H.T. Cook

Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP–/– mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.

Translational Mini-Review Series on Complement Factor H: Renal diseases associated with complement factor H: novel insights from humans and animals

Factor H is the major regulatory protein of the alternative pathway of complement activation. Abnormalities in factor H have been associated with renal disease, namely glomerulonephritis with C3 deposition including membranoproliferative glomerulonephritis (MPGN) and the atypical haemolytic uraemic syndrome (aHUS). Furthermore, a common factor H polymorphism has been identified as a risk factor for the development of age‐related macular degeneration. These associations suggest that alternative pathway dysregulation is a common feature in the pathogenesis of these conditions. However, with respect to factor H‐associated renal disease, it is now clear that distinct molecular defects in the protein underlie the pathogenesis of glomerulonephritis and HUS. In this paper we review the associations between human factor H dysfunction and renal disease and explore how observations in both spontaneous and engineered animal models of factor H dysfunction have contributed to our understanding of the pathogenesis of factor H‐related renal disease.

Expression of the gene for inducible nitric oxide synthase in experimental glomerulonephritis in the rat.

Nitrite, a stable product of nitric oxide (NO), is synthesized in vitro by glomeruli in experimental glomerulonephritis. We have now studied the expression of the gene for inducible NO synthase (iNOS) in accelerated nephrotoxic nephritis (NTN). The purpose of the study was to confirm in vivo induction of iNOS in this model of immune complex disease, and to relate the onset of induction and the level of expression to pathogenic events in the model. Glomeruli from rats with NTN were isolated at 6 h, 24 h and 2, 4 and 7 days and total RNA extracted. RNA (10/μg) was reverse transcribed and polymerase chain reaction (PCR) was performed with primers homologous to rat vascular smooth muscle iNOS and rat β actin. A 222‐base PCR product corresponding to iNOS mRNA was present in all experimental animals. iNOS expression was also found in activated macrophages, neutrophils and IL‐1‐stimulated but not unstimulated mesangial cells. Quantitative competitive PCR was carried out on glomerular samples using a 514‐bp mutant of a 735‐bp PCR product. iNOS expression was present at low levels in normal glomeruli and was markedly enhanced at 6 h after the induction of glomerulonephritis and peaked at 24 h. Increased iNOS expression persisted to day 7. β actin mRNA levels were similar in all glomerular specimens. This study demonstrates that there is in vivo induction of iNOS in immune complex glomerulonephritis, corresponding to the generation of nitrite we have previously reported. iNOS gene expression is detectable within 6 h of induction of NTN, indicating the onset of gene transcription is closely related lo the initial formation of immune complexes.

Identification of chromosome intervals from 129 and C57BL/6 mouse strains linked to the development of systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disease in which complex interactions between genes and environmental factors determine the disease phenotype. We have shown that genes from the non-autoimmune strains 129 and C57BL/6 (B6), commonly used for generating gene-targeted animals, can induce a lupus-like disease. Here, we conducted a genome-wide scan analysis of a cohort of (129 × B6)F2 C1q-deficient mice to identify loci outside the C1qa locus contributing to the autoimmune phenotype described in these mice. The results were then confirmed in a larger dataset obtained by combining the data from the C1q-deficient mice with data from previously reported wild-type mice. Both analyses showed that a 129-derived interval on distal chromosome 1 is strongly linked to autoantibody production. The B6 genome contributed to anti-nuclear autoantibody production with an interval on chromosome 3. Two regions were linked to glomerulonephritis: a 129 interval on proximal chromosome 7 and a B6 interval on chromosome 13. These findings demonstrate that interacting loci between 129 and B6 mice can cause the expression of an autoimmune phenotype in gene-targeted animals in the absence of any disrupted gene. They also indicate that some susceptibility genes can be inherited from the genome of non-autoimmune parental strains.

