Sjögren's syndrome in mice carrying the lprcg gene and the therapeutic efficacy of an immunosuppressive agent FK506

The influence of the lpr^cg gene on the development of Sjögren’s syndrome was followed up to 5 months of age in male and female mice of MRL, CBA and C₃H strains. In MRL-lpr^cg mice, focal mononuclear cell infiltration started at 2 months and became conspicuous after 3 months of age in the lacrimal and submandibular glands but was minimal in the parotid and sublingual glands, even at 5 months of age, without any apparent sex effects found. In CBA and C₃H mice carrying the lpr^cg gene, this manifestation of Sjögren’s syndrome was much less prominent, indicating that the participation of some genes of the MRL strain may be indispensable for the development of Sjögren’s syndrome in mice carrying this gene. In MRL-lpr^cg mice, an immunosuppressive agent, FK506, improved the serological abnormalities (decreased levels of anti-double-stranded DNA antibody of IgG2a and IgG3 subclasses) and proteinuria. It also reduced the manifestations of Sjögren’s syndrome when it was intraperitoneally administered three times weekly at a dose of 2 mg/kg from 6 weeks (before disease onset) until 5 months of age (the termination of the experiment). Although Vβ8.2⁺ T cells have been demonstrated to be responsible for causing several autoimmune diseases, the selective deletion of Vβ8.2⁺ T cells with the superantigen encoded by mouse mammary tumor virus did not affect the disease severity at all, suggesting that this T cell repertoire may not play a crucial role in induction of Sjögren’s syndrome.     

Epigallocatechin gallate inhibits oxidative stress-induced DNA damage and apoptosis in MRL-Faslpr mice with autoimmune sialadenitis via upregulation of heme oxygenase-1 and Bcl-2

Abstract

Pathogenic effects of reactive oxygen species (ROS) in the salivary glands of patients with Sjögren’s syndrome have been demonstrated. Epigallocatechin gallate (EGCG), which is a catechin derivative and exhibits potent antioxidant activity, has been reported to ameliorate autoimmune sialadenitis in a murine model, but the mechanism underlying its protective action remains to be investigated. Herein, we examined the effects of EGCG administration to MRL/MpJ-lpr/lpr (MRL-Fas^lpr) mice on disease severity of autoimmune sialadenitis and protein expression levels of 11 sialadenitis-related molecules – heme oxygenase-1 (HO-1) (antioxidant); thymidine glycol (marker of DNA damage); gp91phox/NADPH oxidase 2 (prooxidant); single-stranded DNA (ssDNA) and cleaved caspase 3 (apoptotic cell markers); p53 and Bax (proapoptotic molecules); Bcl-2 (antiapoptotic molecule); SSA/Ro, SSB/La, and Ifi202 (autoantigens). In EGCG-treated mice, the severity of sialadenitis was substantially decreased. Expression levels of thymidine glycol, gp91phox, ssDNA, cleaved caspase 3, p53, Bax, SSA/Ro, SSB/La, and Ifi202 in duct epithelial cells of salivary glands from EGCG-treated mice were reduced, whereas HO-1 and Bcl-2 were overexpressed. Results of correlation analysis among sialadenitis severity and 11 sialadenitis related-molecules, and those of partial correlation analysis between apoptotic related-molecules and sialadenitis severity or HO-1 suggested that the consecutive pathogenic cycle including activated autoimmune reactions, ROS synthesis, DNA damage and p53-dependent apoptosis was associated with the pathogenesis of autoimmune sialadenitis in MRL-Fas^lpr mice. Overexpression of HO-1 and Bcl-2 mediated by EGCG blocked this pathogenic cycle, subsequently resulting in the inhibition of ROS-mediated DNA damage and apoptosis, and protected salivary gland tissues from oxidative stress. Clinically, green tea catechin may have therapeutic efficacy for Sjögren’s syndrome.     

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.     

