis (DPLN) induced by 2B11.3. Notably, transplantation of the Fkn-AT-producing cells was functionally and histologically protective against this DPLN. Taken together, our findings suggest that Fkn and CD16(+) Mo accumulation are partially associated with the severity and diversity of histology of lupus nephritis.

Evaluating the role of rheumatoid factors for the development of rheumatoid arthritis in a mouse model with a newly established ELISA system.

Enzyme-linked immunosorbent assays (ELISA) have been widely used to determine quantitatively autoantibodies. However, the processes for the purification and immobilization of antigens in conventional ELISA methods include multiple steps, which have hampered the application for screening of autoantibodies. Here, we have developed a novel ELISA system using the plates pre-coated with glutathione casein to capture recombinant proteins fused to N-terminal glutathione S-transferase (GST). The GST-fused proteins were synthesized with the wheat germ cell-free protein production system. Thus, the present system combined the GST-capture ELISA with the cell-free protein production system, which allowed immobilization of the recombinant proteins with one-step purification. Using this ELISA method, we determined whether rheumatoid factors (RF), which have been considered as one of the representative disease-specific autoantibodies for rheumatoid arthritis (RA), were genetically associated with severity of arthritis in a mouse model for RA, MRL/Mp-lpr/lpr (MRL/lpr). GST-fused human IgG1-Fc (GST-Fc), synthesized with the robotic protein synthesizer, were used as reactants for RF. Serum samples for RF were prepared from 11 lines of a recombinant inbred mouse strain, MXH/lpr, which was established from intercrosses between MRL/lpr and non-arthritic C3H/HeJ-lpr/lpr (C3H/lpr) strains, composed of a different genomic recombination derived from the parental strains in each line. A correlation of RF titers with the severity of the arthritis in these lines was not significant, indicating genetic dissociation of RF from arthritis and that RF is not necessarily required for the development of RA. The present method may provide high-throughput screening for determining the disease-specific autoantibodies in autoimmune diseases.

Toll-like receptor 3 signaling induces chronic pancreatitis through the Fas/Fas ligand-mediated cytotoxicity.

Innate immunity plays important roles in host defense against pathogens, but may also contribute to the development of autoimmune diseases under certain conditions. Toll-like receptors (TLRs) recognize various pathogens and induce innate immunity. We herein present a mouse model for chronic pancreatitis, which was induced by TLR3 signaling that generated the Fas/Fas ligand (FasL)-mediated cytotoxicity. An analogue of viral double-stranded RNA, polyinosinic:polycytidylic acid (poly I:C), which is recognized by TLR3, was injected into autoimmune-prone strains: MRL/Mp mice (MRL/+), MRL/Mp mice with a deficit of Fas (MRL/lpr) and MRL/Mp mice with a deficit of functional FasL (MRL/gld). The pancreatitis in MRL/+ mice was initiated by the destruction of pancreatic ductules, and its severity was significantly higher than that in MRL/lpr mice or MRL/gld mice. Using a pancreatic duct epithelial cell line MRL/S-1 newly established from the MRL/gld mouse that lacks FasL, we showed that treatment with poly I:C significantly induced the expression of Fas on the cultured cells. MRL/S-1 cells were destructed when co-cultured with splenocytes bearing intact FasL prepared from MRL/+ or MRL/lpr mice, but the magnitude of cytotoxicity was smaller with splenocytes of MRL/gld mice. Likewise, synthetic FasL protein showed cytotoxicity on MRL/S-1 cells. Furthermore, MRL/S-1 cells expressed higher levels of chemokines after the treatment with poly I:C, suggesting that the poly I:C-mediated induction of chemokines may be responsible for recruitment of lymphoid cells to the pancreatic periductular regions. These findings indicate that TLR3 signaling generates the Fas/FasL-mediated cytotoxicity, thereby leading to the development of chronic pancreatitis.

Genetic dissociation of dacryoadenitis and sialadenitis in a Sjogren's syndrome mouse model with common and different susceptibility gene loci.

