Biomedical subjects

R A Wetsel

Publications and source records attributed to R A Wetsel.

At least 19 recordsLinked to original sources

PubMed2001

Cutting edge: the absence of C3 demonstrates a role for complement in Th2 effector functions in a murine model of pulmonary allergy.

Asthma is a chronic disease of the lung resulting from airway obstruction. Although the initiating causes are not entirely clear, the airway inflammation in asthma is associated with Th2 lymphocytes and their cytokines, particularly IL-4, which play a prominent role in this disease by regulating airway hyperresponsiveness, eosinophil activation, and IgE synthesis. Historically, complement was not thought to contribute to the pathogenesis of asthma. However, using C3-deficient mice in an allergen-induced model of pulmonary allergy, we demonstrate that complement may impact key features of this disease. When challenged with allergen, mice deficient in C3 exhibit diminished airway hyperresponsiveness and lung eosinophilia. Furthermore, these mice also have dramatically reduced numbers of IL-4-producing cells and attenuated Ag-specific IgE and IgG1 responses. Collectively, these results demonstrate that C3-deficient mice have significantly altered allergic lung responses and indicate a role for the complement system in promoting Th2 effector functions in asthma.

Animals↗

PubMed2001

Characterization of mouse carboxypeptidase N small active subunit gene structure.

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease comprised of two small subunits that have enzymatic activity, and two large subunits, which protect the enzyme from degradation. CPN cleaves the carboxyl-terminal amino acids arginine and lysine from biologically active peptides such as complement anaphylatoxins, kinins, and fibrinopeptides. To delineate the murine CPN small subunit coding region, gene structure, and chromosome location, cDNA and genomic clones were isolated, characterized, and used in Northern and fluorescence in situ hybridization analyses. The results from this study demonstrate that the murine CPN small subunit gene is a single copy gene of approximately 29 kb that is transcribed in the liver into a 1793-bp mRNA with an open reading frame of 1371 nucleotides encoding 457 aa. The gene contains nine exons ranging in size from 455 bp (exon 1) to 100 bp (exon 7), and eight introns ranging in size from 6.2 kb (intron 2) to 1.4 kb (intron 4). All intron/exon junctions follow the normal consensus rule. The mouse CPN small subunit gene localized to chromosomal band 19D2, which is syntenic to human chromosome 10q23-25. Primer extension experiments using mouse liver mRNA indicate one major transcriptional initiation site and three minor sites. Sequence analysis of the 5'-flanking region indicated a TATA-less promoter and numerous transcription factor binding sites, which may confer liver-specific expression of the CPN small subunit gene.

5' Untranslated Regions↗

PubMed2001

Expression of the complement anaphylatoxin C3a and C5a receptors on bronchial epithelial and smooth muscle cells in models of sepsis and asthma.

The presence of the complement-derived anaphylatoxin peptides, C3a and C5a, in the lung can induce respiratory distress characterized by contraction of the smooth muscle walls in bronchioles and pulmonary arteries and aggregation of platelets and leukocytes in pulmonary vessels. C3a and C5a mediate these effects by binding to their specific receptors, C3aR and C5aR, respectively. The cells that express these receptors in the lung have not been thoroughly investigated, nor has their expression been examined during inflammation. Accordingly, C3aR and C5aR expression in normal human and murine lung was determined in this study by immunohistochemistry and in situ hybridization. In addition, the expression of these receptors was delineated in mice subjected to LPS- and OVA-induced models of inflammation. Under noninflamed conditions, C3aR and C5aR protein and mRNA were expressed by bronchial epithelial and smooth muscle cells of both human and mouse lung. C3aR expression increased significantly on both bronchial epithelial and smooth muscle cells in mice treated with LPS; however, in the OVA-challenged animals only the bronchial smooth muscle cells showed increased C3aR expression. C5aR expression also increased significantly on bronchial epithelial cells in mice treated with LPS, but was not elevated in either cell type in the OVA-challenged mice. These results demonstrate the expression of C3aR and C5aR by cells endogenous to the lung, and, given the participation of bronchial epithelial and smooth muscle cells in the pathology of diseases such as sepsis and asthma, the data suggest a role for these receptors during lung inflammation.

Aerosols↗

PubMed2001

A role for complement C5 in organism containment and granulomatous response during murine tuberculosis.