Evaluation of an automated chemiluminescent immunoassay kit for antinuclear antibodies in autoimmune diseases

The identification of antinuclear antibodies (ANA) is an essential step in the diagnosis of different autoimmune diseases. The gold standard method for their detection is immunofluorescence assay. However, this is a subjective and laborious method, thus a need for simplified objective methods has aroused. In the current work, we evaluated such automated method, the LIAISON^® (DiaSorin, Italy) for the detection of ANA. A total of 242 sera were analyzed including 67 from healthy subjects, 107 from primary biliary cirrhosis (PBC) patients, 20 from scleroderma patients and 48 from patients with Sjögren’s syndrome. All sera were analyzed using the automated chemiluminescent immunoassays, LIAISON^® for the presence of ANA (kit No. 310300). Positive samples were further analyzed for the presence of antidouble-stranded DNA (dsDNA) and autoantibodies to 6 extractable nuclear antigens (ENA) of the LIAISON^® (kits No. 310330 and 310331). Negative samples were further analyzed by Blueblot ANA assay (D-TEK, Belgium) or BlueDot Liver (D-TEK, Belgium) as appropriate. The LIAISON^® specificity for ANA screening was 97 %. ANA positivity was determined in 80 % of all patients. The sensitivity was 95.5 % in scleroderma, 83 % in PBC and 72.9 % in Sjogren’s syndrome. ENA was positive in all ANA-positive scleroderma and Sjögren’s sera and in 27 % of ANA-positive PBC sera. Among scleroderma or Sjögren patients that were ANA negative, 4 samples were positive for anti-SSA and 2 for RNP-68 utilizing Blueblot assays. M2 protein was found in 1 out of the ANA-negative PBC patients. The LIAISON^® ANA screen is specific and sensitive for the evaluation of ANA in patients with primary biliary cirrhosis, scleroderma and Sjögren’s syndrome.     

T cell‐derived IFN‐γ downregulates protective group 2 innate lymphoid cells in murine lupus erythematosus

Innate lymphoid cells (ILCs) are important regulators of the immune response and play a crucial role in the restoration of tissue homeostasis after injury. GATA‐3⁺ IL‐13‐ and IL‐5‐producing group 2 innate lymphoid cells (ILC2s) have been shown to promote tissue repair in barrier organs, but despite extensive research on ILCs in the recent years, their potential role in autoimmune diseases is still incompletely understood. In the present study, we investigate the role of ILC2s in the MRL/MpJ‐Fas^lpr (MRL‐lpr) mouse model for severe organ manifestation of systemic lupus erythematosus (SLE). We show that in these MRL‐lpr mice, progression of lupus nephritis is accompanied with a reduction of ILC2 abundance in the inflamed renal tissue. Proliferation/survival and cytokine production of kidney‐residing ILC2s was suppressed by IFN‐γ and, to a lesser extent, by IL‐27 which were produced by activated T cells and myeloid cells in the nephritic kidney, respectively. Most importantly, restoration of ILC2 numbers by IL‐33‐mediated expansion ameliorated lupus nephritis and prevented mortality in MRL‐lpr mice. In summary, we show here that development of SLE‐like kidney inflammation leads to a downregulation of the renal ILC2 response and identify an ILC2‐expanding therapy as a promising treatment approach for autoimmune diseases.     

Induction of autoimmune disease by graft-versus host reaction across MHC class II difference: modification of the lesions in IL-6 transgenic mice

We examined the effect of IL-6 on the development of autoimmune diseases (primary biliary cirrhosis. Sjögren's syndrome) employing murine grari-versus-host reaction (GVHR) model with MHC class II disparity. For this purpose, we used IL-6 transgenic(B6.6) mice in which a high level of IL-6 was detected. C57B1/6 (B6) spleen T cells were injected into B6.6 mated with B6.C-H-2^(bml2) mutant mice ((bmi2x B6.6)F^I) and GVHR with MHC class II disparity was induced. The iransgenic hybrid mice with GVHR showed a larger spleen index and contained a higher serum level of IL-6 than those without GVHR. Autoimmune-like lesions in transgenic recipients became weakened compared with those in non-transgenic (bml2 x B6)F₁ recipients. In contrast, levels of antimitochondrial antibodies in (bm 12 x B6.6)FI GVHR group were signiiicantly higher than that of (bml2 X B6)F_I GVHR group. These results indicate that lL-6 excessively produced in vivo might regulate the progression of autotmmune diseases.     

