ABSTRACT
Prostaglandin (PG) D2 is well known as a mediator of inflammation. Hematopoietic PGD synthase (HPGDS) is responsible for the production of PGD2 involved in inflammatory responses. Microglial activation and astrogliosis are commonly observed during neuroinflammation, including that which occurs during demyelination. Using the genetic demyelination mouse twitcher, a model of human Krabbe's disease, we discovered that activated microglia expressed HPGDS and activated astrocytes expressed the DP1 receptor for PGD2 in the brain of these mice. Cultured microglia actively produced PGD2 by the action of HPGDS. Cultured astrocytes expressed two types of PGD2 receptor, DP1 and DP2, and showed enhanced GFAP production after stimulation of either receptor with its respective agonist. These results suggest that PGD2 plays an important role in microglia/astrocyte interaction. We demonstrated that the blockade of the HPGDS/PGD2/DP signaling pathway using HPGDS- or DP1-null twitcher mice, and twitcher mice treated with an HPGDS inhibitor, HQL-79 (4-benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]piperidine), resulted in remarkable suppression of astrogliosis and demyelination, as well as a reduction in twitching and spasticity. Furthermore, we found that the degree of oligodendroglial apoptosis was also reduced in HPGDS-null and HQL-79-treated twitcher mice. These results suggest that PGD2 is the key neuroinflammatory molecule that heightens the pathological response to demyelination in twitcher mice.
Subject(s)
Astrocytes/metabolism , Cell Communication , Demyelinating Diseases/metabolism , Microglia/metabolism , Prostaglandin D2/biosynthesis , Animals , Astrocytes/pathology , Cell Communication/genetics , Cells, Cultured , Demyelinating Diseases/genetics , Demyelinating Diseases/pathology , Leukodystrophy, Globoid Cell/genetics , Leukodystrophy, Globoid Cell/metabolism , Leukodystrophy, Globoid Cell/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Neurologic Mutants , Mice, Transgenic , Microglia/pathology , Prostaglandin D2/geneticsABSTRACT
Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.
Subject(s)
alpha 1-Antitrypsin Deficiency , Animals , Chronic Disease , Chymotrypsin/antagonists & inhibitors , Disease Models, Animal , Female , Fetal Development , Inflammation/etiology , Leukocyte Elastase/antagonists & inhibitors , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Proteins , Serpins , alpha 1-Antitrypsin/physiologyABSTRACT
BACKGROUND: The goal of these studies was to explore the possibility of using gene expression profiles of circulating leukocytes as a functional fingerprint of nephritic disease activity. METHODS: This feasibility study utilized IgA nephropathy (IgAN) as a model system. Genes differentially expressed in IgAN patients were identified by Affymetrix GeneChip microarrays, and compared with gene expression of focal segmental glomerulosclerosis (FSGS), minimal change disease, antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis, and with healthy volunteers. Of the genes identified, 15 transcriptionally up-regulated were validated in a larger cohort of patients using TaqMan polymerase chain reaction (PCR). To test whether increased expression of these genes correlated with disease activity, cluster analyses were performed utilizing the TaqMan PCR values. Taking a mathematical approach, we tested whether gene expression values were correlative with kidney function, as reflected by serum creatinine and creatinine clearance values. RESULTS: We identified 15 genes significantly correlative with disease activity in IgAN. This gene signature of IgAN patients' leukocytes reflected kidney function. This was demonstrated in that mathematically generated theoretical values of serum creatinine and creatinine clearance correlated significantly with actual IgAN patient values of serum creatinine and creatinine clearance. There was no apparent correlation with hematuria and proteinuria. The expression levels of this same gene set in ANCA glomerulonephritis or Lupus nephritis patients were not correlative with serum creatinine or creatinine clearance values. CONCLUSION: These data indicate that leukocytes carry informative disease-specific markers of pathogenic changes in renal tissue.
Subject(s)
Glomerulonephritis, IGA/genetics , Glomerulonephritis, IGA/immunology , Leukocytes/physiology , Oligonucleotide Array Sequence Analysis , Adolescent , Adult , Aged , Biomarkers , Child , Creatinine/blood , Female , Gene Expression Profiling , Humans , Kidney/physiology , Male , Middle Aged , Models, Biological , Regression AnalysisABSTRACT
BACKGROUND: Immunoglobulin A (IgA) nephropathy (IgAN) is a renal disease characterized by glomerular deposition of IgA-dominant immune deposits that cause glomerular inflammation and sclerosis. Gene expression changes induced in renal tissues/cells as a result of the disease are largely uncharacterized. METHODS: A sensitive differential mRNA display technique, restriction endonucleolytic analysis of differentially expressed sequences (READS) compared similarly processed normal renal tissue to renal biopsy RNA from patients with IgAN, minimal change disease, and necrotizing crescentic glomerulonephritis. A subset of genes with altered expression in IgAN as identified by the READS technology was further characterized and expression levels confirmed using real-time quantitative polymerase chain reaction (RT-PCR) analysis (TaqMan) in all RNA. RESULTS: Initial READS analysis showed IgAN samples have lower mRNA levels relative to normal renal tissue mRNA samples based upon total RNA as measured by ribosomal RNA. One hundred seventy-five differentially expressed non-redundant fragments were found from 860 initial candidate fragments. Twenty genes were selected for additional TaqMan analysis, and 13 of 20 genes showed statistically different expression when comparing biopsies from normal individuals and IgAN patients. Expression differences were seen in these genes in biopsies of IgAN of differing clinical activities. Gene expression cluster analysis using the Ward method detailed disease- and gene-related clusters. Detailed examination of the promoter regions of the genes within two gene clusters revealed common gene transcriptional regulatory protein-binding sites. CONCLUSION: IgAN leads to significant changes in overall mRNA transcription levels within the renal tissue, in addition to gene-specific mRNA level changes. Disease-related patterns of expression were identified and gene-specific clusters suggest common mechanisms of transcriptional alteration.