PURPOSE: Sjögren's syndrome (SS) is a systemic autoimmune disease in which the main lesions are dacryoadenitis and sialadenitis. It is unclear whether these lesions develop in a common genetic background. A quantitative trait locus (QTL) analysis was performed in the SS mouse model, MRL/MpJ-lpr/lpr (MRL/lpr), to identify the susceptibility loci to dacryoadenitis and sialadenitis and the association with both loci. METHODS: MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr x C3H/lpr) F1, and (MRL/lpr x C3H/lpr) F2 intercross mice were prepared, and the severity of dacryoadenitis and sialadenitis in individuals was quantified by histopathologic grading. In genomic DNA samples from the F2 mice, the polymorphic microsatellite markers highly associated with each lesion were determined as susceptibility loci. RESULTS: QTLs with significant linkage for dacryoadenitis were mapped on chromosome 1 (the position of maximum logarithm of odds [LOD] score; 64.1 cM), designated Adacm1; chromosome 2 (88.4 cM), Adacm2; and chromosome 5 (63.9 cM), Adacm3. Those for sialadenitis were mapped on chromosome 1 (69.0 cM), Asm3, and chromosome 2 (65.3 cM and 82.1 cM), Asm4 and Asm5. Adacm1/Asm3 and Adacm2/Asm5 seemed to be a common chromosomal region, respectively. MRL-homozygous at Adacm1 and Adacm2 and at Asm3 and Asm5 manifested an additive effect on the development of dacryoadenitis and sialadenitis, respectively, whereas Adacm3 did not. CONCLUSIONS: Dacryoadenitis and sialadenitis in MRL/lpr mice are under the control of common and different susceptibility loci, with an allelic combination that leads to regular variations in pathologic phenotypes.

Increased expression of soluble form of vascular cell adhesion molecule-1 aggravates autoimmune arthritis in MRL-Fas(lpr) mice.

Vascular cell adhesion molecule-1 (VCAM-1, CD106) is important in leukocyte trafficking and its increased expression is associated with a number of chronic inflammatory diseases, including rheumatoid arthritis (RA). A soluble form of VCAM-1 (sVCAM-1) is generated by shedding of the membrane-bound molecule. The concentration of sVCAM-1 is increased in the sera of RA patients, but its pathological role has not been elucidated. The effect of sVCAM-1 relative to protection or aggravation of disease on the development of spontaneous arthritis was examined in an animal model of RA, namely MRL-Fas(lpr) mice (which display a disease resembling human RA), by generation of sVCAM-1 transgenic MRL-Fas(lpr) mice. Transgenic MRL-Fas(lpr) mice that expressed sVCAM-1 had higher incidence and increased severity of arthritis associated with higher levels of serum IgG rheumatoid factor compared with non-transgenic MRL-Fas(lpr) mice. These results suggest that sVCAM-1 plays an arthritogenic role in the development of inflammatory arthritis in MRL-Fas(lpr) mice and may present an important target for therapeutic strategy of RA.

Elevated levels of fractalkine expression and accumulation of CD16+ monocytes in glomeruli of active lupus nephritis.

BACKGROUND: Fractalkine (Fkn) is a chemokine that affects cells expressing its receptor, CX3CR1, including CD16-positive (CD16+) monocytes/macrophages (CD16+ Mos). The relationship of levels of glomerular Fkn expression and infiltration by CD16+ Mos with the severity and diversity of glomerular lesions in human lupus nephritis is not known. STUDY DESIGN: Retrospective cross-sectional analysis of variables observed in biopsy specimens. SETTINGS & PARTICIPANTS: 88 patients with systemic lupus erythematosus. PREDICTOR: Histological class and severity of lupus nephritis according to the International Society of Nephrology/Renal Pathology Society and clinicopathologic factors. OUTCOMES: Outcome variables are assays related to the degree of glomerular Fkn expression and CD16+ Mo infiltration. MEASUREMENTS: Immunohistological grading of Fkn staining, number of CD16+ Mos, and messenger RNA levels of Fkn and CD16 in glomeruli. RESULTS: Patients with proliferative lupus nephritis (class III and IV glomeruli) showed significantly greater glomerular Fkn expression and CD16+ Mo counts than those with other classes. Infiltrating CD16+ Mos within glomeruli expressed CX3CR1. Moreover, glomerular Fkn expression significantly correlated with the histopathologic activity index and CD16+ Mo counts, and CD16+ Mo counts significantly correlated with serum levels of blood urea nitrogen, complement (CH50), and anti-DNA antibody; estimated glomerular filtration rate; and urinary protein excretion. Glucocorticoid therapy had a tendency to decrease both glomerular Fkn expression and CD16+ Mo counts. LIMITATIONS: Only frozen biopsy specimens (from 49 patients) were analyzed for the evaluation of glomerular Fkn expression. CONCLUSION: Disease activity and proliferative glomerular lupus nephritis lesions are associated with the glomerular Fkn expression and CD16+ Mo accumulation.