A possible mouse model for spontaneous cholangitis: serological and histological characteristics of MRL/lpr mice

AIMS: MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop lymphadenopathy, hypergammaglobulinaemia, serum auto-antibodies, and a generalised auto-immune disease including glomerulonephritis and arthritis, and have been used as a model for the study of systemic lupus erythematosus. Recently, MRL/lpr mice were also reported as a potentially suitable animal model of primary biliary cirrhosis (PBC). The aim of this study was to determine the suitability of MRL/Mp-lpr/lpr (MRL/lpr) mice as an experimental auto-immune-mediated cholangitis model for PBC. METHODS: We investigated the serum hepatobiliary enzymes, histopathological findings, and the target antigen of antimitochondrial antibodies (AMA) in MRL/lpr mice. RESULTS: Serum levels of total bilirubin and hepatobiliary enzymes including alanine aminotransferase (ALT), leucine aminopeptidase (LAP), and gamma-glutamyl transpeptidase (G-GTP) in older-aged (over 20 weeks old) MRL/lpr or MRL/Mp-+/+ (MRL/+) mice were not significantly higher than those in younger (8-12 weeks old) MRL/lpr, MRL/+, or older-aged control mice (C3H/HeJ and BALB/C mice). Histopathologically, 24 of 47 (51%) older-aged MRL/lpr mice showed evidence of cholangitis, compared with two of 20 (10%) younger MRL/lpr mice. Especially, epithelioid granuloma and/or bile duct loss were seen in 11 out of 47 (23%) older-aged MRL/lpr mice, whereas such findings were seen in only one of 20 (5%) younger MRL/lpr mice. None of the MRL/+, C3H/HeJ, and BALB/C mice developed cholangitis. The target antigens of AMA were not pyruvate dehydrogenase complex but 2-oxoglutarate dehydrogenase complex and/or branched-chain oxo-acid dehydrogenase complex as confirmed by immunoblotting. There was no significant correlation between the presence of AMA and severity of histological lesions in older-aged MRL/lpr mice, and there were no significant differences in these biochemical data, the proportion of mice with portal inflammation, cholangitis and AMA between male and female MRL/lpr mice. CONCLUSION: Although several clinical features were incompatible with PBC, the serological and histopathological features of MRL/lpr mice indicate that these mice can be used as an experimental immune-mediated cholangitis model for PBC.     

Reduced development of CD4−8−B220+ T cells but normal autoantibody production in lpr/lpr mice lacking major histocompatibility complex class I molecules

The lpr gene has recently been shown to encode a functional mutation in the Fas receptor, a molecule involved in transducing apoptotic signals. Mice homozygous for the lpr gene develop an autoimmune syndrome accompanied by massive accumulation of double-negative (DN) CD4^?8^?B220⁺ T cell receptor-α/β⁺ cells. In order to investigate the origin of these DN T cells, we derived lpr/lpr mice lacking major histocompatibility complex (MHC) class I molecules by intercrossing them with β2-microglobulin (β2m)-deficient mice. Interestingly, these lprβ2m–/– mice develop 13-fold fewer DN T cells in lymph nodes as compared to lpr/lpr wild-type (lprWT) mice. Analysis of anti-DNA antibodies and rheumatoid factor in serum demonstrates that lprβ2m–/– mice produce comparable levels of autoantibodies to lprWT mice. Collectively our data indicate that MHC class I molecules control the development of DN T cells but not autoantibody production in Ipr/lpr mice and support the hypothesis that the majority of DN T cells may be derived from cells of the CD8 lineage.     

Sjögren syndrome: Advances in the pathogenesis from animal models

Sjögren syndrome is an autoimmune disease characterized by hyposecretion of the lacrimal and salivary glands, resulting in dryness of the eyes and mouth. Individuals may experience primary Sjögren syndrome or a secondary form accompanying another rheumatic autoimmune disease, such as rheumatoid arthritis or systemic lupus erythematosus. The pathogenic mechanisms of Sjögren syndrome remain largely unknown, in part a consequence of the heterogeneity of the disease. Animal models have shed light on the connections between specific pathways and symptoms, but an ideal system is wanting. Improved disease models will enable a better understanding of Sjögren syndrome, including how immune tolerance is lost and potential therapeutic interventions. Most importantly, an optimal model will enable detection of disease biomarkers, since injury to the salivary glands may precede lymphocytic infiltration. This review aims to characterize available mice models of Sjögren syndrome, including advantages and disadvantages, from the researcher's perspective.     

