In autoimmune disorders, inactivation of pathogenic antigen-specific T cells, rather than global immunosuppression, would be highly desirable. One way to achieve this would be to deliver the first antigen-specific signal to the T cell in the absence of the second costimulatory signal. Myasthenia gravis (MG) is a well-characterized autoimmune disease in which T cell-dependent autoantibodies are directed against the acetylcholine receptor (A ChR) at the neuromuscular junction. AChR-specific T cells have been cloned from MG patients, and in this study, we have induced long-lasting tolerance in vitro in one particular clone (PM-A1) with a known peptide epitope (alpha 144-163) and MHC class II restriction (DR4 Dw14.2 or 4.2) by using soluble MHC-class II peptide complexes. Preincubation of PM-A1 T cells with such complexes induced death by apoptosis in < or = 40-50% of the AChR-specific cells. Surviving cells remained refractory to stimulation with AChR-derived synthetic peptides or recombinant polypeptides for < or = 38 d after complex treatment. These effects were highly specific, dose-dependent and required > 2 h preincubation. The T cells could be protected from the tolerizing effects of complex by coincubation with DR-matched or -mismatched antigen-presenting cells. This work shows that antigen-specific T cells can be selectively killed or anergized using soluble MHC class II: peptide complexes. Such an antigen-specific therapy offers a rational approach to the immunotherapy of autoimmune or allergic disease in vivo.
M W Nicolle, B Nag, S D Sharma, N Willcox, A Vincent, D J Ferguson, J Newsom-Davis
Angiotensinogen is shown to be produced by the liver and the hepatoma cell line HepG2. As a first step for understanding the molecular relationship between the transcriptional regulation of the angiotensinogen gene and the pathogenesis of hypertension, we have analyzed the basal promoter of the angiotensinogen gene. Chloramphenicol acetyltransferase (CAT) assays with 5'-deleted constructs showed that the proximal promoter region from -96 to +22 of the transcriptional start site was enough to express HepG2-specific CAT activity. Electrophoretic mobility shift assay and DNase I footprinting demonstrated that the liver- and HepG2-specific nuclear factor (angiotensinogen gene-activating factor [AGF2]) and ubiquitous nuclear factor (AGF3) bound to the proximal promoter element from -96 to -52 (angiotensinogen gene-activating element [AGE2]) and to the core promoter element from -6 to +22 (AGE3), respectively. The site-directed disruption of either AGE2 or AGE3 decreased CAT expression, and the sequential titration of AGF3 binding by in vivo competition remarkably suppressed HepG2-specific CAT activity. Finally, the heterologous thymidine kinase promoter assay showed that AGE2 and AGE3 synergistically conferred HepG2-specific CAT expression. These results suggest that the synergistic interplay between AGF2 and AGF3 is important for the angiotensinogen promoter activation.
K Tamura, S Umemura, M Ishii, K Tanimoto, K Murakami, A Fukamizu
Mononuclear phagocytes (Mphi) produce urokinase-type plasminogen activator (uPA) and also express a specific cell-surface receptor for urokinase, uPAR. The concomitant expression of these proteins provides a mechanism by which Mphi can degrade extracellular matrix proteins during directed cell migration. In this study, we sought to determine if uPAR plays a role in Mphi chemotaxis that is distinct from its role in matrix proteolysis. Exposing adherent monocytes to a chemotactic gradient causes plasma membrane uPAR to localize strongly to the leading edge of cell migration. Adherence alone or exposure to FMLP had no effect on uPAR expression. Using Boyden chamber chemotaxis assays, we demonstrate that treating mononuclear cells with an anti-uPAR mAb (either as an intact mAb or F[ab']2) ablates chemotaxis induced by FMLP and monocyte chemotactic peptide-1 (P < 0.001). Inactivating the catalytic activity of uPAR-bound uPA had no effect on chemotaxis. Similarly, blocking uPAR expression with an antisense oligonucleotide to uPAR completely ablates chemotaxis, but blocking uPA expression with an antisense oligonucleotide to uPA has a minimal effect. We therefore demonstrate that expression and unimpeded function of uPAR plays an obligate role in M phi chemotaxis by mechanisms that are largely independent of its ligand, uPA. Combined with its known role in mediating pericellular proteolysis, these observations demonstrate that uPAR is essential for both locomotion and traversing tissue barriers during M phi migration.
M R Gyetko, R F Todd 3rd, C C Wilkinson, R G Sitrin
Thyroglobulin appears in the circulation of vertebrates at species-specific concentrations. We have observed that the clearance of thyroglobulin from the circulation occurs in the liver by macrophages. Here we show that the thyroid hormones T3 and T4 were released by incubation of mouse macrophages (J774) with thyroglobulin. Thyroid hormone release was a fast process, with an initial rate of approximately 20 pmol T4/mg per min and approximately 0.6 pmol T3/mg per min, indicating that macrophages preferentially release T4. The bulk of released thyroid hormones appeared after 5 min of incubation of macrophages with thyroglobulin, whereas degradation of the protein was detectable only after several hours. During internalization of thyroglobulin, endocytic vesicles and endosomes were reached at 5 min and lysosomes at 60 min. T4 release started extracellularly by secreted proteases and continued along the endocytic pathway of thyroglobulin, whereas T3 release occurred mainly intracellularly when thyroglobulin had reached the lysosomes. This shows that the release of both hormones occurred at distinct cellular sites. Our in vitro observations suggest that macrophages in situ represent an extrathyroidal source for thyroid hormones from circulating thyroglobulin.
K Brix, V Herzog
The concept of immunosurveillance against cancer has been an extensively debated question over the last decades. Multiple indirect arguments have supported the view that the immune system may control, at least in certain cases, malignant cell growth while direct demonstration is still lacking in the human. In an attempt to address this issue, we have selected a study model, namely spontaneously regressive melanoma. In previous series of experiments, the variability of T cell receptors (TCRs) in the lymphocytes infiltrating a regressive tumor lesion was investigated. Results demonstrated that clonal T cell populations, precisely defined through their V-D-J junctional sequences, were amplified in situ. One clone was predominant, expressing the V beta 16 variable gene segment. A specific anti-V beta 16 TCR mAb was generated here to purify and functionally characterize the corresponding cells. A tumor-infiltrating lymphocyte-derived V beta 16+ T cell line was developed using this reagent. These in vitro cultured cells were found to express the in vivo predominant TCR sequence exclusively and to display an HLA-B14-restricted cytotoxic activity against the autologous tumor cells. Immunohistochemical experiments, performed with the anti-V beta 16 mAb, showed that the corresponding CTLs are present in the tumor area, some of them being closely opposed to the melanoma cells. Together, these studies demonstrate the existence of a local adaptive immune response clinically associated to tumor regression, thus strongly supporting the validity of the immunosurveillance concept in certain human tumors.
A Mackensen, G Carcelain, S Viel, M C Raynal, H Michalaki, F Triebel, J Bosq, T Hercend
Apolipoprotein E3-Leiden (APOE*3-Leiden) transgenic mice have been used to study the effect of different cholesterol-containing diets on the remnant lipoprotein levels and composition and on the possible concurrent development of atherosclerotic plaques. On high fat/cholesterol (HFC) diet, the high expressing lines 2 and 181 developed severe hypercholesterolemia (up to 40 and 60 mmol/liter, respectively), whereas triglyceride levels remained almost normal when compared with regular mouse diet. The addition of cholate increased the hypercholesterolemic effect of this diet. In lines 2 and 181, serum levels of apo E3-Leiden also increased dramatically upon cholesterol feeding (up to 107 and 300 mg/dl, respectively). In these high expressing APOE*3-Leiden transgenic mice, the increase in both serum cholesterol and apo E3-Leiden occurred mainly in the VLDL/LDL-sized fractions, whereas a considerable increase in large, apo E-rich HDL particles also occurred. In contrast to the high expressing lines, the low expressing line 195 reacted only mildly upon HFC diet. On HFC diets, the high expresser APOE*3-Leiden mice developed atherosclerotic lesions in the aortic arch, the descending aorta, and the carotid arteries, varying from fatty streaks containing foam cells to severe atherosclerotic plaques containing cholesterol crystals, fibrosis, and necrotic calcified tissue. Quantitative evaluation revealed that the atherogenesis is positively correlated with the serum level of cholesterol-rich VLDL/LDL particles. In conclusion, with APOE*3-Leiden transgenic mice, factors can be studied that influence the metabolism of remnant VLDL and the development of atherosclerosis.
B J van Vlijmen, A M van den Maagdenberg, M J Gijbels, H van der Boom, H HogenEsch, R R Frants, M H Hofker, L M Havekes
We have examined the mucosal changes occurring in bronchial biopsies from six atopic asthmatics 5-6 h after local endobronchial allergen challenge and compared them with biopsies from saline-challenged segments from the same subjects at the same time point. All the subjects developed localized bronchoconstriction in the allergen-challenged segment and had a decrease in forced expiratory volume in 1 s (FEV1) (P < 0.01) and a decrease in their methacholine provocative concentration of agonist required to reduce FEV1 from baseline by 20% (P < 0.05) 24 h postchallenge. At 6 h we observed an increase in neutrophils (P = 0.03), eosinophils (P = 0.025), mast cells (P = 0.03), and CD3+ lymphocytes (P = 0.025), but not in CD4+ or CD8+ lymphocyte counts. We also detected an increase in endothelial intercellular adhesion molecule type 1 (P < 0.05) and E-selectin (P < 0.005), but not vascular cell adhesion molecule type 1 expression with a correlative increase in submucosal and epithelial LFA+ leucocytes (P < 0.01). Thus, in sensitized asthmatics, local endobronchial allergen instillation leads to an increased inflammatory cell infiltrate of the airway mucosa that involves upregulation of specific adhesion molecules expressed on the microvasculature.
S Montefort, C Gratziou, D Goulding, R Polosa, D O Haskard, P H Howarth, S T Holgate, M P Carroll
We investigated the role of intracellular iron on the capacity of Histoplasma capsulatum (Hc) yeasts to multiply within human macrophages (Mphi). Coculture of Hc-infected Mphi with the iron chelator deferoxamine suppressed the growth of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by iron-free transferrin (apotransferrin). Chloroquine, which prevents release of iron from transferrin by raising endocytic and lysosomal pH, induced human Mphi to kill Hc. The effect of chloroquine was reversed by iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. Chloroquine (40-120 mg/kg) given intraperitoneally for 6 d to Hc-infected C57BL/6 mice significantly reduced the growth of Hc in a dose-dependent manner. At 120 mg/kg there was a 17- and 15-fold reduction (P < 0.01) in CFU in spleens and livers, respectively. The therapeutic effect of chloroquine also correlated with the length of treatment. As little as 2 d of chloroquine therapy (120 mg/kg), when started at day 5 after infection, reduced CFU in the spleen by 50%. Treatment with chloroquine for 10 d after a lethal inoculum of Hc protected six of nine mice; all control mice were dead by day 11 (P = 0.009). This study demonstrates that: (a) iron is of critical importance to the survival and multiplication of Hc yeasts in human Mphi; (b) in vitro, chloroquine induces Mphi killing of Hc yeasts by restricting the availability of intracellular iron; and (c) in vivo, chloroquine significantly reduces the number of organisms in the spleens and livers of Hc-infected mice and can protect mice from a lethal inoculum of Hc yeasts. Thus, chloroquine may be effective in the treatment of active histoplasmosis and also may be useful in preventing relapse of histoplasmosis in patients with acquired immunodeficiency syndromes.
