Increased susceptibility to immunologically mediated glomerulonephritis in IFN-gamma-deficient mice.

It is postulated that IFN-gamma confers susceptibility to immunologically mediated tissue injury. To test this hypothesis, we compared the intensity of accelerated anti-glomerular basement membrane glomerulonephritis between wild-type (IFN-gamma+/+) and IFN-gamma gene knockout (IFN-gamma-/-) mice. This disease model is initiated by binding of heterologous (sheep) anti-glomerular basement membrane Abs to the glomeruli of mice preimmunized with sheep IgG. The secondary cellular and humoral immune responses to the planted Ag then lead to albuminuria and glomerular pathology. We found that IFN-gamma-/- mice or IFN-gamma+/+ mice injected with IFN-gamma-neutralizing Ab develop worse albuminuria and glomerular pathology than IFN-gamma+/+ mice. The humoral response to sheep IgG (serum mouse anti-sheep IgG titers and intraglomerular mouse IgG deposits) was comparable in the IFN-gamma+/+ and IFN-gamma-/- groups. In contrast, IFN-gamma-/- mice mounted a stronger cellular immune response (cutaneous delayed-type hypersensitivity reaction) to sheep IgG than IFN-gamma+/+ mice. These findings provide evidence that endogenous IFN-gamma has a protective role in immunologically mediated glomerulonephritis initiated by foreign Ags.     

Macrophage metalloelastase as a major factor for glomerular injury in anti-glomerular basement membrane nephritis

Rat anti-glomerular basement membrane (GBM) nephritis is a model of crescentic glomerulonephritis induced by injection of anti-GBM antiserum. To elucidate the mechanism of glomerular injury, we analyzed the gene expression patterns in the kidneys of anti-GBM nephritis rats using DNA arrays, and found that macrophage metalloelastase/matrix metalloproteinase (MMP)-12 was one of the highly expressed genes in the kidneys on days 3 and 7 after the injection of anti-GBM antiserum. Enhancement of MMP-12 mRNA expression was confirmed by Northern blot analysis, and in situ hybridization revealed that MMP-12 mRNA was expressed in ED-1-positive macrophages and multinuclear giant cells in the glomeruli with crescent. Moreover, these cells were positive with anti-rat rMMP-12 Ab on the section of the kidneys of anti-GBM nephritis rats on day 7. To clarify the role of MMP-12, we conducted a neutralization experiment using anti-rat rMMP-12 Ab, which had an ability to inhibit rMMP-12 activity of degrading natural substrate such as bovine elastin or human fibronectin in vitro. Anti-rat rMMP-12 Ab or control Ig was injected in each of six rats on days 0, 2, 4, and 6 after the injection of anti-GBM antiserum. Consequently, crescent formation and macrophage infiltration in the glomeruli were significantly reduced in the rats treated with anti-rat rMMP-12 Ab, and the amount of urine protein was also decreased. These results disclosed that MMP-12 played an important role in glomerular injury in a crescentic glomerulonephritis model, and inhibition of MMP-12 may lead to a new therapeutic strategy for this disease.     

Increased susceptibility to complement attack due to down-regulation of decay-accelerating factor/CD55 in dysferlin-deficient muscular dystrophy

Dysferlin is expressed in skeletal and cardiac muscles. However, dysferlin deficiency results in skeletal muscle weakness, but spares the heart. We compared intraindividual mRNA expression profiles of cardiac and skeletal muscle in dysferlin-deficient SJL/J mice and found down-regulation of the complement inhibitor, decay-accelerating factor/CD55, in skeletal muscle only. This finding was confirmed on mRNA and protein levels in two additional dysferlin-deficient mouse strains, A/J mice and Dysf-/- mice, as well as in patients with dysferlin-deficient muscular dystrophy. In vitro, the absence of CD55 led to an increased susceptibility of human myotubes to complement attack. Evidence is provided that decay-accelerating factor/CD55 is regulated via the myostatin-SMAD pathway. In conclusion, a novel mechanism of muscle fiber injury in dysferlin-deficient muscular dystrophy is demonstrated, possibly opening therapeutic avenues in this to date untreatable disorder.     