Altered Circadian Rhythms of the Stress Hormone and Melatonin Response in Lupus-prone MRL/MP-fas 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^Iprmice. The results show that lupus-prone MRL/MP- fas^Iprmice 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^Iprmice 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.     

Epigallocatechin gallate stimulates the neuroreactive salivary secretomotor system in autoimmune sialadenitis of MRL-Faslpr mice via activation of cAMP-dependent protein kinase A and inactivation of nuclear factor κB

Abstract

The water channel aquaporin 5 (AQP5) plays a crucial role in regulating salivary flow rates. Xerostomia is often observed in patients with Sjögren's syndrome, and this is attributed to reduced AQP5 expression in the salivary glands. Recently, anti-type 3 muscarinic cholinergic receptors (M₃R) autoantibodies and nuclear factor κB (NF-κB) have been found to be negative regulators of AQP5 expression in the salivary gland. Anti-M₃R autoantibodies desensitize M₃R to salivary secretagogues in Sjögren's syndrome, while activated NF-κB translocates to nuclei and binds to the AQP5 gene promoter, resulting in the suppression of AQP5 expression. We previously documented that epigallocatechin gallate (EGCG), which is a robust antioxidant contained in green tea, ameliorates oxidative stress-induced tissue damage to the salivary glands of MRL/MpJ-lpr/lpr (MRL-Fas^lpr) mice, which are widely used as a model of Sjögren's syndrome. Reactive oxygen species (ROS) can activate NF-κB and inactivate protein kinase A (PKA), which is a key driver of AQP5 expression. In this study, we examined the effects of administering EGCG to MRL-Fas^lpr mice with autoimmune sialadenitis on the levels of AQP5, activated NF-κB p65 subunit, activated PKA, activated c-Jun N-terminal kinase (JNK) (an activator of NF-κB), inhibitor κB (IκB) and histone deacetylase 1 (HDAC1) (an inhibitor of NF-κB). In EGCG-treated mice, intense aster-like immunostaining for AQP5 was observed on the apical plasma membranes (APMs) of submandibular gland acinar cells. Likewise, PKA, IκB and HDAC1 were highly expressed in salivary gland tissues, whereas the expression of JNK and NF-κB p65 was negligible. Rank correlation and partial correlation analyses revealed that treatment with EGCG upregulated AQP5 expression on the APM of acinar cells through activation of PKA and inactivation of NF-κB, while IκB and HDAC1 played a pivotal role in the induction of AQP5 expression by PKA. Our study indicates that EGCG may have therapeutic potential for Sjögren's syndrome patients.     

Lupus-prone MRL/faslpr/lpr mice display increased AID expression and extensive DNA lesions,comprising deletions and insertions,in the immunoglobulin locus: Concurrent upregulation of somatic hypermutation and class switch DNA recombination

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/fas^lpr/lpr mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/fas^lpr/lpr mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/fas^lpr/lpr B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/fas^lpr/lpr greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/fas^lpr/lpr mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (AID), which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) θ, pol η and pol ζ, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone MRL/fas^lpr/lpr mice, SHM and CSR are upregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.     

Primary biliary cirrhosis: lessons learned from an organic-specific disease

Primary biliary cirrhosis is an autoimmune liver disease that predominantly affects women and is characterized by chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and subsequent fibrosis. The serological hallmark is the presence of antimitochondrial antibodies are directed against the 2-oxo-acid dehydrogenase complexes located on the inner membrane of mitochondria. Although the role of antimitochondrial antibodies in the pathogenesis is unknown, the presence of antibodies has allowed detailed immunological definition of the antigenic epitopes, the autoantibodies, and the T-cell response. Theories have been proposed regarding the mechanism of immune-mediated bile duct damage in primary biliary cirrhosis, including the possible role of T-cell-mediated cytotoxicity and molecular mimicry. Primary biliary cirrhosis is usually diagnosed based on the triad of elevated alkaline phosphatase, antimitochondrial antibodies, and characteristic histological changes on liver biopsy. Biochemical liver abnormalities are consistent with the presence of cholestasis and include an elevation of both serum alkaline phosphatase and γ-glutamyl transpeptidase, with or without elevation of aminotransferase levels. Ursodeoxycholic acid, a dihydroxy biel acid, appears to be the only effective therapy in preventing or delaying the need for liver transplantation. However, a number of patients receiving ursodeoxycholic acid still develop progressive disease and require transplantation; at present, liver transplantation is the only effective therapy for end-stage primary biliary cirrhosis. Received: 13 September 2001 / Accepted: 14 November 2001     