S L Newman, L Gootee, G Brunner, G S Deepe Jr
A unique feature of the choroid plexus as a single-layer epithelium is its localization of Na+K(+)-ATPase at its apical (lumenal) surface. In contrast, a band 3 (AE1)-related anion exchanger protein has been localized to the basolateral surface of the choroid plexus. Both Na+K(+)-ATPase and AE1 in other tissues have been shown to bind via ankyrin to the spectrin-actin-based membrane cytoskeleton. Since linkage of integral membrane proteins to the membrane cytoskeleton is important for their restriction to specialized domains of the cell surface, we investigated the polarity of the choroid plexus membrane cytoskeleton. We developed isoform-specific antibodies to confirm the identity of choroid plexus band 3-related polypeptide as AE2. We demonstrated that ankyrin, fodrin/spectrin, actin, myosin, and alpha-actinin are predominantly apical in choroid plexus and preferentially colocalize with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. Colchicine administration did not alter the polarity of apical cytoskeletal and transport proteins or basolateral AE2 in choroid plexus, suggesting that biosynthetic targeting of these proteins is not microtubule dependent. In choroid plexus papilloma, Na+K(+)-ATPase and AE2 were decreased in amount and failed to preserve their polarized distributions.
S L Alper, A Stuart-Tilley, C F Simmons, D Brown, D Drenckhahn
Studies were performed to determine the effects of intracerebroventricular norepinephrine (NE) or neuropeptide Y (NPY) on the ovine hypothalamic-pituitary-adrenal (HPA) axis. NE (50 micrograms) increased mean hypophysial-portal corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) levels (1 h, 1.3- and 2.9-fold; 4 h, 2.2- and 5.7-fold) and caused acute and sustained increases in mean plasma ACTH and cortisol. NPY (50 microgram) also increased mean CRF and AVP levels (1 h, 1.4- and 4.2-fold; 4 h, 1.1- and 1.9-fold), increased pituitary-adrenal activity at 1 h, and caused ACTH hypersecretion at 4 h. When added to cultured ovine anterior pituitary cells, NPY neither increased basal ACTH release nor augmented CRF- or AVP-induced ACTH release. We conclude that: (a) activation of either the central noradrenergic or NPY pathways causes an acute and sustained stimulation of the ovine HPA axis; (b) such activation increases the AVP/CRF ratio, suggesting a dominant role for AVP in the ovine stress response; and (c) the central noradrenergic or NPY systems may cause sustained HPA activation by attenuating or disrupting the glucocorticoid negative feedback on those brain areas concerned with regulation of the HPA axis. The possible roles of the central noradrenergic and NPY systems in the etiology of the hypercortisolemia of endogenous depression and anorexia nervosa are discussed.
J P Liu, I J Clarke, J W Funder, D Engler
Direct contact between hematopoietic cells and viral packaging cell lines or other sources of stroma has been shown to increase the efficiency of retroviral-mediated gene transfer into these target cells compared with infection with viral supernatant. We have investigated the role of defined bone marrow extracellular matrix molecules (ECM) in this phenomenon. Here we report that infection of cells adhering to the carboxy-terminal 30/35-kD fragment of the fibronectin molecule (30/35 FN), which contains the alternatively spliced CS-1 cell adhesion domain, significantly increases gene transfer into hematopoietic cells. Two retroviral vectors differing in recombinant viral titer were used. Gene transfer into committed progenitor cells and long-term culture-initiating cells, an in vitro assay for human stem cells, was significantly increased when the cells were infected while adherent to 30/35 FN-coated plates compared with cells infected on BSA-coated control plates or plates coated with other bone marrow ECM molecules. Although gene transfer into committed progenitor cells and to a lesser degree into long-term culture-initiating cells was increased on intact fibronectin as well, increased gene transfer efficiency into hematopoietic cells on 30/35 FN was dependent on CS-1 sequence since infection on a similar FN fragment lacking CS-1 (42 FN) was suboptimal. 30/35 FN has previously been shown by our laboratory and other investigators to mediate adhesion of primitive murine and human hematopoietic stem cells to the hematopoietic microenvironment. Additional studies showed that neither soluble 30/35 FN nor nonspecific binding of hematopoietic cells to poly-L-lysine-coated plates had any appreciable effect on the infection efficiency of these cells. Our findings indicate that hematopoietic stem cell adhesion to specific ECM molecules alters retroviral infection efficiency. These findings should aid in the design of gene transfer protocols using hematopoietic progenitor and stem cells for somatic gene therapy.
T Moritz, V P Patel, D A Williams
The cell cycle regulatory enzyme, cdk (cyclin-dependent kinase) 2 kinase, is activated in the rat carotid artery after balloon angioplasty injury, and may mediate smooth muscle proliferation. To test the hypothesis that inhibition of the expression of this key enzyme can inhibit intimal hyperplasia, we studied the effect of antisense phosphorothioate oligodeoxynucleotides (ODN) against cdk 2 kinase administered by intraluminal delivery using hemagglutinating virus of Japan (HVJ)-liposome-mediated transfer. The specificity of antisense cdk 2 ODN was confirmed by the observation that mRNA level of cdk 2 kinase in injured vessels was markedly diminished by the antisense ODN treatment. At 2 wk after transfection, antisense cdk 2 ODN treatment (15 microM) resulted in a significant inhibition (60%) in neointima formation, compared with sense ODN-treated and untreated vessels. Since we have previously observed that cell division cycle 2 kinase mRNA was also activated after vascular injury, we administered the combination of antisense cdc 2 and cdk 2 ODN in this study. Antisense cdc 2 ODN alone (15 microM) only reduced intimal formation by 40%. Combined antisense treatment resulted in near complete inhibition of neointima formation. To understand the mechanism of the sustained effect of a single antisense ODN administration, we examined kinetics of ODN in the vessel wall. Using phosphorothioate FITC-labeled ODN, we transfected carotid artery using the HVJ-liposome method. Fluorescence localized immediately to the medial layer, and persisted up to 2 wk after transfection. These results demonstrate that a single intraluminal administration of antisense ODN directed to cell cycle regulatory genes (e.g., cdk 2 kinase) using the HVJ method can result in a sustained inhibition of neointima formation after balloon angioplasty in rat carotid injury model.
R Morishita, G H Gibbons, K E Ellison, M Nakajima, H von der Leyen, L Zhang, Y Kaneda, T Ogihara, V J Dzau
Nitric oxide (NO) has been suggested to be involved in the regulation of osteoclast activity. Since osteoblasts, through the release of various factors, are the main regulators of osteoclastic resorption, first we have investigated whether osteoblast-like cells and fetal mouse long bone explants are able to produce NO. Second, we have assessed the effect of NO on osteoclastic resorption in whole bone cultures. In this study we show that primary rat osteoblast-like cells as well as the clonal rat osteoblast-like cell line UMR-106, stimulated with IFN-gamma together with TNF-alpha and LPS, produce NO, measured as nitrite production. IL-1 alpha enhanced while TGF-beta 2 inhibited TNF-alpha + IFN-gamma + LPS-stimulated NO production in UMR-106 cells dose dependently. Both the cytokines, however, had no effect when given alone. The competitive inhibitor of NO production, NG-monomethyl-arginine (L-NMMA), and cycloheximide abolished the increase in nitrite production induced by TNF-alpha + IFN-gamma + LPS, while hydrocortisone had no effect, as previously reported for chondrocytes. Calciotropic hormones had either no effect [1,25(OH)2D3] or had a small inhibitory effect (parathyroid hormone) on stimulated NO production. Furthermore, we found that in cultured fetal mouse long bone explants the combination of TNF-alpha + IFN-gamma + LPS as well as the NO donor sodium nitroprusside could inhibit osteoclastic resorption, measured as 45Ca release. The inhibition of resorption was prevented by concurrent administration of L-NMMA. Histological evaluation revealed that the TNF-alpha + IFN-gamma + LPS-induced inhibition of 45Ca release was associated with a decrease in the number of tartrate-resistant acid phosphatase-positive osteoclasts. We propose that the NO production by osteogenic cells (osteoblasts and chondrocytes) may represent an important regulatory mechanism of osteoclastic activity especially under pathological conditions characterized by release of bone-resorbing inflammatory cytokines.
C W Löwik, P H Nibbering, M van de Ruit, S E Papapoulos
The eosinophilia-myalgia syndrome (EMS) is a recently described disease that has been associated with the ingestion of L-tryptophan containing trace amounts of several impurities. The first such contaminant to be identified and linked epidemiologically to the EMS epidemic was 1,1'-ethylidenebis(L-tryptophan) (EBT), but its role in the etiology and pathogenesis of the syndrome has been controversial. We report the development of inflammation and fibrosis affecting the dermis and subcutis, including the fascia and perimyseal tissues, after the daily intraperitoneal administration of EBT to female C57BL/6 mice. Such changes are accompanied by increased numbers of mast cells, many of which appear to be degranulating. Plasma levels of quinolinic acid, a metabolic product of L-tryptophan via the kynurenine pathway, are reduced initially, and then become elevated when inflammation and fibrosis are more pronounced. The nature and location of the inflammatory cell infiltrate and fibrosis, as well as the presence of mast cells and alterations of L-tryptophan metabolism, are consistent with findings reported in patients with EMS. This murine model suggests that EBT may have been one of the mediators of EMS and should facilitate studies of the pathogenesis of EMS.