The molecular mechanisms underlying protective granuloma formation and control of bacterial growth during infection with Mycobacterium tuberculosis (MTB) are not yet completely understood. MTB-infected mice with natural deficiency in complement component C5 are unable to develop productive granulomatous responses, and are impaired in limiting organism growth within the lung. To address the molecular basis for this histologic dysfunction, congenic complement C5-sufficient (B10.D2-H2d H2-T18c Hcl/nSnJ) and complement C5-deficient strains (B10.D2-H2d H2-T18c Hco/oSnJ) congenic mice were infected with Mycobacterium tuberculosis, and cytokine and chemokine responses were examined. Twelve and 28 days after infection, lungs showed elevated messages for multiple inflammatory cytokines in both congenic strains. Interleukin (IL)-12(p40) mRNA was also induced during infection in C5-deficient mice, although levels were significantly decreased compared to C5-sufficient congenics. C5-deficient mice also demonstrated reduced KC, MIP-2, IP-10, and MCP-1 mRNA. The defect may directly involve C5-mediated effects on macrophage responses; C5-deficient bone marrow derived macrophages had significantly reduced secretion of KC, MIP-1 alpha and MIP-2 compared to C5-sufficient macrophages following in vitro infection. These findings indicate a role for C5 in mediation of chemotactic and activation events that are the basis for granulomatous responses during murine tuberculosis.

Animals↗

PubMed2000

Cutting edge: targeted disruption of the C3a receptor gene demonstrates a novel protective anti-inflammatory role for C3a in endotoxin-shock.

The complement anaphylatoxin C3a, on binding the C3aR, mediates numerous proinflammatory activities. In addition, recent in vitro studies with C3a have implicated C3aR as a possible anti-inflammatory receptor. Because of its possible dual role in modulating the inflammatory response, it is uncertain whether C3aR contributes to the pathogenesis of endotoxin shock. Here, the targeted-disruption of the C3aR in mice is reported. These mice exhibit an enhanced lethality to endotoxin shock with a pronounced gene dosage effect. In addition, the plasma concentration of IL-1beta was significantly elevated in the C3aR(-/-) mice compared with their littermates following LPS challenge. These findings demonstrate an important protective role for the C3aR in endotoxin shock and indicate that, in addition to its traditionally accepted functions in mediating inflammation, the C3aR also acts in vivo as an anti-inflammatory receptor by attenuating LPS-induced proinflammatory cytokine production.

Amino Acid Sequence↗

PubMed2000

Attenuation of experimental autoimmune demyelination in complement-deficient mice.

The exact mechanisms leading to CNS inflammation and myelin destruction in multiple sclerosis and in its animal model, experimental allergic encephalomyelitis (EAE) remain equivocal. In both multiple sclerosis and EAE, complement activation is thought to play a pivotal role by recruiting inflammatory cells, increasing myelin phagocytosis by macrophages, and exerting direct cytotoxic effects through the deposition of the membrane attack complex on oligodendrocytes. Despite this assumption, attempts to evaluate complement's contribution to autoimmune demyelination in vivo have been limited by the lack of nontoxic and/or nonimmunogenic complement inhibitors. In this report, we used mice deficient in either C3 or factor B to clarify the role of the complement system in an Ab-independent model of EAE. Both types of complement-deficient mice presented with a markedly reduced disease severity. Although induction of EAE led to inflammatory changes in the meninges and perivascular spaces of both wild-type and complement-deficient animals, in both C3(-/-) and factor B(-/-) mice there was little infiltration of the parenchyma by macrophages and T cells. In addition, compared with their wild-type littermates, the CNS of both C3(-/-) and factor B(-/-) mice induced for EAE are protected from demyelination. These results suggest that complement might be a target for the therapeutic treatment of inflammatory demyelinating diseases of the CNS.

Animals↗

PubMed2000

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B.

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.

Animals↗

PubMed2000

The C5a receptor is expressed by human renal proximal tubular epithelial cells.

The C5a receptor is expressed by a variety of cell types. These studies demonstrate by immunohistochemistry that the receptor is present on the surface of proximal and distal tubular epithelial cells from normal kidney. In addition, the receptor was detected on transitional epithelial cells of the ureter and bladder. Primary proximal tubular cultures and a proximal tubular cell line both also expressed the C5a receptor, as demonstrated by immunofluorescence and by FACS analysis. The presence of mRNA encoding the receptor was confirmed by reverse transcriptase-polymerase chain reaction analysis. As opposed to its effect on glomerular mesangial cells, the receptor did not mediate a proliferative response by the proximal tubular cells. C5a also did not enhance the synthesis/secretion of transforming growth factor-beta 1, monocyte chemoattractant protein-1, platelet-derived growth factor-AB or tumour necrosis factor-alpha by cultured proximal tubular cells. Therefore, although the C5a receptor clearly is expressed by proximal tubular cells, clarification of its functional relevance on this cell type awaits further studies.