Increased concentrations of basement membrane collagen fragment in urine of diabetic mice

Levels of a urinary component of basement membrane collagen (type IV) were compared in diabetic (db/db) and normal (db/+) C57BL/Ks inbred mice. A modification of the enzyme-linked immunosorbent assay (ELISA) technique was used with antigen and antibody preparations previously defined to be specific for a fragment from human basement membrane collagen. Significantly elevated levels of basement membrane collagen were found in the urine of homozygous diabetic (db/db) mice as soon as the fourth week after birth. These levels were consistently higher than those found in age-matched normal (db/+) mice throughout the 28 weeks in which measurements were made. Results described here support previous evidence that increased basement membrane collagen synthesis occurs in the db/db model of diabetic nephropathy.     

Role of cathepsin B and L in anti-glomerular basement membrane nephritis in rats

We have examined the potential role of the cysteine proteinases, cathepsin B and L, in renal tubular protein degradation and increased permeability of the glomerular basement membrane (GBM) which occurs in a neutrophil- and complement-independent model of anti-GBM antibody disease. The specific activity of cathepsin L, but not cathepsin B, was significantly increased (157%, p greater than 0.01) in cortical homogenates (85-90% tubules) prepared from anti-GBM-treated rats compared to saline-treated controls. Using highly purified cathepsin B and L, we documented the ability of these proteinases to degrade albumin in vitro (Km 5.92 and 0.22 microM for B and L, respectively). In two separate studies, treatment of rats with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, (E-64), a specific and irreversible inhibitor of cysteine proteinases, significantly reduced proteinuria (-45 and -41%, p less than 0.01) in the 24-hour period following injection of the anti-GBM IgG. Taken together, these data suggest an important role for cysteine proteinases in the increased tubular protein degradation which occurs in response to increased filtered protein loads and in the increased GBM permeability (proteinuria) characteristic of glomerular disease.     

Unaltered susceptibility to scrapie in serotonin transporter deficient mice

The serotonergic system has been hypothesized to play an important role in prion diseases. Specifically, hyperactivity of the serotonergic system in prion diseases is suggested by an increase in the turnover rate of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) in human and experimental prion diseases. The 5-HT transporter (5-HTT) determines the duration of serotonergic neurotransmission by way of reuptake of 5-HT from the extracellular space. 5-HTT availability is reduced in brains of patients with the human prion disease familial fatal insomnia. To further clarify a possible role of the 5-HTT in prion diseases we investigated whether mice lacking the 5-HTT display an altered susceptibility to experimental scrapie infection. Surprisingly, 5-HTT knockout mice developed mouse scrapie in a time course similar to wildtype control mice with accumulation of the pathological prion protein, PrP(Sc) and with typical pathological hallmarks of the disease. These findings argue against a major role of the 5-HTT in the pathogenesis of prion diseases in mice.     

Increased glycosylation of glomerular basement membrane collagen in diabetes

Basement membrane was purified from glomeruli isolated from normal and streptozotocin-diabetic rats. After extraction of non-collagen protein with 8M urea, the extent of glycosylation in glomerular basement membrane collagen was determined with a specific colorimetric reaction that detects carbohydrate in ketoamine linkage with proteins. The level of glycosylation of glomerular basement membrane collagen purified from diabetic rats was significantly greater than that in non-diabetic animals. Increased basement membrane glycosylation may alter structure-function relationships of the capillary filtration barrier.     