R M Silver, A Ludwicka, M Hampton, T Ohba, S A Bingel, T Smith, R A Harley, J Maize, M P Heyes
Plasma lipoprotein(a) (Lp(a)) concentrations vary considerably between individuals. To examine the variation for products of the same and different apolipoprotein(a) (apo(a)) alleles, conditions were established whereby phenotyping immunoblots could be used to estimate the concentration of Lp(a) associated with the constituent apo(a) isoforms. In these studies 28 distinct isoforms were identified, each differing by a single kringle IV unit. Tracking the isoforms through 10 families showed that there could be up to 200-fold difference in the Lp(a) concentration associated with the same-sized isoform produced from different alleles. In contrast there was typically < 2.5-fold variation in the Lp(a) concentration associated with the same allele. However, there were four occasions where the concentration associated with a particular allele was reduced below the typical range from one generation to the next. A nonlinear, inverse trend with isoform size was apparently superimposed upon the other factors that determine Lp(a) concentration. Inheritance of familial hypercholesterolemia or familial-defective apoB100 had little consistent effect upon Lp(a) concentration. In both the families and in other unrelated individuals the distribution of isoforms and their associated concentrations provided evidence for the presence of at least two and possibly more subpopulations of apo(a) alleles with different sizes and expression.
Y F Perombelon, A K Soutar, B L Knight
Ceruloplasmin is a plasma protein that carries most of the copper found in the blood. Although its elevation after inflammation and trauma has led to its classification as an acute phase protein, its physiological role is uncertain. A frequently reported activity of ceruloplasmin is its ability to suppress oxidation of lipids. In light of the intense recent interest in the oxidation of plasma LDL, we investigated the effects of ceruloplasmin on the oxidation of this lipoprotein. In contrast to our expectations, highly purified, undegraded human ceruloplasmin enhanced rather than suppressed copper ion-mediated oxidation of LDL. Ceruloplasmin increased the oxidative modification of LDL as measured by thiobarbituric acid-reacting substances by at least 25-fold in 20 h, and increased electrophoretic mobility, conjugated dienes, and total lipid peroxides. In contrast, ceruloplasmin that was degraded to a complex containing 115- and 19-kD fragments inhibited cupric ion oxidation of LDL, as did commercial preparations, which were also degraded. However, the antioxidant capability of degraded ceruloplasmin in this system was similar to that of other proteins, including albumin. The copper in ceruloplasmin responsible for oxidant activity was not removed by ultrafiltration, indicating a tight association. Treatment of ceruloplasmin with Chelex-100 removed one of seven copper atoms per molecule and completely blocked oxidant activity. Restoration of the copper to ceruloplasmin also restored oxidant activity. These data indicate that ceruloplasmin, depending on the integrity of its structure and its bound copper, can exert a potent oxidant rather than antioxidant action on LDL. Our results invite speculation that ceruloplasmin may be in part responsible for oxidation of LDL in blood or in the arterial wall and may thus have a physiological role that is quite distinct from what is commonly believed.
E Ehrenwald, G M Chisolm, P L Fox
The effect of nonsense mutations on mRNA levels is variable. The levels of some mRNAs are not affected and truncated proteins are produced, while the levels of others are severely decreased and null phenotypes are observed. The effect on mRNA levels is important for the understanding of phenotype-genotype association. Cystic fibrosis (CF) is a lethal autosomal recessive disease with variable clinical presentation. Recently, two CF patients with mild pulmonary disease carrying nonsense mutations (R553X, W1316X) were found to have severe deficiency of mRNA. In the Jewish Ashkenazi CF patient population, 60% of the chromosomes carry a nonsense mutation, W1282X. Patients homozygous for this mutation have severe disease presentation with variable pulmonary disease. The presence of CF transcripts in a group of patients homozygous and heterozygous for this mutation was studied by reverse transcriptase PCR of various regions of the gene. Subsequent hybridization to specific CF PCR probes and densitometry analysis indicated that the CF mRNA levels in patients homozygous for the W1282X mutation are not significantly decreased by the mutation. mRNA levels were compared for patients heterozygous for the W1282X mutation. The relative levels of mRNA with the W1282X, and the delta F508 or the normal alleles, were similar in each patient. These results indicate that the severe clinical phenotype of patients carrying the W1282X mutation is not due to a severe deficiency of mRNA. In addition, the severity, progression, and variability of the pulmonary disease are affected by other, as yet unknown factors.
T Shoshani, E Kerem, A Szeinberg, A Augarten, Y Yahav, D Cohen, J Rivlin, A Tal, B Kerem
The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.
H Asako, I Kurose, R Wolf, S DeFrees, Z L Zheng, M L Phillips, J C Paulson, D N Granger
Stromal cells of the bone marrow control the development of osteoclasts through the production of cytokines capable of promoting the proliferation and differentiation of hematopoietic progenitors. Moreover, the deregulated production of the cytokine IL-6 in the bone marrow mediates an increase in osteoclastogenesis after estrogen loss. IL-6, however, does not influence osteoclastogenesis in the estrogen-replete state, suggesting that other cytokines might be responsible for osteoclast development under physiologic circumstances. We report here that IL-11, a newly discovered cytokine that is produced by marrow stromal cells, induced the formation of osteoclasts exhibiting an unusually high degree of ploidy in cocultures of murine bone marrow and calvarial cells. Osteoclasts formed in the presence of IL-11 were capable of bone resorption, as evidenced by the formation of resorption pits, as well as the release of 45Ca from prelabeled murine calvaria. Further, an antibody neutralizing IL-11 suppressed osteoclast development induced by either 1,25-dihydroxyvitamin D3, parathyroid hormone, interleukin-1, or tumor necrosis factor; whereas inhibitors of IL-1 or TNF had no effect on IL-11-stimulated osteoclast formation. The effects of IL-11 on osteoclast development were blocked by indomethacin; more important, however, they were independent of the estrogen status of the marrow donors.
G Girasole, G Passeri, R L Jilka, S C Manolagas
Fibroblast heterogeneity is known to exist in chronically inflamed tissue such as pulmonary fibrosis (IPF) and scleroderma. We have previously shown differences in proliferation rates in primary lines and cloned lines of fibroblasts derived from IPF tissue compared with normal lung. In this study, we report that cell lines derived from fibrotic tissue demonstrate anchorage-independent growth in soft agarose culture whereas normal lung fibroblast lines do not. We also show that fibroblast lines derived from neonatal lung tissue form colonies at about the same frequency as the fibrotic cells. Colonies from both fibrotic and neonatal lines were shown to be positive for vimentin, laminin, fibronectin, fibronectin receptor, beta-actin, and tropomyosin by immunohistochemistry but were negative for desmin, keratin, Factor VIII, alpha-smooth muscle cell actin, and tenascin. Treatment with cytokines TGF-beta and PDGF or with corticosteroid modified the colony-forming capacity of fibrotic and neonatal cell lines, however, none of these treatments induced normal lung cell lines to form colonies. The presence of cells in adult fibrotic tissue with growth characteristics similar to those exhibited by neonatal cells is further evidence of fibroblast heterogeneity and suggests newly differentiated fibroblasts may be prevalent in fibrotic tissue and contribute directly to the matrix disorder seen in this disease.
D J Torry, C D Richards, T J Podor, J Gauldie
Epidermolytic hyperkeratosis (EH) is a skin disease caused by mutations in the genes encoding K1 and K10, the differentiation-specific keratins of epidermis. To explore the heterogeneity of mutations and to assess whether a correlation exists between disease severity and the extent to which a mutation interferes with keratin network formation, we determined the genetic bases of four severe incidences of EH and one unusually mild case. Two severe cases have the same mutation, K10-R156:C, at a conserved arginine that we previously showed was mutated to a histidine in two unrelated EH families. An additional severe case has a mutation six residues away, still within the amino end of the alpha-helical rod domain of K10. The other severe case has a mutation in the conserved carboxy end of the K1 rod. In contrast, affected members of the atypically mild family have a mutation just proximal to the conserved carboxy end of the K10 rod. By genetic engineering and gene transfection, we demonstrate that each mutation is functionally responsible for the keratin filament aberrations that are typical of keratinocytes cultured from these patients. Moreover, we show that the mild EH mutation less severely affects filament network formation. Taken together, our studies strengthen the link between filament perturbations, cell fragility, and degeneration.
A J Syder, Q C Yu, A S Paller, G Giudice, R Pearson, E Fuchs
This study was designed to assess the potential relationship between the late loss of angiographic luminal diameter and biochemical abnormalities of arterial wall collagen in rabbits subjected to angioplasty, and to test the hypothesis that beta-aminopropionitrile (beta APN), an inhibitor of lysyl oxidase, would inhibit such changes when administered orally for 1 mo after angioplasty. Endovascular injury was induced in rabbit iliac arteries by ipsilateral balloon angioplasty (BA) and by contralateral balloon angioplasty accompanied by exposure to continuous wave neodymium: yttrium aluminum garnet laser radiation (LBA). Computer measurement of angiographic luminal diameter demonstrated significant vessel narrowing at 1 and 6 mo after both procedures. By quantitative histology, the majority of the 1-mo loss in angiographic diameter could not be attributed to neointimal thickening. Analysis of collagen cross-linking by HPLC in collagen obtained from the LBA-injured segments of the arteries 1 mo after angioplasty revealed a significant increase, relative to values from uninjured arteries (P < 0.05), in the difunctional cross-link dihydroxylysinonorleucine (DHLNL). 6 mo after angioplasty, the content of hydroxypyridinium, the trifunctional maturational product of DHLNL, was significantly elevated in both BA- and LBA-treated arteries compared with values from uninjured arteries (P < 0.05). In animals administered beta APN, luminal narrowing at 1 mo, compared with controls, was attenuated (P < 0.01) and DHLNL content was decreased (P < 0.05) in arteries subjected to LBA, but not in arteries subjected to BA. The results suggest that lathyrogenic agents may be efficacious in favorably modulating LBA-induced alterations in vessel diameter and mural connective tissue.
J R Spears, H Zhan, S Khurana, R L Karvonen, K M Reiser
Previous investigations of cutaneous delayed hypersensitivity (DHR) in humans and animals have demonstrated that lymphocyte recruitment from blood is temporally and spatially associated with the de novo, asynchronous expression of both vascular cell adhesion molecule 1 (VCAM-1) and E-selectin on dermal endothelium. In this study, DHR was induced in rhesus monkeys sensitized against tuberculin in order to investigate the contribution of E-selectin and VCAM-1 in lymphocyte recruitment to skin. Intravenous infusions of neutralizing doses of F(ab')2 fragments of murine antibodies to either E-selectin or VCAM-1 during the early inductive phases of DHR showed that murine IgG localized to dermal endothelium at the site of DHR in a pattern kinetically similar to the expression of each endothelial adhesion protein. Most importantly, the relative numbers of lymphocytes localized to the inflammatory site were significantly reduced in DHR modified with infusions of antibodies to either VCAM-1 or E-selectin, while the numbers of lymphocytes recruited to skin in the animal given F(ab')2 fragments of an irrelevant murine monoclonal antibody of the same isotype and at the same dose were not changed. Moreover, in individual animals, the relative inhibition achieved with a particular antibody was proportional to the magnitude of expression of the targeted adhesion protein. Therefore, both VCAM-1 and E-selectin are functionally relevant in the genesis of cutaneous DHR, and each appears to contribute to lymphocyte recruitment in relation to its relative degree of expression in any one particular animal.