C3 opsonization regulates endocytic handling of apoptotic cells resulting in enhanced T-cell responses to cargo-derived antigens

Significance Activation of the complement system, a network of circulating and surface-bound molecules, is known to enhance humoral immunity. However, paradoxically, the lack of some complement components (mainly C1q and C4), but not C3, is associated with the development of autoimmunity. Prior studies have suggested that this association could be explained by the role of complement in the disposal of dying cells that are a potential source of autoantigens. This study demonstrates that C3 bound to dying cells can direct the intracellular route of the cargo and modulate the subsequent T-cell response to antigens displayed on dying cells. These results uncover a new role of C3 and have important implications for our understanding of the role of complement in health and disease. Apoptotic cells are a source of autoantigens and impairment of their removal contributes to the development of autoimmunity in C1q deficiency. However, the lack of complement component 3 (C3), the predominant complement opsonin, does not predispose to autoimmunity, suggesting a modifying role of C3 in disease pathogenesis. To explore this hypothesis, here we investigated the role of C3 in the T-cell response to apoptotic cell-associated antigens. By comparing the phagosome maturation and the subsequent MHC class II presentation of a peptide derived from the internalized cargo between C3-deficient or C3-sufficient dendritic cells, we found that C3 deficiency accelerated the fusion of the apoptotic cargo with lysosomes. As a result, C3 deficiency led to impaired antigen-specific T-cell proliferation in vitro and in vivo. Notably, preopsonization of the apoptotic cells with C3 activation fragments rectified the trafficking and T-cell stimulation defects. These data indicate that activated C3 may act as a “chaperone” in the intracellular processing of an apoptotic cargo and, thus, may modulate the T-cell response to self-antigens displayed on dying cells.

Bacille Calmette–Guérin (BCG)-associated inflammation and fibrosis: modulation by recombinant BCG expressing interferon-gamma (IFN-γ)

Immunization with existing BCG vaccines has failed to confer consistent protection against tuberculosis. One of the ways to improve the efficacy of BCG is by enhancing its ability to induce a type‐1 T cell response. However, this approach carries the risk that enhanced immunoreactivity may exacerbate tissue pathology associated with vaccination. The aim of the present study was to determine whether use of a recombinant BCG expressing IFN‐γ (BCG‐IFN) would result in an alteration in the pattern of inflammation and local tissue fibrosis. A murine intravenous BCG infection model was used in which there was a time‐ and dose‐dependent increase in the weight and number of granulomas in the liver. Infection was associated with increased inflammatory activity in the liver, as shown by the increase in expression of inducible nitric oxide synthase (iNOS) assessed by immunochemistry and by measurement of specific mRNA, and in fibrosis measured by hydroxyproline content of the liver and percentage of granuloma cells staining positively for type 1 procollagen. Infection with BCG‐IFN resulted in a reduction in organ weight and bacterial load on day 21 compared with infection with control BCG transformed with vector alone (BCG‐plasmid). By day 21, there was also a reduction in iNOS mRNA and iNOS+ cells in granulomas in mice infected with BCG‐IFN compared with infection with BCG‐plasmid, and a similar reduction in both total number of granulomas and liver hydroxyproline content. These results demonstrate that the granulomas in the areas of mycobacterial infection are active sites of both inflammation and fibrosis, and that the local expression of IFN‐γ by the recombinant BCG results in more efficient bacterial clearance which is accompanied by a reduction in tissue pathology.

Interleukin-10- and corticosteroid-induced reduction in type I procollagen in a human ex vivo scar culture

Fibroblasts act as the effector cells of the fibrotic response via production of collagen. In an attempt to understand the regulation of fibroblasts from areas of active human tissue fibrosis, we have developed an ex vivo model in which biopsies of scars from patients 6 weeks post thoracotomy were cultured. This model has been used to investigate whether interleukin‐10 (IL‐10) and triamcinolone acetonide modulate the expression of type I procollagen mRNA and protein. In situ hybridization and a quantitative competitive RT‐PCR were used to measure type I procollagen mRNA. Type I procollagen protein was evaluated by immunochemistry. Viability of biopsies in culture using 3H‐uridine incorporation into RNA was observed to be > 80% for at least 96 hours. Following addition of either IL‐10 or triamcinolone acetonide there was a modest but significant decrease (P<0.05) in type I procollagen mRNA expression. Similarly, each agent added individually to biopsies reduced the proportion of cells staining positively for type I procollagen when compared to biopsies treated with medium alone (P<0.05). These results extend in vitro data that IL‐10 and corticosteroids down‐regulate collagen synthesis in skin fibroblast cell lines and suggest that this ex vivo model may offer a closer approximation to the post‐operative scarring process when testing new therapeutic agents for reducing an over‐exuberent fibrotic response.