IK cytokine ameliorates the progression of lupus nephritis in MRL/lpr mice.

OBJECTIVE: IK cytokine has been isolated as a factor that inhibits interferon-gamma (IFNgamma)-induced expression of class II major histocompatibility complex (MHC) antigens. Aberrant expression of class II MHC antigens has reportedly been recognized in the target organs of autoimmune diseases and been associated with disease activity. In this study, we investigated whether IK cytokine can ameliorate the progression of lupus nephritis in MRL/lpr mice. METHODS: A truncated IK analog was prepared and transfected into a nonmetastatic fibroblastoid cell line, and then injected subcutaneously into MRL/lpr mice at ages 8 weeks (before the onset of lupus nephritis) and 12 weeks (at the early stage of the disease). RESULTS: An IK cytokine, when it was translated from methionine at position 316, acted as a secretory protein. This truncated IK cytokine (tIK) reduced IFNgamma-induced class II MHC expression in various cells through decreased expression of class II MHC transcription activator. Treatment of MRL/lpr mice with tIK significantly reduced renal damage as compared with control mice. A significant decrease in macrophage and T cell infiltration was found in the kidneys of tIK-treated mice, resulting in decreased production of IFNgamma and interleukin-2. Mice treated with tIK also showed significant reduction of anti-DNA antibodies and circulating immune complexes. A specific reduction of class II MHC expression was observed on B cells and monocytes as well as in the kidney. CONCLUSION: We prepared a potent IK analog and demonstrated its ability to ameliorate the progression of lupus nephritis. This agent may therefore provide a new therapeutic approach for lupus nephritis.

Cappuccino mutation in an autoimmune-prone strain of mice suggests a role of platelet function in the progression of immune complex crescentic glomerulonephritis.

OBJECTIVE: Crescent formation in the renal glomerulus is a typical manifestation of progressive glomerulopathy associated with fatal renal failure; therefore, its prevention is of clinical importance. Little is known about the pathogenic mechanism for crescent formation. This study was undertaken in an attempt to identify the events that are critical for crescent formation in immune complex crescentic glomerulonephritis (CGN) by analyzing a novel mutant strain of mice. METHODS: A spontaneous mutant strain of mice was isolated from the autoimmune-prone strain EOD, which stably develops fatal CGN. The mutant phenotypes were assessed histopathologically, hematologically, and immunologically. The mutation was searched for with positional cloning using microsatellite markers. RESULTS: Compared with wild-type EOD (WT-EOD) mice, mutant EOD (mut-EOD) mice showed marked improvement in CGN in conjunction with an improvement in spontaneous mortality. In WT-EOD mice, an inverse correlation between blood urea nitrogen concentration and blood platelet count and massive accumulation of platelets in the glomerulus were evident, suggesting that an accumulation of platelets in the glomerulus contributes to the progression of CGN. The mutant platelets showed an abnormal aggregation in response to collagen and thrombin, associated with a bleeding tendency in mut-EOD mice. Genetic analysis revealed a deleterious mutation in the cappuccino gene (cno), which encodes a protein that belongs to a complex called the biogenesis of lysosome-related organelle complex 1 and is profoundly involved in platelet function. Morphologic examination revealed a partial defect in dense body formation in the delta-granule of platelets. CONCLUSION: The present findings suggest that platelet functions have a critical role in crescent formation in autoimmune GN.