A Silber, W Newman, V G Sasseville, D Pauley, D Beall, D G Walsh, D J Ringler
Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of IL-8 antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to IL-8. Production of IL-8 by hypoxic ECs occurred concomitantly with both increased levels of IL-8 mRNA, based on polymerase chain reaction analysis, and increased IL-8 transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for macrophage chemotactic protein-1, another member of the chemokine superfamily of proinflammatory cytokines. IL-8 gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of IL-8 antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased myeloperoxidase activity in tissue homogenates. In parallel, increased levels of transcripts for IP-10, a murine homologue in the chemokine family related to IL-8, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis.
M Karakurum, R Shreeniwas, J Chen, D Pinsky, S D Yan, M Anderson, K Sunouchi, J Major, T Hamilton, K Kuwabara
M G O'Sullivan, D C Anderson, J D Fikes, F T Bain, C S Carlson, S W Green, N S Young, K E Brown
We determined the capacity of transplanted beta cells to modify their replication and mass when stimulated by changes in metabolic demand. Five groups of Lewis rats were studied: group 1 (Tx-Px) had a 95% pancreatectomy 14 d after transplantation of 500 islets; group 2 (Px-Tx) had a 95% pancreatectomy 14 d before transplantation of 500 islets; group 3 (Tx) was transplanted with 500 islets; group 4 (Px) had a 95% pancreatectomy; and group 5 (normal) was neither transplanted nor pancreatectomized. Blood glucose was normal in Tx-Px and Tx groups at all times. Px-Tx and Px groups developed severe hyperglycemia after pancreatectomy that was corrected in Px-Tx group in 83% of rats 28 d after transplantation. Replication of transplanted beta cells increased in Tx-Px (1.15 +/- 0.12%) and Px-Tx (0.85 +/- 0.12%) groups, but not in Tx group (0.64 +/- 0.07%) compared with normal pancreatic beta cells (0.38 +/- 0.05%) (P < 0.001). Mean beta cell size increased in Tx-Px (311 +/- 14 microns2) and Px-Tx (328 +/- 13 microns2) groups compared with Tx (252 +/- 12 microns2) and normal (239 +/- 9 microns2) groups (P < 0.001). Transplanted beta cell mass increased in Tx-Px (1.87 +/- 0.51 mg) and Px-Tx (1.55 +/- 0.21 mg) groups compared with Tx group (0.78 +/- 0.17 mg) (P < 0.05). In summary, changes in transplanted beta cells prevented the development of hyperglycemia in Tx-Px rats. Transplanted beta cells responded to increased metabolic demand increasing their beta cell mass.
E Montaña, S Bonner-Weir, G C Weir
Production of leukotriene B4 (LTB4) by human neutrophils (PMN) in response to different stimuli is increased after pretreatment with lipopolysaccharides (LPS). We have analyzed the steps in arachidonic acid (AA) metabolism affected by LPS by examining release of AA and its metabolites from [3H]AA prelabeled PMN. Pretreatment of PMN for 60 min with up to 1 microgram/ml of LPS alone had no effect, but release of [3H]AA was stimulated up to fivefold during subsequent stimulation with a second agent. In the absence of LPS-binding protein (LBP), priming was maximal after pretreatment of PMN with 10 ng of LPS/ml for 60 min; in the presence of LBP maximal priming occurred within 45 min at 0.1 ng of LPS/ml and within 15 min at 100 ng of LPS/ml. Treatment of PMN with 10 ng of LPS/ml also increased uptake of opsonized zymosan by up to 60%. Phospholipids are the source of released [3H]AA. No release was observed from [14C]oleic acid (OA)-labeled PMN suggesting that phospholipolysis may be specific for [3H]AA-labeled phospholipid pools. Cytosol from PMN primed with LPS contains two to three times the phospholipase A2 (PLA2) activity of control PMN, against 1-palmitoyl-[2-14C]arachidonoyl-phosphatidylcholine. This activity is Ca2+ dependent and dithiothreitol resistant. LPS priming is accompanied by reduced migration during SDS-PAGE of an 85-kD protein, identified as a cytosolic PLA2. The extent and kinetics of this effect of LPS on cPLA2 parallel the priming of [3H]AA release, both depending on LPS concentration either with or without LBP. These findings suggest that priming by LPS of AA metabolism by PMN includes phosphorylation of an AA-phospholipid-selective cytosolic PLA2 that is dissociated from activation until a second stimulus is applied.
M E Doerfler, J Weiss, J D Clark, P Elsbach
Although both rat cardiac nonmyocytes (mostly fibroblasts) and cardiomyocytes have a functional angiotensin II (AngII) receptor, the regulation mechanism of its subtype expression in the rat heart remains unknown. In this study, by using a binding assay and a competitive reverse-transcriptase polymerase chain reaction, we examined the regulation of AngII types 1a and 1b (AT1a-R and AT1b-R) and type 2 receptor (AT2-R) expression in embryonal day 19 (E19) and neonatal (1-d) rat cardiac fibroblasts and cardiomyocytes. The number of AT2-R in E19 fibroblasts was dramatically decreased (from 305 to 41 fmol/mg protein) in 1-d fibroblasts, whereas that of AT1-R and the mRNA levels remained unchanged. The ratio of AT1a-R to AT1b-R mRNA in both E19 and 1-d fibroblasts was 9:1. The number of AT2-R in E19 cardiomyocytes was also significantly decreased (from 178 to 87 fmol/mg protein) in 1-d cardiomyocytes, whereas the magnitude was less prominent compared with that in fibroblasts. AT1-R expression remained unaltered in E19 and 1-d cardiomyocytes. In E19 and 1-d cardiomyocytes, the AT1b-R mRNA level was 1.5-fold higher than that of AT1a-R mRNA. Dexamethasone induced significant increases in AT1a-R mRNA (2.1-fold) and numbers (1.8-fold) without changing the affinity, whereas neither AT1b-R mRNA nor the number of AT2-R was affected by dexamethasone. The AT1a-R gene transcription rate, determined by means of a nuclear run-off assay, was increased (2-fold) by dexamethasone. The half-life of AT1a-R mRNA (18 h) was unchanged by dexamethasone. These data indicate that AngII receptor subtype expression in the rat heart is regulated in a cell- and subtype-specific manner.
H Matsubara, M Kanasaki, S Murasawa, Y Tsukaguchi, Y Nio, M Inada
I Y Gluzman, S E Francis, A Oksman, C E Smith, K L Duffin, D E Goldberg
Although the activation of calcium-independent phospholipase A2 (PLA2) enzymes has been described in the heart, the pathogenetic role of this enzyme(s) in hypoxic cell injury has not been previously examined in any tissue. Therefore, we characterized the time course of activation of calcium-independent PLA2 using both plasmalogen and diacylglycerophospholipid substrates during hypoxia in rabbit proximal tubules and examined whether inhibition of calcium-independent PLA2 activity is associated with a cytoprotective effect. Subjecting rabbit proximal tubules to hypoxia for 5 min resulted in at least a threefold increase in cytosolic calcium-independent PLA2, which was selective for plasmalogen substrates (control 444 +/- 69 vs hypoxia 1,675 +/- 194 pmol.mg protein-1.min-1, n = 5). In contrast, no changes in PLA2 activity were observed in the presence of 4 mM EGTA in the membrane fraction using plasmenylcholine substrates. 20 min of hypoxia resulted in an increase in arachidonate from 3 +/- 1 to 28 +/- 4 ng/mg protein and lactate dehydrogenase release from 7.5 +/- 2% to 38 +/- 5%, n = 4. Pretreatment of proximal tubules with 10 microM Compound I, a specific inhibitor of calcium-independent PLA2, resulted in reduction in the magnitude of both hypoxia-induced arachidonic acid release (11 +/- 3 ng/mg protein) and lactate dehydrogenase release (18 +/- 4%). Our data indicate that a significant fraction of PLA2 activity in the proximal tubule is calcium-independent and selective for plasmalogen substrates. Furthermore, the activation of this enzyme plays an important role in the pathogenesis of membrane injury during hypoxia in the proximal tubule.
D Portilla, S V Shah, P A Lehman, M H Creer
Fetuses of eight pregnant ewes (114-117 d of gestation) were used to study whether fetal insulin concentration affects fetal protein accretion and, if so, whether such changes are caused by effects on protein synthesis or protein breakdown. Fetal leucine kinetics were measured by infusion of [1-14C]leucine during each of three protocols: (I) low vs. normal insulin concentration; (II) low vs. high insulin concentration; and (III) low vs. high insulin concentration during amino acid infusion to keep leucine concentration constant. Fetal leucine concentration (233 +/- 20 vs. 195 +/- 18 microM) and clearance (48.3 +/- 4.4 vs. 54.2 +/- 5.5 ml/kg per min) were the only aspects of fetal leucine kinetics that changed during protocol I. During protocol II, insulin infusion decreased fetal leucine concentration (222 +/- 22 vs. 175 +/- 22), decreased fetal leucine disposal (11.63 +/- 0.89 vs. 12.55 +/- 0.89 mumol/kg per min), increased leucine clearance (48.0 +/- 4.2 vs. 57.6 +/- 6.5 ml/kg per min), decreased leucine decarboxylation (1.77 +/- 0.17 vs. 2.04 +/- 0.21 mumol/kg per min), decreased nonoxidative leucine disposal (9.81 +/- 0.78 vs. 10.51 +/- 0.74 mumol/kg per min), decreased release of leucine from fetal protein (7.43 +/- 1.08 vs. 8.38 +/- 0.84 mumol/kg per min), but did not change the accretion of leucine into protein. In contrast, when leucine concentrations (205 +/- 25 vs. 189 +/- 23) were maintained (protocol III), insulin infusion did not change fetal leucine disposal, decarboxylation, or nonoxidative disposal although leucine clearance still rose (55.4 +/- 5.0 vs. 64.4 +/- 5.9 ml/kg/min). Fetal release of leucine from protein, however, decreased (7.46 +/- 0.83 vs. 8.57 +/- 0.71 mumol/kg per min) and the accretion of leucine into protein increased (3.27 +/- 0.30 vs. 1.80 +/- 0.32 mumol/kg/min). These findings show that insulin decreases fetal protein breakdown. If insulin-induced hypoaminoacidemia occurs, protein synthesis decreases so that no net accretion of protein occurs. If fetal amino acid concentrations are maintained, however, insulin itself does not affect protein synthesis, and fetal protein accretion increases.