Do Autoantibodies Predict Autoimmune Liver Disease in Primary Sjögren's Syndrome? Data of 180 Patients upon a 5 Year Follow‐Up

Objective: To evaluate the clinical value of autoantibodies as serological markers to predict autoimmune liver diseases in primary Sjögren's syndrome (SS). Materials and methods: 180 patients who met the European diagnostic criteria for SS but without a history of liver disease were studied upon a 5 year follow‐up. Sera taken at enrolment were evaluated by immunofluorescence analysis (IF‐AMA) on rat liver, stomach and kidney sections, enzyme‐linked immunosorbent assay using rat mitochondrial, microsomal and soluble liver antigens and Western blot (WB) analysis using rat mitochondrial antigens. Results: At presentation, 152 (84%) sera had autoantibodies. Antinuclear antibodies (ANA) were expressed in 58% of patients and displayed three distinct patterns (speckled, homogenous and anticentromere). Smooth muscle autoantibodies (SMAs) and parietal cell autoantibodies were found in 39 and 4.5% of patients, respectively. Three patients presented antimitochondrial antibodies by IF‐AMA, and two of them developed symptomatic primary biliary cirrhosis (PBC). Two patients without IF‐AMA and without evidence of cholestasis had PBC‐specific AMA (anti‐PDC–E2 and anti‐BCKADC–E2). However, these two patients and the third IF‐AMA‐positive woman remained free from symptoms and biochemical signs of PBC. Autoimmune hepatitis (AIH) (n = 2), ‘overlap syndrome’ of AIH and chronic hepatitis C (n = 1) and autoimmune cholangiopathy (AIC) (n = 1) were diagnosed in four patients. Conclusions: Patients with IF‐AMA usually develop symptomatic PBC upon a 5 year follow‐up. Our findings support the idea that patients without IF‐AMA, who express PBC‐specific AMA, are in early, asymptomatic stage of the disease. High‐titre SMA and IF‐AMA are the most specific indicators for AIH and PBC.     

Reversible lacrimal gland‐protective regulatory T‐cell dysfunction underlies male‐specific autoimmune dacryoadenitis in the non‐obese diabetic mouse model of Sjögren syndrome

CD4⁺ CD25⁺ Foxp3⁺ regulatory T (Treg) cells are required to maintain immunological tolerance; however, defects in specific organ‐protective Treg cell functions have not been demonstrated in organ‐specific autoimmunity. Non‐obese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity and are a well‐characterized model of Sjögren syndrome. Lacrimal gland disease in NOD mice is male‐specific, but the role of Treg cells in this sex‐specificity is not known. This study aimed to determine if male‐specific autoimmune dacryoadenitis in the NOD mouse model of Sjögren syndrome is the result of lacrimal gland‐protective Treg cell dysfunction. An adoptive transfer model of Sjögren syndrome was developed by transferring cells from the lacrimal gland‐draining cervical lymph nodes of NOD mice to lymphocyte‐deficient NOD‐SCID mice. Transfer of bulk cervical lymph node cells modelled the male‐specific dacryoadenitis that spontaneously develops in NOD mice. Female to female transfers resulted in dacryoadenitis if the CD4⁺ CD25⁺ Treg‐enriched population was depleted before transfer; however, male to male transfers resulted in comparable dacryoadenitis regardless of the presence or absence of Treg cells within the donor cell population. Hormone manipulation studies suggested that this Treg cell dysfunction was mediated at least in part by androgens. Surprisingly, male Treg cells were capable of preventing the transfer of dacryoadenitis to female recipients. These data suggest that male‐specific factors promote reversible dysfunction of lacrimal gland‐protective Treg cells and, to our knowledge, form the first evidence for reversible organ‐protective Treg cell dysfunction in organ‐specific autoimmunity.     