Antigens, CD↗

PubMed2000

Hypersusceptibility of A/J mice to tuberculosis is in part due to a deficiency of the fifth complement component (C5).

Mycobacterium tuberculosis (MTB) causes tuberculosis in man, which occurs as an acute, chronic or dormant disease reactivating over several years. The mechanisms of persistence and reactivation are not well understood and there is a need for animal models. Moderate-dose, aerosol infection killed A/J mice earlier than partially resistant C57Bl/6 mice, whereas a low-dose, aerosol-induced chronic infection exacerbated earlier in A/J mice. A/J mice lethally infected with MTB but drug cured of disease underwent reactivation of tuberculosis at least 100 days before similarly infected C57Bl/6 mice. Because A/J mice were C5 deficient, congenic B10 mice sufficient and deficient for C5 were infected intravenously with MTB to define the role of C5. C5-deficient mice again showed enhanced growth of MTB in the lungs. MTB-infected macrophages from C5-deficient mice showed enhanced growth of MTB coinciding with a reduced secretion of both cytokines (TNF-alpha, IL-1beta, IL-6, IL-12) and chemokines (KC, MIP-2 and MIP-1alpha) in A/J and TNF-alpha and chemokines in C5-deficient mice. Because C5-deficient macrophages could be activated from extraneous C5 and TNF-alpha we suggest that both play a role in the macrophage-mediated killing as well as containment mechanisms in tuberculosis.

Animals↗

PubMed1999

Genetic deficiency of acylation stimulating protein (ASP(C3ades-Arg)) does not cause hyperapobetalipoproteinemia in mice.

The acylation stimulating protein (ASP) is a 76-amino acid peptide that has been proposed as a potent mediator of triglyceride synthesis and, when functionally impaired, as a major cause of hyperapobetalipoproteinemia (HyperapoB). Purification and sequence analysis of ASP from human sera have revealed that ASP is identical to the complement C3-derived activation peptide C3ades-Arg. Because C3 is the precursor for C3ades-Arg and therefore ASP, a deficiency in C3 would be predicted to result in a phenotype characteristic of HyperapoB. To test this hypothesis in vivo, the current study was undertaken in which ASP(C3ades-Arg)-deficient mice were used as a model system. No significant differences were found in the triglyceride, cholesterol, or free fatty acid concentrations in the plasma of fasted normal and ASP(C3ades-Arg)-deficient animals. In addition, plasma lipoprotein analyses indicated that the very low density lipoprotein, low density lipoprotein, and high density lipoprotein cholesterol and triglyceride concentrations as well as the apolipoprotein B-48 and B-100 levels were not significantly different in the plasma of ASP(C3ades-Arg)-deficient and wild type mice. Furthermore, when challenged with an oral fat load, the ASP(C3ades-Arg)-deficient mice showed no impaired ability to clear triglycerides and free fatty acids from their circulation when compared with their wild-type littermates. Collectively, these results indicate that ASP(C3ades-Arg) deficiency does not cause HyperapoB in mice and that the physiological importance of impaired ASP(C3ades-Arg) function as a cause of hyperapobetalipoproteinemia needs to be reevaluated.

Animals↗

PubMed1999

Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.

Genetic deficiencies of the complement protein C3 occur naturally in humans and animal models and have been induced in mice by targeted deletion of the C3 gene. The study of these deficiencies has provided evidence for C3 functions in immune responses. C3 deficient mice were generated by replacing the 5'-flanking region of the C3 gene with the neomycin-resistance (neo) gene. Serum from these mice had no detectable C3 protein or complement activity. Challenge with Streptococcus pneumoniae revealed approximately 2000-fold increase in bacteremia as compared to littermate controls. C3 mRNA was absent in the liver, but it was detected in the lung, kidney, fat tissue, heart and spleen. Metabolic labeling of the lung tissue and peritoneal macrophages showed synthesis of pro-C3, but no post-synthetic intracellular processing of the protein and no secretion of mature C3. cDNA analysis at the cap site indicated that extrahepatic transcription of the targeted gene was initiated in the neo cassette, close to the C3/neo junction and predicted a primary translation product lacking the leader peptide. The data indicate that these mice provide a good animal model for the study of complete C3 deficiencies and a potential probe for tissue-specific C3 gene regulatory elements.