J R Milley
Some human small cell lung carcinomas (SCLC) secrete proopiomelanocortin (POMC) derived peptides, but in contrast to the pituitary, glucocorticoids fail to inhibit this hormone production. We have previously described an in vitro model using human SCLC cell lines that express POMC and are resistant to glucocorticoids. We have now identified the glucocorticoid receptor (GR) in the SCLC cell line COR L24 using a whole cell ligand binding assay (Kd = 5.7 nM; Bmax = 11 fmol/million cells), while another cell line, DMS 79, lacked significant glucocorticoid binding. To analyze GR function both positive (GMCO) and negative (TRE)3-tkCAT), glucocorticoid-regulated reporter gene constructs were transfected into COR L24 cells. In the SCLC cell line, neither hydrocortisone nor dexamethasone (500-2,000 nM) significantly induced chloramphenicol acetyltransferase expression from GMCO; in addition, they did not suppress chloramphenicol acetyltransferase expression from (TRE)3-tkCAT. Similar results were obtained with two other POMC-expressing SCLC cell lines. Expression of wild type GR in COR L24 cells restored glucocorticoid signaling, with marked induction of GMCO reporter gene expression by dexamethasone (9,100 +/- 910%; n = 3), and an estimated EC50 of 10 nM. This failure of the GR explains the resistance of the POMC gene to glucocorticoid inhibition and may have implications for cell growth in SCLC.
D W Ray, A C Littlewood, A J Clark, J R Davis, A White
Migration plays an important role in the formation of tumor metastases. Nonetheless, little is known about electrophysiological phenomena accompanying or underlying migration. Previously, we had shown that in migrating alkali-transformed Madin-Darby canine kidney focus (MDCK-F) cells a Ca(2+)-sensitive 53-pS K+ channel underlies oscillations of the cell membrane potential. The present study defines the role this channel plays in migration of MDCK-F cells. We monitored migration of individual MDCK-F cells by video imaging techniques. Under control conditions, MDCK-F cells migrated at a rate of 0.90 +/- 0.03 microns/min (n = 201). Application of K+ channel blockers (1 and 5 mmol/liter Ba2+, 5 mmol/liter tetraethylammonium, 100 mumol/liter 4-aminopyridine, 5 nmol/liter charybdotoxin) caused marked inhibition of migration, pointing to the importance of K+ channels in migration. Using patch-clamp techniques, we demonstrated the sensitivity of the Ca(2+)-sensitive 53-pS K+ channel to these blockers. Blockade of this K+ channel and inhibition of migration were closely correlated, indicating the necessity of oscillating K+ channel activity for migration. Migration of MDCK-F cells was also inhibited by furosemide or bumetanide, blockers of the Na+/K+/2Cl- cotransporter. We present a model for migration in which oscillations of cell volume play a central role. Whenever they are impaired, migration is inhibited.
A Schwab, L Wojnowski, K Gabriel, H Oberleithner
Ro small ribonucleoprotein complexes (RoRNPs) are thought to comprise several proteins, including the 60-kD Ro and the 52-kD Ro proteins, and several small RNAs, designated Y RNAs. Although RoRNPs are fairly ubiquitous in nature, their precise composition remains unknown, their function has been elusive, and their intracellular localization has been controversial. We have analyzed HeLa cell extracts by glycerol density gradient fractionation in order to determine the distribution of the individual protein and RNA components of RoRNPs. We found that 52-kD Ro was not detectable in an RNP complex with the 60-kD protein under a variety of conditions. Pretreatment of cell extracts with ribonuclease affected gradient migration of the 60-kD but not the 52-kD protein, suggesting that the latter is not complexed with RNA. The migration of the hY RNAs in these gradients closely followed that of 60-kD and not 52-kD Ro. Immunofluorescence analysis of two different cell lines with monospecific antibodies against 52- and 60-kD proteins strongly suggests that these two proteins are not present on overlapping sets of structures in vivo. We conclude that the 52-kD Ro protein is not a detectable component of the RoRNP complex under these conditions despite its reactivity with Ro autoimmune antisera.
A Kelekar, M R Saitta, J D Keene
The strategic location of mast cells at the host-environment interface and their ability to release potent mediators of inflammation have suggested that these cells may play a pivotal role in host defense against bacterial infection. The ability of the opportunistic pathogen, Escherichia coli, to induce degranulation of mast cells obtained from the mouse peritoneum was investigated. We determined that unlike a mutant derivative deficient in the FimH subunit of the fimbriae or nonfimbriated E. coli, type 1 fimbriated E. coli induced mast cell degranulation in vitro. The magnitude of mast cell degranulation was directly proportional to the number of adherent bacteria on the cell surface in the initial period of the interaction. Using a mouse model of bacterial peritonitis, we demonstrated mast cell degranulation and histamine release by type 1 fimbriated bacteria in vivo. Furthermore, beads coated with FimH but not with FimA, the major subunit of type 1 fimbriae, evoked mast cell release of histamine in vivo in amounts comparable to that elicited by type 1 fimbriated E. coli. These studies reveal that mast cells can be degranulated by interaction with type 1 fimbriated E. coli and that FimH, the mannose-binding component of the fimbriae, is a potent mast cell stimulant.
R Malaviya, E Ross, B A Jakschik, S N Abraham
Isometric exercise increases sympathetic nerve activity and blood pressure. This exercise pressor reflex is partly mediated by metabolic products activating muscle afferents (metaboreceptors). Whereas adenosine is a known inhibitory neuromodulator, there is increasing evidence that it activates afferent nerves. We, therefore, examined the hypothesis that adenosine stimulates muscle afferents and participates in the exercise pressor reflex in healthy volunteers. Intraarterial administration of adenosine into the forearm, during venous occlusion to prevent systemic effects, mimicked the response to exercise, increasing muscle sympathetic nerve activity (MSNA, lower limb microneurography) and mean arterial blood pressure (MABP) at all doses studied (2, 3, and 4 mg). Heart rate increased only with the highest dose. Intrabrachial adenosine (4 mg) increased MSNA by 96 +/- 25% (n = 6, P < 0.01) and MABP by 12 +/- 3 mmHg (P < 0.01). Adenosine produced forearm discomfort, but equivalent painful stimuli (forearm ischemia and cold exposure) increased MSNA significantly less than adenosine. Furthermore, adenosine receptor antagonism with intrabrachial theophylline (1 microgram/ml forearm per min) blocked the increase in MSNA (92 +/- 15% vs. 28 +/- 6%, n = 7, P < 0.01) and MABP (38 +/- 6 vs. 27 +/- 4 mmHg, P = 0.01) produced by isometric handgrip (30% of maximal voluntary contraction) in the infused arm, but not the contralateral arm. Theophylline did not prevent the increase in heart rate produced by handgrip, a response mediated more by central command than muscle afferent activation. We propose that endogenous adenosine contributes to the activation of muscle afferents involved in the exercise pressor reflex in humans.
F Costa, I Biaggioni
Recent work conducted in our laboratory has been directed towards understanding the role of TNF alpha in stimulating the synthesis of two macrophage gene products, namely IGF-1, a growth factor implicated in wound repair and fibrosis, and complement component factor B (Bf), an alternative pathway complement component. The expression of these proteins is induced by hyaluronic acid and poly (I:C), respectively, although TNF alpha plays a requisite role in the expression of both proteins. The objective of this study was to determine the mechanism governing the dichotomy in the expression of IGF-1 and Bf by TNF alpha. First, we questioned if the diversity in IGF-1 and Bf synthesis was regulated at the level of TNF receptor usage. Second, based on earlier findings that IFNs contribute to the initiation of Bf expression, we determined if IFNs modulate the response of macrophages to TNF alpha. Our data show that differences in TNF receptor usage cannot fully explain the dichotomy in the expression of IGF-1 and Bf. However, prior exposure to IFN-beta or IFN-gamma was found to be a dominant factor controlling the expression of these proteins, suppressing IGF-1, and enhancing Bf. These findings indicate that IFNs mediate a functional "switch" in the response of macrophages to TNF alpha and suggest that the pattern of cytokine expression by diverse macrophage stimuli is an important determinant of the eventual responses of macrophages to TNF alpha.
F R Lake, P W Noble, P M Henson, D W Riches
Prevention of red cell K+ and water loss is a therapeutic strategy for sickle cell disease. We have investigated in vitro and in vivo the effects of clotrimazole (CLT) and miconazole (MIC) on transgenic mice red cells expressing hemoglobin SAD. CLT blocked the Gardos channel (ID50 75 +/- 22 nM; n = 3) and the A23187-induced dehydration of Hbbs/Hbbthal SAD 1 mouse erythrocytes in vitro. Oral treatment with CLT (160 mg/kg per d) and MIC (100 mg/kg per d) inhibited the Gardos channel in both SAD 1 and control (Hbbs/Hbbthal) mice. In the SAD 1 mice only, cell K+ content increased, and mean corpuscular hemoglobin concentration and cell density decreased. After 7 d of treatment, the hematocrit of SAD 1, CLT-treated animals also increased. All changes were fully reversible. Long-term treatments of SAD 1 mice with oral CLT (80 mg/kg per d for 28 d) lead to sustained increases in cell K+ content and hematocrit and sustained decreases in mean corpuscular hemoglobin concentration and cell density, with no changes in animals treated with vehicle alone. Thus, CLT and MIC can reverse dehydration and K+ loss of SAD 1 mouse erythrocytes in vitro and in vivo, further supporting the potential utility of these drugs in the treatment of sickle cell anemia.
L De Franceschi, N Saadane, M Trudel, S L Alper, C Brugnara, Y Beuzard
The central nervous system has been implicated in the activation of counterregulatory hormone release during hypoglycemia. However, the precise loci involved are not established. To determine the role of the ventromedial hypoglycemia, we performed hypoglycemic clamp studies in conscious Sprague-Dawley rats with bilateral VMH lesions produced by local ibotenic acid injection 2 wk earlier. Rats with lesions in the lateral hypothalamic area, frontal lobe, sham operated (stereotaxic needle placement into hypothalamus without injection), and naive animals served as control groups. The clamp study had two phases. For the first hour plasma glucose was fixed by a variable glucose infusion at euglycemia (approximately 5.9 mM). Thereafter, for an additional 90 min, glucose was either allowed to fall to (a) mild hypoglycemia (approximately 3.0 mM) or (b) more severe hypoglycemia (approximately 2.5 mM). Glucagon and catecholamine responses of lateral hypothalamic area-, frontal lobe-lesioned, sham operated, and naive animals were virtually identical at each hypoglycemic plateau. In contrast, glucagon, epinephrine, and norepinephrine responses in the VMH-lesioned rats were markedly inhibited; hormones were diminished by 50-60% during mild and by 75-80% during severe hypoglycemia as compared with the other groups. We conclude that the VMH plays a crucial role in triggering the release of glucagon and catecholamines during hypoglycemia.