Detection of glomerular-binding immune elements in murine lupus using a tissue-based ELISA

The glomerulonephritis in systemic lupus erythemalosus (SLE) is presumably triggered by ihe binding of circulating immune elements (autoantibodies and immune complexes) to the glomerulus; however, the nature of these elements is unclear. In order to detect and characterize such elements, we developed an ELISA using whole intact glomeruli as the substrate. With this assay, glomerular binding activity (GBA) was detected in the serum of MRLlpr, NZB × W, and B × SB mice, but not in non-autoimmune BALB/c mice. Less activity was present in the serum of C3H lpr, C57B1/6J Ipr and AKR lpr animals which develop signs of autoimmunity but only modest renal disease. The GBA in MRLlpr mice contained IgG (subclasses 1, 2a and 2b), but not IgG3. IgM. igA, orC3. GBA was not significantly decreased by preadsorption of MRL lpr serum by DNA-agarose (although anti-DNA antibodies were). Binding activity in serum, however, was diminished by DNAase treatment. Fractionation of MRL lpr serum over a molecular sizing column showed that GBA eluled in a broad peak. GBA bound to the glomerulus ex vivo in a tissue-specific fashion and was enriched in renal eluates relative to serum in vivo. In sum, the binding activity detected by this assay appeared to be a heterogeneous entity (possibly in part immune complexes containing DNA) which bound specifically to the glomerulus and which appeared to parallel the presence of renal disease. This novel assay system may help elucidate the pathogenesis of SLE nephritis and have utility as a disease marker.     

Antibody to carbonic anhydrase II is present in primary biliary cirrhosis (PBC) irrespective of antimitochondrial antibody status

Antibody to carbonic anhydrase II, an enzyme abundantly present in biliary epithelium, has been proposed as a diagnostic marker for antimitochondrial antibody-negative PBC. In this study we determine its prevalence and clinical significance in a large series of patients with antimitochondrial antibody-positive and -negative PBC. Reactivity to carbonic anhydrase II was sought by Western immunoblotting in sera from 215 consecutive patients with PBC (26 antimitochondrial antibody-negative), 13 with autoimmune hepatitis, 25 with primary Sjögren's syndrome (pSS), 12 with systemic sclerosis, 19 with systemic lupus erythematosus and 73 healthy subjects. The prevalence of antibody to carbonic anhydrase II (titre 1:100) in PBC was 8%. No specific reactivity to carbonic anhydrase II was found in antimitochondrial antibody-negative PBC (7% versus 8% in antimitochondrial antibody-positive PBC). Ascites (P = 0.006) and Sjögren's syndrome (SS) (P = 0.022) in PBC were significantly associated with presence of the antibody. In patients with SS associated with PBC, the prevalence (19%) was similar to that observed in pSS (16%). At a serum dilution of 1:40, the prevalence of positive sera in PBC rose to 27% but disease specificity was reduced. Our findings in a large population of PBC patients rule out a relation between presence of antibody to carbonic anhydrase II and lack of antimitochondrial antibody. The higher prevalence of ascites found in positive patients warrants further evaluation.     

Antilactoferrin antibodies in autoimmune liver disease

Antilactoferrin antibodies have been reported in patients with several autoimmune disorders, including primary biliary cirrhosis, autoimmune hepatitis and autoimmune cholangitis. We investigated the prevalence and the clinical significance of such autoreactivity in patients with autoimmune and viral chronic liver disease. Sera from 39 patients with autoimmune hepatitis, 51 with primary biliary cirrhosis, 17 with autoimmune cholangitis, 24 with primary sclerosing cholangitis and 28 with HCV-related chronic hepatitis were studied. Positivity for antilactoferrin antibodies was evaluated by Western immunoblotting with purified human lactoferrin. Antilactoferrin antibodies were detected more often in autoimmune liver disorders (25% autoimmune hepatitis, 25% primary biliary cirrhosis, 35% autoimmune cholangitis, 29% primary sclerosing cholangitis) than in HCV-related chronic hepatitis (3.5%, P < 0.02 versus all). Positivity for antilactoferrin antibodies was not associated with a particular clinical or biochemical profile of the underlying liver disease. No correlation was observed between antilactoferrin reactivity and perinuclear antineutrophil cytoplasmic antibodies. Antilactoferrin antibodies are present significantly more often in autoimmune than in viral liver disorders, but they cannot be considered the serological marker of a specific autoimmune liver disease.