Antagonist of interferon-inducible protein 10/CXCL10 ameliorates the progression of autoimmune sialadenitis in MRL/lpr mice.

OBJECTIVE: Mononuclear cell infiltration of the salivary glands is a major feature of Sjögren's syndrome (SS) and its animal model. Local generation of chemokines and the presence of chemokine receptors on the infiltrating cells may be involved in this process. We undertook the present study to investigate the expression of chemokines during the development of autoimmune sialadenitis in MRL/lpr mice and the therapeutic effect of chemokine antagonists on sialadenitis. METHODS: NH2-terminal-truncated interferon-inducible protein 10 (IP-10)/CXCL10 analogs were transfected into a nonmetastatic fibroblastoid cell line, MRL/N-1, and injected subcutaneously into MRL/lpr mice, and the effects on sialadenitis were monitored. RESULTS: IP-10 analogs truncated by 5 or more amino acid residues from the N-terminal failed to induce chemotaxis and calcium influx by CXCR3-expressing cells. Of these, the most potent antagonist (AT) (IP-10-AT) was a molecule with methionine added after removal of the 5 N-terminal amino acid residues. Significantly increased expression of the Th1-associated chemokines IP-10, monokine induced by interferon-gamma/CXCL9, and interferon-inducible T cell chemoattractant/CXCL11 was induced in the ductal epithelium by interferon-gamma produced in the salivary glands, whereas expression of the Th2-associated chemokines thymus and activation-regulated chemokine (TARC)/CCL17 and monocyte-derived chemokine/CCL22 was almost undetectable during sialadenitis. Inoculation of IP-10-AT into MRL/lpr mice during the early stage of sialadenitis significantly reduced periductal mononuclear cell infiltration and parenchymal destruction compared with these features in control and TARC-AT-bearing mice. This was due to a significant reduction in infiltration of CXCR3+ T cells, predominantly Th1 cells, resulting in decreased interferon-gamma production. CONCLUSION: We prepared a novel potent IP-10 antagonist and demonstrated its ability to ameliorate the progression of autoimmune sialadenitis. This agent may provide a new therapeutic approach to SS.

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 x 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 fractalkine (CX3CL1) delays the initiation and ameliorates the progression of lupus nephritis in MRL/lpr mice.

OBJECTIVE: Lupus nephritis is characterized by immune complex deposition and inflammatory cell infiltration into the renal glomeruli. Local generation of chemokines and the presence of chemokine receptors on the infiltrating cells may be involved in this process. Fractalkine (Fkn)/CX3CL1 and its receptor, CX3CR1, form one such chemokine system. We therefore undertook this study to investigate whether Fkn antagonist inhibits the initiation and progression of lupus nephritis in MRL/lpr mice. METHODS: NH(2)-terminally truncated Fkn/CX3CL1 analogs were transfected into a nonmetastatic fibroblastoid cell line, MRL/N-1, and injected subcutaneously into MRL/lpr mice. RESULTS: Fkn analogs truncated by >/=4 amino acid residues from the N-terminus failed to induce chemotaxis and calcium influx by CX3CR1-expressing cells. Of these, the most potent antagonist (Fkn-AT) lacked the 4 N-terminal amino acid residues. Fkn expression in the glomerulus was significantly increased in 12-week-old MRL/lpr mice. Expression was localized predominantly in the glomerular endothelial cells, but was occasionally observed in the mesangial cells and, to a lesser extent, in the interstitial microvasculature. Inoculation of MRL/lpr mice with Fkn-AT before the onset or during the early stages of lupus nephritis significantly reduced glomerular hypercellularity, glomerulosclerosis, crescent formation, and vasculitis compared with control mice. This seemed to be due to a marked reduction in macrophage accumulation. In contrast, Fkn antagonist did not affect pneumonitis, sialadenitis, lymphadenopathy, or splenomegaly. CONCLUSION: We prepared a novel potent Fkn antagonist and demonstrated its ability to delay the initiation and ameliorate the progression of lupus nephritis. This agent may therefore provide a new therapeutic approach to lupus nephritis.

Endothelial adhesion molecules in glomerular lesions: association with their severity and diversity in lupus models.