Animals↗

PubMed1998

Cloning, expression, sequence determination, and chromosome localization of the mouse complement C3a anaphylatoxin receptor gene.

The complement C3a anaphylatoxin receptor (C3aR) is a seven-transmembrane G-protein coupled chemoattractant receptor that on binding the C3a peptide ligand mediates numerous cellular responses, including histamine release from mast cells. smooth muscle contraction, and the directed migration of eosinophils. To delineate the murine C3aR coding sequence, gene structure, 5'-flanking region, and chromosome location, cDNA and genomic clones encoding the mouse C3a receptor were isolated, characterized, and used in fluorescence in situ hybridization experiments. The results from this study indicate that the murine C3a receptor structural gene is a single copy gene of approximately 8 kb comprised of 2 exons which are separated by a large intervening intron of 4724 bp. The first exon encodes 97 bp of 5'-untranslated sequence. Exon 2 encodes the remaining 8 bp of 5'-untranslated sequence and the entire coding and 3'-untranslated sequences. This genomic organization is typical of most other chemoattractant receptor genes in that the entire coding sequence is contained on a single exon. The human and mouse C3a receptor genes were localized to syntenic chromosomal bands 12q13.2-3 and 6F1, respectively. No other seven-transmembrane receptor genes, to date, have been localized to these chromosomal regions. Primer extension experiments using mouse macrophage RNA indicated a single transcriptional initiation site. Sequence analysis 5' of the transcriptional site indicated a TATA-less promoter with possible cis-acting motifs that may regulate C3a receptor gene expression. These included the recognition sequence for the nuclear transcription factor SP1 and the phorbol ester response sequence which binds the Fos/Jun heteromeric transcription factor AP1.

Amino Acid Sequence↗

PubMed1998

Molecular heterogeneity in deficiency of complement protein C2 type I.

Deficiency of the complement protein C2 (C2D), one of the most common genetic deficiencies of the complement system, is associated with rheumatological disorders and increased susceptibility to infection. Two types of C2D have been recognized, each in the context of specific major histocompatibility complex (MHC) haplotypes; type I, a deletion, frameshift and premature stop codon resulting in absence of detectable C2 protein synthesis, and type II, missense mutations resulting in a block in secretion of C2 proteins. Analysis of C2 expression in a child with C2 deficiency, a MHC haplotype different from those associated with type I or II C2D, and recurrent infections revealed additional molecular heterogeneity among C2 deficient patients. No detectable C2 protein was synthesized in the child's fibroblasts under conditions supporting C2 synthesis and secretion in normals and the child's C2 mRNA was reduced to 42% of normal. Nucleotide sequencing of RT-PCR fibroblast mRNA and genomic DNA revealed a type I C2 deficiency (28 base-pair deletion) on one allele and a previously unrecognized two base-pair deletion in exon 2 on the other. Expression of the closely linked factor B gene was markedly decreased (Bf mRNA 25% of normal), though Bf was up-regulated appropriately by interferon-gamma and the flanking sequence containing the Bf promoter was normal in this C2-deficient patient. Moreover, the concentration of Bf protein was normal in the patient's plasma.

Bacteremia↗

PubMed1998

Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo.

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.

Animals↗

PubMed1997

TNF-alpha-mediated expression of the receptor for anaphylatoxin C5a on neurons in experimental Listeria meningoencephalitis.

The anaphylatoxin C5a has been implicated in the pathogenesis of bacterial meningitis as a potent mediator of inflammation in the subarachnoid space. We investigated the expression of the receptor for C5a (C5aR) in brains of mice with experimental Listeria monocytogenes (LM) meningoencephalitis. In the course of the disease, infiltrating cells in the meninges and the ventricles were found to express C5aR mRNA and protein. In the brain parenchyma, very low constitutive C5aR expression was seen on pyramidal neurons and Purkinje cells. However, in LM-infected mice, a dramatic increase in C5aR expression occurred on neurons starting 6 h after infection and was maximal between 24 and 36 h. TNF-alpha was identified as an essential mediator of neuronal C5aR expression, since mice lacking the genes for TNF and lymphotoxin-alpha (TNF/lymphotoxin-alpha -/- mice) showed significantly attenuated C5aR expression after LM infection. Furthermore, i.p. injection of recombinant TNF-alpha induced enhanced C5aR expression in the brains of TNF/lymphotoxin-alpha -/- mice and in normal animals even in the absence of a bacterial infection. We also assessed the levels of anaphylatoxin C5a in the cerebrospinal fluid of patients with infectious meningitis. C5a was detected in all patients with bacterial meningitis (n = 9), in 6 of 18 patients with aseptic meningitis, and in 1 of 66 control patients. The finding of TNF-alpha-mediated C5aR expression on neurons in experimental Listeria meningitis and the detection of the ligand, C5a, in the cerebrospinal fluid of human patients with infectious meningitis present new directions in the investigation of the pathophysiologic sequelae leading to secondary brain damage.