T cell infiltration is associated with kidney injury in patients with anti-glomerular basement membrane disease

Cell-mediated autoimmunity, particularly that involving autoreactive T cells, participates in mediating anti-glomerular basement membrane(GBM) disease. However, direct kidney injury mediated by renal infiltrated T cells has not been clearly elucidated in humans. The T cell profile(CD3, CD4, CD8, IL-17, and foxp3) and macrophage(CD68) were examined by immunohistochemistry on renal biopsy tissues from 13 patients with anti-GBM disease. The correlation between cell infiltration and clinical data was also analyzed. We found that the distribution of T cell infiltration was predominant in the peri-glomerular and interstitial areas. CD3~+ T cell infiltratrion around the glomeruli with cellular crescent formations was significantly higher than that around the glomeruli with mild mesangial proliferation. CD8~+ T cells significantly accumulated around the glomeruli with cellular crescents without Ig G deposits compared to those with Ig G deposits. The prevalence of infiltrating CD8~+ T cells was correlated with the percentage of ruptured Bowman's capsules. In conclusion, cellular immunity may play a crucial role in the inflammatory kidney injury in anti-GBM patients. The periglomerular infiltration of T cells, especially CD8~+ T cells, may participate in the pathogenic mechanism of glomerular damage.     

A new pentapeptide compound, PLNPK, ameliorates anti-glomerular basement membrane nephritis in Wistar rats

PLNPK is a pentapeptide compound extracted from pig spleen with a Pro-Leu-Asn-Pro-Lys molecular structure. The spleen is the biggest immune organ in the body, in which there are lots of immunocytes and immune molecules. Our pilot study showed that PLNPK could suppress the transformation and proliferation of T lymphocytes and the production of antibodies in mice. It is widely accepted that most types of glomerulonephritis are immunological diseases caused by the reaction of antigen and antibody. Both humoral immunity and cell-mediated immunity contribute to the progress of these diseases, and suppression of immunoreactions and inflammation is important to ameliorate nephritis. After the immunosuppressive effects of this compound were discovered, this study also examined whether PLNPK had beneficial effects on a rat model of glomerulonephritis. The results suggested PLNPK (200microg/kg/d and 400microg/kg/d) reduced urinary protein excretion, lessened the deposit of autoantibodies along the glomerular basement membrane (GBM), reduced formation of crescent and protein casts, and ameliorated glomerular fibrosis and GBM injury. After treatment with PLNPK (200microg/kg/d and 400microg/kg/d) for 7 days, macrophage infiltration in the glomeruli was markedly reduced. Our results suggest that PLNPK has a beneficial effect on rat anti-GBM nephritis.     

Formation of basement membrane in extracapillary proliferates in rapidly progressive glomerulonephritis.

In the extracapillary proliferations (crescents) of the glomeruli in glomerulonephritis, basement membranes appear and in addition "secretory bodies" are formed in the cisternae of the rough endoplasmatic reticulum. The findings permit the conclusion that proliferated visceral epithelial cells are involved in the crescent formation to a greater extent than previously assumed.     

Increased susceptibility to 6-aminonicotinamide-induced cleft lip of heterozygote Dancer mice

Dancer heterozygotes (Dc/+) very rarely have cleft lip and show a dancing behaviour due to inner ear defects while homozygotes (Dc/Dc) have cleft lip. Males of the two genotypes Dc/+ and +/+ were mated to C3H strain and R stock females and Dc/+ males to Dc/+ females. On day 10/8 of gestation females were treated with 6-aminonicotinamide (6AN) at either 19 mg/kg or 28.5 mg/kg followed 3 h later by a protective dose of nicotinamide. Controls were untreated. Both 6AN treatments caused a significant increase in cleft lip to between 25% and 29% for crosses of Dc/+ males to C3H and R females whereas crosses with +/+ males gave 0% cleft lip. In the controls the cleft lip frequency was: for Dc/+ X Dc/+ 14%, Dc/+ X C3H 1.4%, and for the other three crosses 0%. The four crosses given the high dose of 6AN and the +/+ X C3H and Dc/+ X R cross at the low dose showed significantly increased resorption rates to between 23% and 47% over the control rates of from 5% to 11%. The presence of the Dc gene increased the susceptibility to cleft lip caused by 6AN.     