W P Borg, M J During, R S Sherwin, M A Borg, M L Brines, G I Shulman
We have generated transgenic mice expressing the human apolipoprotein CII (apoCII) gene under the transcriptional control of the human cytochrome P-450 IA1 (CYPIA1) promoter. Human apoCII transgenic (HuCIITg) mice exhibited significant basal expression of the transgene (plasma apoCII level = 26.1 +/- 4 mg/dl) and showed further induction of transgene expression after treatment with beta-naphthoflavone. Unexpectedly, HuCIITg mice were hypertriglyceridemic and human apoCII levels correlated strongly to triglyceride levels (R = 0.89, P < 0.0001). Triglyceride levels (mg/dl +/- SEM) were elevated compared to controls in both the fed (804 +/- 113 vs 146 +/- 18, P < 0.001) and fasted (273 +/- 39 vs 61 +/- 4, P < 0.001) states. HuCIITg mice accumulated triglyceride-rich very low density lipoproteins (VLDL) with an increased apoC/apoE ratio. Tracer kinetic studies indicated delayed clearance of VLDL-triglyceride, and studies using Triton inhibition of VLDL clearance showed no increase in VLDL production. Plasma from these mice activated mouse lipoprotein lipase normally and radiolabeled VLDL were normally hydrolyzed. However, HuCIITg VLDL showed markedly decreased binding to heparin-Sepharose, suggesting that apoCII-rich, apoE-poor lipoprotein may be less accessible to cell surface lipases or receptors within their glycosaminoglycan matrices. HuCIITg mice are a promising model of hypertriglyceridemia that suggests a more complex role for apoCII in the metabolism of plasma triglycerides.
N S Shachter, T Hayek, T Leff, J D Smith, D W Rosenberg, A Walsh, R Ramakrishnan, I J Goldberg, H N Ginsberg, J L Breslow
Increased production of EGF or TGF-alpha by the respiratory epithelial cells has been associated with the pathogenesis of various forms of lung injury. Growth factors and cytokines are thought to act locally, via paracrine and autocrine mechanisms, to stimulate cell proliferation and matrix deposition by interstitial lung cells resulting in pulmonary fibrosis. To test whether TGF-alpha mediates pulmonary fibrotic responses, we have generated transgenic mice expressing human TGF-alpha under control of regulatory regions of the human surfactant protein C (SP-C) gene. Human TGF-alpha mRNA was expressed in pulmonary epithelial cells in the lungs of the transgenic mice. Adult mice bearing the SP-C-TGF-alpha transgene developed severe pulmonary fibrosis. Fibrotic lesions were observed in peribronchial, peribronchiolar, and perivascular regions, as well as subjacent to pleural surfaces. Lesions consisted of fibrous tissue that included groups of epithelial cells expressing endogenous SP-C mRNA, consistent with their identification as distal respiratory epithelial cells. Peripheral fibrotic regions consisted of thickened pleura associated with extensive collagen deposition. Alveolar architecture was disrupted in the transgenic mice with loss of alveoli in the lung parenchyma. Pulmonary epithelial cell expression of TGF-alpha in transgenic mice disrupts alveolar morphogenesis and produces fibrotic lesions mediated by paracrine signaling between respiratory epithelial and interstitial cells of the lung.
T R Korfhagen, R J Swantz, S E Wert, J M McCarty, C B Kerlakian, S W Glasser, J A Whitsett
An adoptive transfer model of insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic mouse was used to examine the roles of alpha 4-integrin, vascular cell adhesion molecule 1 (VCAM-1); and intercellular adhesion molecule 1 (ICAM-1) in the pathogenesis of autoimmune diabetes. Antibodies specific for both alpha 4-integrin and one of its ligands, VCAM-1, were able to delay onset of diabetes and decrease the incidence of the disease in adoptive transfer studies. This blocking of disease was accompanied by a marked decrease in lymphocytic infiltration of the islets of Langerhans. Furthermore, these antibodies preferentially block entrance of CD4 T cells into the tissue. Antibodies specific for ICAM-1 had little effect on the onset or incidence of IDDM. Thus, we conclude that an alpha 4-integrin-VCAM-1 interaction is important in T cell entry into the islets of Langerhans and in the pathogenesis of IDDM. In addition, the cascade of events leading to T cell transit across endothelium may be different for CD4 and CD8 cells, and may differ depending on the endothelium involved. Our results support the more general conclusion that an alpha 4-integrin-VCAM-1 interaction may be crucial in allowing activated effector CD4T cells to leave the blood and enter tissue to clear infection.
J L Baron, E P Reich, I Visintin, C A Janeway Jr
A hallmark of systemic sclerosis (SSc) is the development of tissue fibrosis. Excessive production of several connective tissue components normally present in the dermis, including type I, III, V, and VI collagens as well as fibronectin and proteoglycans, is a consistent finding in the skin of SSc patients. Type VII collagen is a major constituent of anchoring fibrils, present in the skin at the dermal-epidermal basement membrane zone. TGF-beta has been shown to upregulate the expression of the type VII collagen gene. In this study, we assessed the expression of type VII collagen and TGF-beta in the skin of patients with SSc. Indirect immunofluorescence showed an abundance of type VII collagen in the patients' skin, including the dermis. Ultrastructural analysis of SSc skin revealed an abundance of fibrillar material, possibly representing type VII collagen. The increased expression of type VII collagen epitopes was accompanied by the elevated expression of immunodetectable TGF-beta 1 and TGF-beta 2. Dermal fibroblasts cultured from the affected individuals showed a statistically significant (P < 0.02) increase in the expression of type VII collagen at the mRNA level, as detected by reverse transcription-PCR with a mutated cDNA as an internal standard, and increased deposition of the protein as assessed by indirect immunofluorescence. Thus, type VII collagen is abundantly present in SSc patients' dermis, a location not characteristic of its normal distribution, and its aberrant expression may relate to the presence of TGF-beta in the same topographic distribution. The presence of type VII collagen in the dermis may contribute to the tightly bound and indurated appearance of the affected skin in SSc patients.
L Rudnicka, J Varga, A M Christiano, R V Iozzo, S A Jimenez, J Uitto
In the present study, we have isolated and sequenced the complementary DNAs of two mutant alleles for lysyl hydroxylase (LH) in fibroblasts from one patient (AT750) with Ehlers-Danlos syndrome type VI (EDS VI). We have identified a putative mutation in each allele which may be responsible for the patient's decreased LH (normalized to prolyl hydroxylase) activity (24% of normal). Intermediate levels of LH activity were measured in the patient's parents, who are clinically normal (father 52%; mother 86%). After the cloning of cDNAs and amplification by PCR, sequence analysis revealed two equally distributed populations of cDNAs for LH in the AT750 cell line. Each allele revealed different but significant changes from the normal sequence. In one allele (allele 1), the most striking change was a triple base deletion that would result in the loss of residue Glu532. The most significant difference in the other allele (allele 2) was a G-->A change which would produce a Gly678-->Arg codon change in a highly conserved region of the enzyme. Restriction analysis identified that allele 1 was inherited from the proband's mother and allele 2 from the father. This study represents the first example of compound heterozygosity for the LH gene in an EDS VI patient, and it appears that there is an additive effect of each mutant allele on clinical expression in this patient.
V T Ha, M K Marshall, L J Elsas, S R Pinnell, H N Yeowell
A new immunoassay was developed to detect denaturation of type II collagen in osteoarthritis (OA). A peptide, alpha 1 (II)-CB11B, located in the CB11 peptide of type II collagen, was synthesized and used to produce a monoclonal antibody (COL2-3/4m) of the IgG1 (kappa) isotype. This reacts with a defined epitope in denatured but not native type II collagen and the alpha 3 chain of type XI collagen. The latter is present in very small amounts (about 1% wt/wt) in cartilage relative to the alpha 1 (II) chain. By using an enzyme-linked immunosorbent assay, type II collagen denaturation and total type II collagen content were determined. The epitope recognized by the antibody was resistant to cleavage by alpha-chymotrypsin and proteinase K which were used to extract alpha 1 (II)-CB11B from the denatured (alpha-chymotrypsin soluble) and residual native (proteinase K soluble) collagen alpha-chains, respectively, present in human femoral articular cartilage. Type II collagen content was significantly reduced from a mean (range) of 14% (9.2-20.8%) of wet weight in 8 normal cartilages to 10.3% (7.4-15.0%) in 16 OA cartilages. This decrease, which may result in part from an increased hydration, was accompanied by an increase in the percent denaturation of type II collagen in OA to 6.0% of total type II collagen compared with 1.1% in normal tissue. The percent denaturation was ordinarily greater in the more superficial zone than in the deep zone of OA cartilage.
A P Hollander, T F Heathfield, C Webber, Y Iwata, R Bourne, C Rorabeck, A R Poole
Interleukin 12 (IL-12), a heterodimeric cytokine composed of p40 and p35 chains, has potent immunologic effects in vitro. We used tuberculous pleuritis as a model to study the immunoregulatory potential of IL-12 in vivo at the site of human infectious disease. Messenger RNAs for p40 and p35 were detected in pleural fluid from six of six patients by reverse-transcription polymerase chain reaction. By using an ELISA that detected both free p40 and heterodimeric IL-12, we found that mean concentrations were 585 +/- 89 pg/ml in pleural fluid of patients with tuberculous pleuritis, which were significantly higher than those in serum of the same patients (54 +/- 36 pg/ml), or in malignant pleural effusions (123 +/- 35 pg/ml). By using an ELISA specific for heterodimeric IL-12, we found that mean concentrations in pleural fluid of patients with tuberculous pleuritis were 165 +/- 28 pg/ml and undetectable in serum of the same patients, or in malignant pleural effusions. Bioactive IL-12 was detectable in five of five supernatants of pleural fluid cells stimulated with Mycobacterium tuberculosis. Addition of anti-IL-12 antibodies suppressed proliferative responses of pleural fluid cells to M. tuberculosis by 36 +/- 7%. These data indicate that IL-12 may play a role in the human immune response to infectious agents in vivo. We hypothesize that IL-12 contributes to the antimycobacterial immune response by enhancing production of interferon-gamma, facilitating development of Th1 cells and augmenting cytotoxicity of antigen-specific T cells and natural killer cells.