Animals↗

PubMed1996

Type II human complement C2 deficiency. Allele-specific amino acid substitutions (Ser189 --> Phe; Gly444 --> Arg) cause impaired C2 secretion.

Type II complement protein C2 deficiency is characterized by a selective block in C2 secretion. The Type II C2 null allele (C2Q0) is linked to two major histocompatibility haplotypes (MHC) that differ from the MHC of the more common Type I C2 deficiency. To determine the molecular basis of Type II deficiency the two Type II C2Q0 genes were isolated and transfected separately into L-cells. Subsequent molecular biology, biosynthetic, and immunofluorescence studies demonstrated that C2 secretion is impaired in Type II C2 deficiency because of different missense mutations at highly conserved residues in each of the C2Q0 alleles. One is in exon 5 (nucleotide C566 --> T; Ser189 --> Phe) of the C2Q0 gene linked to the MHC haplotype A11,B35,DRw1,BFS, C4A0B1. The other is in exon 11 (G1930 --> A; Gly444 --> Arg) of the C2Q0 gene linked to the MHC haplotype A2,B5, DRw4,BFS,C4A3B1. Each mutant C2 gene product is retained early in the secretory pathway. These mutants provide models for elucidating the C2 secretory pathway.

Alleles↗

PubMed1996

Inherited complement C3 deficiency: reduced C3 mRNA and protein levels in a Laotian kindred.

To determine the molecular basis of complement C3 deficiency in a Laotian kindred, the homozygous C3-deficient male propositus was studied. By ELISA, this individual's serum was determined to contain approximately 4 microg/ml C3 (0.3% of normal). In accord with this result, anti-C3 immunoprecipitation of [35S]-methionine-labeled fibroblasts from this C3D individual revealed pro-C3 of normal size (180,000 Mr), but in significantly reduced amounts (approximately 1% of normal fibroblasts), that was processed and secreted with normal-size alpha- and beta-chains. In addition, C3-specific mRNA of normal size (5.2 kb) but in reduced quantity (approximately 1% of normal) was detected in this individual's fibroblasts by Northern analysis. The nucleotide sequence of the transcriptional initiation site, the promoter, and the IL-1beta/IL-6 cis-regulatory elements of the C3-deficient gene are normal in this C3-deficient individual, indicating that the low C3 mRNA and protein levels are not caused by reduced C3 transcription that is the result of a cis-mutation. Moreover, cDNA sequencing studies revealed no defect in the C3-deficient mRNA, including the areas mutated in four previously characterized C3-deficient patients. These data indicate that (1) C3 protein deficiency in this Laotian patient results from reduced levels of C3-specific mRNA, (2) the small amount of expressed C3 protein is processed and secreted normally from the deficient cells, and (3) the molecular genetic defect(s), although not yet delineated, is different from those described in other C3-deficient individuals, thereby providing additional evidence for numerous mutations that cause inherited C3 deficiency in humans.

Cells, Cultured↗

PubMed1996

Enhanced expression of chemotactic receptors in multiple sclerosis lesions.

We have previously shown that astrocytes and microglia express the receptors for C5a, interleukin-8 (IL-8) and N-formyl-Met-Leu-Phe (FMLP) in vitro. The expression and function of chemotactic receptors in the central nervous system (CNS) is, however, largely unexplored. In this study, we examined tissue sections from normal human brain and active, chronic active and chronic silent multiple sclerosis (MS) lesions for the expression of the receptors for C5a, IL-8 and FMLP by immunohistochemistry. In normal brain tissue, the expression of all three receptors was seen at low levels on astrocytes and microglia. In contrast, expression for all three receptors was markedly elevated on foamy macrophages in the acute and chronic active MS lesions. In addition, fibrous astrocytes stained intensely for the C5a receptor in the chronic active disease. Receptor expression in the chronic silent lesion was low and similar to that seen in normal brain, with staining confined to a few hypertrophic astrocytes and foamy macrophages. These are the first studies to demonstrate expression of these receptors in the CNS and elevated receptor expression in inflammatory MS lesions. The data suggest that these chemotactic receptors may play a role in inflammatory responses in MS and possibly in other CNS diseases.

Acute Disease↗