Increased susceptibility to autoimmune gastritis in thymic stromal lymphopoietin receptor-deficient mice

Thymic stromal lymphopoietin (TSLP), mainly produced by epithelial cells, activates a variety of cell types, including dendritic cells, mast cells, T cells, and B cells. It is involved in the pathogenesis of allergic inflammation in the lung, skin, and gastrointestinal tract. In addition, TSLP promotes Th2-type intestinal immunity against helminth infection and regulates Th1-type inflammation in a mouse model of colitis, suggesting that it plays crucial roles in intestinal immune homeostasis. Although autoimmune gastritis (AIG), mediated by inflammatory Th1 responses, develops in the gastric mucosa, it is not clear whether TSLP is involved in regulating these responses in AIG. The aim of this study was to examine the roles of TSLP in the development of AIG. Because BALB/c mice thymectomized 3 d after birth (NTx mice) develop AIG, we used this model to test the role of TSLP in the development of AIG. We found that in AIG-bearing mice, TSLP was expressed in the inflamed stomach and that the serum anti-parietal cell Ab levels in neonatal thymectomized TSLPR-deficient mice (NTx-TSLPR(-/-) mice) were significantly elevated over those in NTx-TSLPR(+/+) mice. In addition, NTx-TSLPR(-/-) mice exhibited an earlier onset of AIG than that observed in NTx-TSLPR(+/+) mice. The rapid development of AIG in NTx-TSLPR(-/-) mice resulted in more aggressive CD4(+) T cell infiltration and more severe loss of parietal and chief cells in the progression phase of AIG, accompanied by enhanced production of IL-12/23p40 and IFN-γ. Taken together, these data suggested that TSLP negatively regulates the development of AIG.     

Increased susceptibility to Toxoplasma gondii infection in SAG-1 transgenic mice

SAG-1, one of the major surface proteins of Toxoplasma gondii, has been reported to play an important role in immune and pathogenic mechanisms of the parasites but its exact function is still unclear. We investigated the time courses of T. gondii infection in B6C3F1 transgenic mice carrying the SAG-1 gene. SAG-1 transgenic mice were infected intraperitoneally with a high virulent RH strain or a low virulent Beverley strain of T. gondii. When infected with RH strain tachyzoites, no significant differences in time courses of survivals between SAG-1 transgenic and wild-type mice were observed. Both groups succumbed to an acute infection within 8 days after infection. However, a lower survival rate (20%) was observed in SAG-1 transgenic mice than in wild-type (80%), when infected with Beverley strain cysts. This result indicates that SAG-1 transgenic mice are more susceptible to T. gondii infection as compared with their wild-type counterpart. ELISA using recombinant SAG-1 protein indicates that SAG-1 transgenic mice do not produce antibodies to the SAG-1 molecule. These findings may provide a critical tool for analysing the molecular mechanisms of pathogenesis and host immune responses during toxoplasmosis.     

Immune complex glomerulonephritis following bone marrow transplantation in C3 deficient mice

Background

The role of circulating complement in host defense and immune disease is well established. Although a number of cells and tissues are capable of synthesizing complement components locally, the importance of such local synthesis in immune disease has been difficult to establish.

Methodology/Principal Findings

We used bone marrow transplantation (BMT) between C3 knockout (C3KO) and wild type (WT) mice to construct animals that were discordant for systemic (hepatic) and local (monocytic) C3 synthetic capacity. An immune complex glomerulonephritis (GN) was then induced using intraperitoneal injections of horse spleen apoferritin (HSA) with a lipopolysaccharide (LPS) adjuvant. All HSA/LPS animals developed a proliferative GN with glomerular infiltration by monocytes. By sensitive ELISA, monocyte C3 synthesis could be detected in C3KO animals transplanted with WT bone marrow cells. Despite this, there were no significant differences among groups of mice in measures of clinical (proteinuria, renal function) or histologic (glomerular cellularity, crescents) disease severity.

Conclusions/Significance

In this model of GN, local synthesis of C3 by infiltrating cells does not appear to be of pathologic importance.