M Zhang, M K Gately, E Wang, J Gong, S F Wolf, S Lu, R L Modlin, P F Barnes
We examined the enzyme protein and biosynthesis of human trifunctional protein harboring enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase activity in cultured skin fibroblasts from two patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The following results were obtained. (a) In cells from patient 1, immunoblot analysis and pulse-chase experiments indicated that the content of trifunctional protein was < 10% of that in control cells, due to a very rapid degradation of protein newly synthesized in the mitochondria. The diminution of trifunctional protein was associated with a decreased activity of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase, when measured using medium-chain to long-chain substrates. (b) In cells from patient 2, the rate of degradation of newly synthesized trifunctional protein was faster than that in control cells, giving rise to a trifunctional protein amounting to 60% of the control levels. The 3-hydroxy-acyl-CoA dehydrogenase activity with medium-chain to long-chain substrates was decreased drastically, with minor changes in activities of the two other enzymes. These data suggest a subtle abnormality of trifunctional protein in cells from patient 2. Taken together, the results obtained show that in both patients, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is caused by an abnormality in the trifunctional protein, even though there is a heterogeneity in both patients.
T Kamijo, R J Wanders, J M Saudubray, T Aoyama, A Komiyama, T Hashimoto
Acute salpingitis complicating cervical gonococcal infection is a significant cause of infertility. Relatively little data are available concerning the pathophysiologic mechanisms of this disease. A cohort of 243 prostitutes residing in Nairobi were followed between March 1985 and April 1988. Gonococcal cultures were performed at each visit, and acute salpingitis was diagnosed clinically. Serum at enrollment was tested by immunoblot for antibody to gonococcal outer membrane proteins. 8.6% (146/1689) of gonococcal infections were complicated by salpingitis. Increased risk of salpingitis was associated with younger age, shorter duration of prostitution, HIV infection, number of gonococcal infections, and episodes of nongonococcal salpingitis. Rmp antibody increased the risk of salpingitis. Antibody to Opa decreased the risk of salpingitis. By logistic regression analysis, antibody to Opa was independently associated with decreased risk of gonococcal salpingitis (adjusted odds ratio [OR], 0.35; 95% confidence interval [95%CI], 0.17-0.76); HIV infection (adjusted OR, 3.5; 95% CI, 0.96-12.8) and episodes of nongonococcal salpingitis (adjusted OR, 3.4; 95% CI, 1.8-6.4) were independently associated with an increased risk of salpingitis. Antibody to Opa appears to protect against ascending gonococcal infection, perhaps by interfering with Opa mediated adherence and endocytosis. The demonstration of natural immunity that protects against upper genital tract infection in women suggests that a vaccine to prevent gonococcal salpingitis is possible.
F A Plummer, H Chubb, J N Simonsen, M Bosire, L Slaney, N J Nagelkerke, I Maclean, J O Ndinya-Achola, P Waiyaki, R C Brunham
Hydrolysis of glucosylceramide by beta-glucocerebrosidase results in ceramide, a critical component of the intercellular lamellae that mediate the epidermal permeability barrier. A subset of type 2 Gaucher patients displays ichthyosiform skin abnormalities, as do transgenic Gaucher mice homozygous for a null allele. To investigate the relationship between glucocerebrosidase deficiency and epidermal permeability barrier function, we compared the stratum corneum (SC) ultrastructure, lipid content, and barrier function of Gaucher mice to carrier and normal mice, and to hairless mice treated topically with bromoconduritol B epoxide (BrCBE), an irreversible inhibitor of glucocerebrosidase. Both Gaucher mice and BrCBE-treated mice revealed abnormal, incompletely processed, lamellar body-derived sheets throughout the SC interstices, while transgenic carrier mice displayed normal bilayers. The SC of a severely affected type 2 Gaucher's disease infant revealed similarly abnormal ultrastructure. Furthermore, the Gaucher mice demonstrated markedly elevated transepidermal water loss (4.2 +/- 0.6 vs < 0.10 g/m2 per h). The electron-dense tracer, colloidal lanthanum, percolated between the incompletely processed lamellar body-derived sheets in the SC interstices of Gaucher mice only, demonstrating altered permeability barrier function. Gaucher and BrCBE-treated mice showed < 1% and < 5% of normal epidermal glucocerebrosidase activity, respectively, and the epidermis/SC of Gaucher mice demonstrated elevated glucosylceramide (5- to 10-fold), with diminished ceramide content. Thus, the skin changes observed in Gaucher mice and infants may result from the formation of incompetent intercellular lamellar bilayers due to a decreased hydrolysis of glucosylceramide to ceramide. Glucocerebrosidase therefore appears necessary for the generation of membranes of sufficient functional competence for epidermal barrier function.
W M Holleran, E I Ginns, G K Menon, J U Grundmann, M Fartasch, C E McKinney, P M Elias, E Sidransky
Phenotype variability and incomplete penetrance are frequently observed in human monogenic diseases such as osteogenesis imperfecta. Here an inbred strain of transgenic mice expressing an internally deleted gene for the pro alpha 1(I) chain of type I procollagen (COL1A1) was bred to wild type mice of the same strain so that the inheritance of a fracture phenotype could be examined in a homogeneous genetic background. To minimize the effects of environmental factors, the phenotype was evaluated in embryos that were removed from impregnated females 1 d before term. Examination of stained skeletons from 51 transgenic embryos from 11 separate litters demonstrated that approximately 22% had a severe phenotype with extensive fractures of both long bones and ribs, approximately 51% had a mild phenotype with fractures of ribs only, and approximately 27% had no fractures. The ratio of steady-state levels of the mRNA from the transgene to the level of mRNA from the endogenous gene was the same in all transgenic embryos. The results demonstrated that the phenotypic variability and incomplete penetrance were not explained by variations in genetic background or levels in gene expression. Instead, they suggested that phenotypic variation is an inherent feature of expression of a mutated collagen gene.
R Pereira, K Halford, B P Sokolov, J S Khillan, D J Prockop
In phagocytes, superoxide generation by the NADPH oxidase is accompanied by metabolic acid production. Cytoplasmic acidification during this metabolic burst is prevented by a combination of H+ extrusion mechanisms, including a unique H+ conductance. NADPH oxidase is deficient in chronic granulomatous disease (CGD) patients. The burst of acid production is absent in CGD patients lacking the 47-kD (p47-phox) or the 91-kD (gp91-phox) subunits of the oxidase. Activation of the H+ conductance is also defective in these patients suggesting that (a) the oxidase itself undertakes H+ translocation or (b) oxidase assembly is required to stimulate a separate H+ conducting entity. To discern between these possibilities, three rare forms of CGD were studied. In neutrophils expressing nonfunctional cytochrome b, the conductance was activated to near-normal levels, implying that functional oxidase is not required to activate H+ extrusion. CGD cells expressing diminished amounts of cytochrome displayed H+ conductance approaching normal levels, suggesting that the oxidase itself does not translocate H+. Finally, the conductance was only partially inhibited in patients lacking the 67-kD subunit, indicating that this component is not essential for stimulation of H+ transport. We propose that normal assembly of the oxidase subunits is required for optimal activation of a closely associated but distinct H+ conducting entity.
A Nanda, J T Curnutte, S Grinstein
Two transgenic mouse lines, expressing low or high amounts of human apo A-IV were created. In low and high expressor HuAIVTg mice on a chow diet, serum human apo A-IV levels were 6 and 25 times the normal human level and on a high fat diet, they were 12 and 77 times higher. Human apo A-IV was equally distributed between lipoprotein (mainly HDL) and lipid-free fractions. Intestinal absorption of radiolabeled cholesterol and triglycerides was unaffected in HuAIVTg mice. Vitamin A, carried exclusively in chylomicrons and their remnants, was catabolized normally. When an intragastric vitamin E bolus is given to the HuAIVTg mice, the initial absorption and appearance in triglyceride-rich lipoproteins was similar to that observed in normal mice. However, elevated amounts of vitamin E were subsequently observed in the VLDL of the HuAIVTg mice. Furthermore, in the fed state, serum VLDL triglycerides were markedly elevated in HuAIVTg mice. This effect was greater in high expressor mice. Serum total cholesterol was not elevated, but the distribution was altered in the HuAIVTg mice; VLDL-C was increased at the expense of VLDL-C. Kinetic studies suggested a delayed clearance of VLDL in HuAIVTg mice. Apo A-IV has been suggested to be a satiety factor, but no effect on feeding behavior or weight gain was observed in these HuAIVTg mice. In summary, our studies with HuAIVTg mice show that additional apo A-IV does not effect intestinal absorption of fat and fat-soluble vitamins, and at least chronic elevation of plasma apo A-IV does not effect feeding behavior in this model system.
K Aalto-Setälä, C L Bisgaier, A Ho, K A Kieft, M G Traber, H J Kayden, R Ramakrishnan, A Walsh, A D Essenburg, J L Breslow
This study analyzed the distribution of an idiotype, B3-Id, in patients with active SLE, classified according to organ involvement, normal controls, and other autoimmune rheumatic diseases. A polyclonal anti-idiotype was raised by immunizing a rabbit with a monoclonal IgG anti-double-stranded (ds) DNA antibody, B3, generated from a patient with SLE who had active arthritis. The idiotype is present on the lambda chain and is at or near the binding site for double-stranded DNA. The lambda chain, which was characterized by nucleotide sequencing, was 90% homologous to the V lambda 2.1 germline, which is known to be involved in coding for nephritogenic anti-DNA antibodies carrying the 8.12 idiotype. There were four changes to positively charged amino acids, known to be involved in DNA binding, in the complementarity determining regions of B3 lambda chain compared with a non-DNA binding, 8.12 positive antibody, PV11. Only one change to a positively charged amino acid occurs in the heavy chain of B3, which is 93.5% homologous to VH-26. The B3-Id was present on IgG antibodies in the serum of 20% of patients with SLE but was not found in the normal controls. Within the SLE group, there is a statistically significant association of B3-Id on IgG in the arthritis group (42%) compared to the other manifestations (9%) (P < 0.001). In four B3-Id-positive SLE patients tested serially, the level of B3-Id reflected the arthritis disease activity more closely than the overall disease activity (P < 0.05). The B3-Id was also present on IgM antibodies in one third of patients with rheumatoid arthritis. This idiotype is the first to be derived from a human monoclonal anti-DNA antibody of the IgG class, the isotype associated with active disease. Sequence analysis shows that positively charged amino acids on the lambda chain may contribute to DNA binding.