Prostaglandin E1 suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage‐dependent glomerulonephritis

Prostaglandin E1 (PGE1) suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage dependent glomerulonephritis. Macrophages are mediators of glomerular injury in models of proliferative glomerulonephritis. We have recently shown that macrophages in glomerulonephritis have low prostaglandin E2 (PGE) generation, and other evidence implicates eicosanoids as regulators of macrophage activation. Here we have studied in rats the effect of 15(S)‐15‐methyl PGE1 (M‐PGE1) on accelerated nephrotoxic nephritis, a model of acute macrophage‐dependent glomerular injury. M‐PGE1 ameliorated proteinuria (day 4; 61 ± 13 mg/24 h, n=9; vehicle treated, 164.17 mg/24 h, n=11; P<0.002) and glomerular hypercellularity; quantification of infiltrating macrophages by isolating glomerular cells showed reduction in the numbers of macrophages (44.9/glomerulus; vehicle treated, 119.15/glomerulus; P <0.02) with inhibition of Ia antigen expression on infiltrating macrophages (8.5%; vehicle treated 25.4%P < 0.05). Glomerular binding of nephrotoxic globulin and levels of autologous antibodies were not affected by M‐PGE1. Thus the mechanism of suppression involves inhibition of macrophage accumulation and activation. M‐PGE1 administered to normal rats did not affect numbers of resident leucocytes (12.6±1.5/glomerulus; vehicle treated, 13.2±1.3/glomerulus) or alter Ia antigen expression (4.1±0.2 Ia + cells/glomerulus; vehicle treated, 3.9±0.6/glomerulus). This study suggests a therapeutic role for PGE1 in this type of glomerulonephritis, and has implications for the pathophysiology of macrophage‐mediated inflammation.

Shiga toxin-2 results in renal tubular injury but not thrombotic microangiopathy in heterozygous factor H-deficient mice.

Haemolytic uraemic syndrome (HUS) is characterized by microangiopathic haemolytic anaemia, thrombocytopenia and renal failure because of thrombotic microangiopathy (TMA). It may be caused by infection with Shiga toxin‐producing enteropathic bacteria (Stx‐associated HUS) or with genetic defects in complement alternative pathway (CAP) regulation (atypical HUS). We hypothesized that defective complement regulation could increase host susceptibility to Stx‐associated HUS. Hence, we studied the response of mice with heterozygous deficiency of the major CAP regulator, factor H, to purified Stx‐2. Stx‐2 was administered together with lipopolysaccharide to wild‐type and Cfh+/− C57BL/6 animals. Forty‐eight hours after administration of the first Stx‐2 injection all animals developed significant uraemia. Renal histology demonstrated significant tubular apoptosis in the cortical and medullary areas which did not differ between wild‐type or Cfh+/− Stx‐2‐treated mice. Uraemia and renal tubular apoptosis did not develop in wild‐type or Cfh+/− animals treated with lipopolysaccharide alone. No light microscopic evidence of TMA or abnormal glomerular C3 staining was demonstrable in the Stx‐2 treated animals. In summary, Stx‐2 administration did not result in TMA in either Cfh+/− or wild‐type C57BL/6 mice. Furthermore, haploinsufficiency of factor H did not alter the development of Stx‐2‐induced renal tubular injury.

Murine glomerular mesangial cell uptake of apoptotic cells is inefficient and involves serum-mediated but complement-independent mechanisms

An increased number of apoptotic bodies have been detected in glomeruli of non‐nephritic kidneys of C1q‐deficient mice. In these mice an in vivo impaired uptake of apoptotic cells by peritoneal macrophages was also demonstrated. Here we investigated whether C1q plays a role in the in vitro clearance of apoptotic cells by glomerular mesangial cells. Phagocytosis was assessed using a novel flow cytometric assay that was validated by immunofluorescence studies. The uptake of apoptotic cells by mesangial cells, measured as percentage of mesangial cells ingesting apoptotic cells, was ∼25%, 10% and 10% for a T cell lymphoma line (RMA), thymocytes and neutrophils, respectively. The uptake reached a plateau phase after 3 h, was specific for apoptotic cells and was mediated by serum but not by complement components C1q or C3. The phagocytosis of apoptotic cells was significantly inhibited by Arg‐Gly‐Asp‐Ser (RGDS), a peptide capable of blocking the interaction of thrombospondin with CD36 or the vitronectin receptor. Pretreatment of the mesangial cells with dexamethasone (200 nm) but not with LPS increased the uptake markedly. These findings indicate that murine mesangial cells are capable of taking up syngeneic apoptotic cells, although much less efficiently than professional phagocytic cells. They also show that serum proteins other than complement components mediate the removal of apoptotic cells by murine mesangial cells in vitro.