BACKGROUND: To clarify whether vascular endothelial adhesion molecules in glomeruli might contribute to the severity and diversity of glomerular lesions in lupus nephritis, their expression in lupus models was analyzed. METHODS: The expression levels of E- and P-selectin and vascular cell adhesion molecule-1 (VCAM-1) in glomeruli of different histopathologic grades of MRL/MpJ-lpr/lpr (MRL/lpr) lupus mice was studied using laser-capture microdissection of the glomeruli, followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The glomerular lesions in SCID mice injected with the 2B11.3 and 7B6.8 clones, which are derived from an MRL/lpr mouse and induce endocapillary proliferative and wire loop type of glomerular lesions, respectively, were analyzed. To investigate the effect of a soluble form of E-selectin (sE-selectin) on the development of glomerular lesions, sE-selectin-producing L cells were prepared by transfection of the cDNA encoding sE-selectin and injected into SCID mice. RESULTS: The glomeruli in MRL/lpr mice showed increased expression of these adhesion molecules, corresponding to the severity of the glomerular lesions. The endocapillary proliferative type lesions in SCID mice induced by the 2B11.3 clone showed significantly increased expression of the adhesion molecules, especially E-selectin and P-selectin, but the wire loop type lesion induced by the 7B6.8 clone expressed only VCAM-1. Formation of the endocapillary proliferative type lesions induced by the 2B11.3 clone was markedly prevented in association with elevation of the serum level of sE-selectin produced by the tansfected L cells. CONCLUSION: The severity and diversity of the histopathology of lupus nephritis are partially associated with the expression of vascular endothelial adhesion molecules in glomeruli.

Internalization of antibodies by endothelial cells via fibronectin implicating a novel mechanism in lupus nephritis.

BACKGROUND: One of the crucial events in lupus nephritis is the glomerular deposition of immunoglobulins (Igs), of which pathogenic properties have been proposed mostly to be either type IIor type III allergic reactions. Some of IgG3-producing hybridoma clones established from an MRL/MpTn-gld/gld (MRL/gld) lupus mouse generate wire loop-like lesions in glomeruli resembling lupus nephritis when injected into SCID mice. These clones are useful for analyzing the mechanisms of glomerular deposition of antibodies in lupus nephritis at the monoclonal level. METHODS: Glomerular lesions of SCID mice injected with the hybridoma clones, 17H8a or 1G3 as control were analyzed by light and electron microscopy. Interaction of the antibodies with human glomerular endothelial cells (HGECs) and human umbilical vein endothelial cells (HUVECs) in vitro was studied by fluorescence microscopy, electron microscopy, and flow cytometry. RESULTS: Both antibodies did not show any antigen specificity for mouse glomeruli. The glomerular lesions generated by 17H8a, but not by 1G3, contained electron-dense deposits not only in subendothelial regions but also in the cytoplasm of endothelial cells, suggesting internalization of the 17H8a antibodies by endothelial cells. In cell culture studies, internalization of only 17H8a antibodies by HGECs and HUVECs was observed, but the antibodies did not have antigen specificity for both types of endothelial cells. The internalization by HUVECs was mediated by actin polymerization, and it was inhibited by RGDS (Arg-Gly-Asp-Ser) tetrapeptide, antihuman fibronectin and antihuman integrin beta1 monoclonal antibodies. CONCLUSION: The interaction between particular antibodies and endothelial cell surface integrins via fibronectin may be involved in their subsequent internalization by endothelial cells leading to antibody deposition in glomeruli. This may be one of the mechanisms of glomerular injury in lupus nephritis.

Antagonist of monocyte chemoattractant protein 1 ameliorates the initiation and progression of lupus nephritis and renal vasculitis in MRL/lpr mice.

OBJECTIVE: To examine whether chemokine antagonists inhibit the initiation and progression of lupus nephritis in MRL/lpr mice. METHODS: NH(2)-terminal-truncated monocyte chemoattractant protein 1 (MCP-1)/CCL2 or thymus and activation-regulated chemokine (TARC)/CCL17 analogs were inserted into the pCXN2 expression vector and transfected into a nonmetastatic fibroblastoid cell line, MRL/N-1, established from an MRL/gld mouse. RESULTS: MCP-1 antagonist- or TARC antagonist-transfected MRL/N-1 cells were injected subcutaneously into MRL/lpr mice ages 7 weeks (before the onset of lupus nephritis) and 12 weeks (at the early stage of the disease). After 8 weeks, mice bearing the MCP-1 antagonist showed markedly diminished infiltration of macrophages and T cells, glomerular hypercellularity, glomerulosclerosis, crescent formation, and vasculitis compared with control mice. This seemed to be due to decreased production of interferon-gamma and interleukin-2 in the kidney. In contrast, there was no significant difference in renal damage between mice bearing TARC antagonist and control mice. CONCLUSION: We established a new system using MRL/N-1 cells that allows long-term observation of the effects of chemokine antagonists on lupus nephritis in MRL/lpr mice. We also showed that the MCP-1 antagonist ameliorated the initiation and progression of lupus nephritis and of renal vasculitis, which might provide a new approach to the treatment of the disease.

Genetic basis of tissue specificity of vasculitis in MRL/lpr mice.

OBJECTIVE: To clarify the mode of inheritance of the tissue distribution of vasculitis in MRL/Mp-lpr/lpr (MRL/lpr) lupus-prone mice and to identify the susceptibility loci. METHODS: Vasculitis in individual MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr x C3H/lpr)F(1), and (MRL/lpr x C3H/lpr)F(2) intercross mice was analyzed by histopathologic grading of main branches of the aorta and of medium-sized arteries in the lower limbs. Genomic DNA samples from F(2) intercross mice were examined by simple sequence-length polymorphism analysis, and the polymorphic microsatellite markers highly associated with vasculitis in each tissue were determined as vasculitis susceptibility loci. RESULTS: A susceptibility locus with significant linkage to vasculitis of main branches of the aorta was mapped on chromosome 4 at D4Mit213 (map position 13.3cM) selectively in males, while vasculitis of medium-sized arteries in the lower limbs was mapped to different chromosomes: at D8Mit31 on chromosome 8 (map position 33.0) selectively in females and at D5Mit36 on chromosome 5 (map position 65.0). All of these were different from the previously defined loci governing susceptibility to vasculitis involving the kidneys. CONCLUSION: Systemic vasculitis in MRL/lpr mice is genetically controlled with cumulative effects of multiple gene loci, each of which has tissue specificity.

Arthritis in MRL/lpr mice is under the control of multiple gene loci with an allelic combination derived from the original inbred strains.

OBJECTIVE: To clarify the mode of inheritance and the genome origins of arthritis in a lupus-prone strain of mice, MRL/MpJ, bearing a Fas deletion mutant gene, lpr (MRL/lpr). METHODS: Using non-lupus-prone strains of mice, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr x C3H/lpr)F(1) intercross and MRL/lpr x (MRL/lpr x C3H/lpr)F(1) backcross mice were prepared. Arthritis in individual mice was analyzed by histopathologic grading, and the genomic DNA of the backcross mice was examined by simple sequence-length polymorphism analysis to determine the polymorphic microsatellite markers highly associated with arthritis. RESULTS: Arthritis-susceptibility loci with significant linkage were mapped between D15Mit111 and D15Mit18 (map position 17.8-18.7 cM) on chromosome 15 and between D19Mit112 and D19Mit72 (map position 43.0-55.0) on chromosome 19 (logarithm of odds scores 3.5 and 4.3, respectively). Three other loci, one mapped to each of chromosomes 1, 2, and 7, showed suggestive linkage. Loci homozygous for MRL alleles on chromosomes 1 and 19 enhanced arthritis in both sexes, whereas other loci on chromosomes 2 and 15 selectively affected males. A locus homozygous for MRL alleles on chromosome 7 inhibited arthritis in both sexes. Three of these loci were found to originate from an LG/J strain and 1 from an AKR/J strain. Some combinations of these loci showed an additive effect in a hierarchical manner on the development of arthritis. CONCLUSION: Arthritis in MRL/lpr mice is a complex pathologic manifestation resulting from the cumulative effect of multiple gene loci with an allelic combination derived from the original inbred strains.