M R Ehrenstein, C M Longhurst, D S Latchman, D A Isenberg
To characterize quantitatively the quinidine (QUIN)-induced conduction delay (CD) in vivo, canine ventricular activation times were examined with an epicardial mapping technique. A high-resolution index of normalized (N) QUIN CD, derived from all 56 recording sites, was used to quantify QUIN effect. Repetitive stimulation elicited monoexponential increases in CD(N), the rates of which were a linear function of interpulse recovery interval, tr. Steady-state CD(N) was also linearly related to an exponential function of tr and drug uptake rates. The frequency-dependent properties of QUIN in 14 dogs were characterized by apparent binding and unbinding rates of ka = 7.1 +/- 3.5 x 10(6) M-1 s-1, la = 81 +/- 51 s-1 for activated, and kr = 12.6 +/- 11.3 x 10(3) M-1 s-1, lr = 0.51 +/- 0.26 s-1 for resting states. ka and la were similar to values previously derived in canine Purkinje fibers. Drug unbinding at resting potentials was faster in vivo than previously observed in vitro. The time constant of recovery from QUIN block extracted from the interpulse recovery rate was also identical to that determined from post-mature stimulus diastolic scanning. As predicted by the two-state model, similar binding rates were also derived from declining CD(N) elicited by step decreases in heart rate. These findings represent a complete quantitative description of use-dependent QUIN CD in vivo and provide a firm foundation for characterizing antiarrhythmic drug action under physiologic and pathologic conditions.
F N Haugland, S B Johnson, D L Packer
The mut0 mutation resulting in methylmalonyl CoA mutase (MCM) apoenzyme deficiency and methylmalonic aciduria is characterized by undetectable enzyme activity in cell extracts and low incorporation of propionate into cultured cells which is not stimulated by hydroxycobalamin. A mut0 fibroblast cell line (WG1681) from an African-American male infant complemented another mut0 cell line (WG 1130). Cloning and sequencing of cDNA from WG 1681 demonstrated compound heterozygosity for two novel changes at highly conserved sites: G623R and G703R. In addition, two previously described homozygous polymorphisms, H532R and V671I, were found. Hybridization of allele-specific oligonucleotides to PCR amplified MCM exons from the proband and family members identified a clinically normal mother, half-sister, and half-brother as carriers of the G703R change in cis with both polymorphisms. Transfection of each change into a mut0 cell line with very low MCM mRNA (GM1673) demonstrated a lack of stimulation of propionate uptake in the absence and presence of hydroxycobalamin. Cotransfection of each mutation with the previously identified R93H mutation of WG 1130 stimulated propionate uptake, indicating that G623R and G703R are independently capable of complementing the R93H mutation.
A A Qureshi, A M Crane, N V Matiaszuk, I Rezvani, F D Ledley, D S Rosenblatt
The important intracellular mechanisms of astrocyte growth are not well defined. Using an inhibitor of astrocyte proliferation, atrial natriuretic peptide (ANP), and the glial mitogen endothelin (ET-3), we sought a common pathway for growth regulation in these neural cells. In cultured fetal rat diencephalic astrocytes, ANP selectively and rapidly inhibited the Tis 8 immediate early gene and protein. After 4 h, ANP selectively inhibited the basic fibroblast growth factor (bFGF) gene and protein. ET-3 significantly stimulated both Tis 8 and bFGF mRNAs and protein, but also stimulated several other immediate early and growth factor/receptor genes. An antisense oligonucleotide to Tis 8 strongly prevented ET-stimulated thymidine incorporation, while the inhibitory action of ANP was enhanced. The Tis 8 antisense oligonucleotide also significantly reversed ET-stimulated bFGF transcription and enhanced the bFGF inhibition caused by ANP. In addition, an antisense oligonucleotide to bFGF significantly reversed the ET-stimulated thymidine incorporation and enhanced the ANP inhibition of DNA synthesis. The sequential modulation of Tis 8, followed by bFGF, provides a novel mechanism for both positive and negative regulation of astrocyte growth by endogenous neuropeptides.
R M Hu, E R Levin
The regulation of ACTH receptor binding sites and mRNA by ACTH and angiotensin II (A-II) was studied using cultured human adrenal fasciculata reticularis cells (HAC). These cells expressed two major ACTH receptor transcripts of 1.8 and 3.4 kb and three minor ones of 4, 7, and 11 kb. ACTH increased the levels of all these transcripts in a time- and dose-dependent manner. At a maximal concentration of 10(-8) M, ACTH enhanced 21- and 4-fold the level of ACTH receptor mRNA and the number of receptors per cell, respectively. Pretreatment of HAC with A-II produced a dose-dependent enhancement of ACTH receptor mRNA that was associated with an increase of both ACTH receptor number and responsiveness to this hormone. The effects of A-II were completely blocked by an AT1 receptor subtype antagonist but not by an AT2 antagonist. The effects of ACTH together with A-II on ACTH receptor mRNA were greater than those induced by each hormone alone. These results show that ACTH receptor number and mRNA are positively regulated by the two main hormones (ACTH and A-II) which, in vivo, regulate adrenocortical functions. In addition, they also show that HAC are a target for A-II. Thus, regulation of ACTH receptors may be one mechanism by which ACTH and A-II regulate adrenocortical functions under both normal and pathological conditions.
M C Lebrethon, D Naville, M Begeot, J M Saez
A recombinant prostaglandin G/H (PGH) synthase gene has been expressed in vitro in bovine pulmonary artery endothelial cells and in vivo in rabbits by transfection with a plasmid using cationic liposomes. Transfection of bovine pulmonary artery endothelial cells with the PGH synthase cDNA resulted in increased intracellular PGH synthase protein (determined by Western blot analysis) and increased release of prostacyclin. Rabbits intravenously transfected with the PGH synthase gene had increased plasma levels of prostacyclin and PGE2, and their lungs produced increased amounts of the same eicosanoids. In an in situ, perfused preparation of PGH synthase transfected rabbit lungs, the pressor response to endotoxin was markedly attenuated. In addition, pulmonary edema and release of thromboxane B2 into the perfusate after endotoxin infusion were markedly decreased in transfected lungs compared to controls (animals transfected with a pCMV4 construct that did not contain a cDNA insert). The data suggest that augmented endogenous production of prostacyclin and PGE2, achieved by liposome-mediated gene transfer, protects the lungs from endotoxin. This may be caused in part by suppression of endotoxin-stimulated thromboxane B2 production. Modification of lipid mediator responses by in vivo transfection is a potential approach to the therapy of acute lung injury.
J T Conary, R E Parker, B W Christman, R D Faulks, G A King, B O Meyrick, K L Brigham
Using the Xenopus oocyte expression system, we have previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathione (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-2328). Starting with a cDNA library constructed from this fraction, we have now isolated a single clone that expresses GSH transporter activity. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 kb with an open reading frame of 2,505 nucleotides encoding for a polypeptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An approximately 96-kD protein is generated in in vitro translation of cRNA for RcGshT. Northern blot analysis reveals a single 4-kb transcript in liver, kidney, intestine, lung, and brain. The abundance of mRNA for RcGshT in rat liver increased 3, 6, and 12 h after a single dose of phenobarbital. Insensitivity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the canalicular GSH transporter.
J R Yi, S Lu, J Fernandez-Checa, N Kaplowitz
Thrombomodulin is an endothelial cell surface glycoprotein that inhibits the procoagulant activities of thrombin and accelerates activation of the anticoagulant protein C. Because protein C deficiency is associated with cutaneous thrombosis, we investigated the expression of thrombomodulin in human skin. Thrombomodulin was detected by immunohistochemical staining both in dermal endothelial cells and in epidermal keratinocytes. Within the epidermis, thrombomodulin staining was limited to keratinocytes of the spinous layer, suggesting that thrombomodulin is induced when basal keratinocytes begin to terminally differentiate. Thrombomodulin expression also correlated with squamous differentiation in epidermal malignancies; little or no thrombomodulin staining was seen in five basal cell carcinomas, whereas strong thrombomodulin staining was observed in each of five squamous cell carcinomas. Human foreskin keratinocytes cultured in medium containing 0.07 mM calcium chloride synthesized functional thrombomodulin with cofactor activity comparable to thrombomodulin in human umbilical vein endothelial cells. Stimulation of keratinocyte differentiation with 1.4 mM calcium chloride for 48 h produced 3.5-, 3.2-, and 5.6-fold increases in thrombomodulin cofactor activity, antigen, and mRNA, respectively. These observations suggest that thrombin is regulated by keratinocyte thrombomodulin at sites of cutaneous injury, and indicate a potential role for thrombomodulin in epidermal differentiation.
T J Raife, D J Lager, K C Madison, W W Piette, E J Howard, M T Sturm, Y Chen, S R Lentz
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We report on a novel nonsense mutation that leads to exon skipping and the activation of a cryptic exon. Screening of genomic DNA from 700 German patients with CF uncovered four cases with the nonsense mutation E92X, a G-->T transversion that creates a termination codon and affects the first base of exon 4 of the CFTR gene. Lymphocyte RNA of two CF patients heterozygous for E92X was found to contain the wild type sequence and a differentially spliced isoform lacking exon 4. In RNA derived from nasal epithelial cells of E92X patients, a third fragment of longer size was observed. Sequencing revealed the presence of E92X and an additional 183-bp fragment, inserted between exons 3 and 4. The 183-bp sequence was mapped to intron 3 of the CFTR gene. It is flanked by acceptor and donor splice sites. We conclude that the 183-bp fragment in intron 3 is a cryptic CFTR exon that can be activated in epithelial cells by the presence of the E92X mutation. E92X abolishes correctly spliced CFTR mRNA and leads to severe cystic fibrosis.
K Will, T Dörk, M Stuhrmann, T Meitinger, R Bertele-Harms, B Tümmler, J Schmidtke
To determine the molecular defect accounting for the deficiency of pulmonary surfactant protein B (SP-B) in full-term neonates who died from respiratory failure associated with alveolar proteinosis, the sequence of the SP-B transcript in affected infants was ascertained. A frameshift mutation consisting of a substitution of GAA for C in codon 121 of the SP-B cDNA was identified. The three affected infants in the index family were homozygous for this mutation, which segregated in a fashion consistent with autosomal recessive inheritance of disease. The same mutation was found in two other unrelated infants who died from alveolar proteinosis, one of whom was also homozygous, and in the parents of an additional unrelated, affected infant, but was not observed in 50 control subjects. We conclude that this mutation is responsible for SP-B deficiency and neonatal alveolar proteinosis in multiple families and speculate that the disorder is more common than was recognized previously.
L M Nogee, G Garnier, H C Dietz, L Singer, A M Murphy, D E deMello, H R Colten
Tight regulation of foreign genes expressed in vivo would facilitate studies of many biologic processes and would be useful for gene transfer-based therapies. To test the ability of a tetracycline-regulated gene expression system to function in vivo, we directly injected chimeric tet repressor-VP16 transactivator expression plasmids and luciferase target genes into the hearts of adult rats. Cardiac luciferase activity increased over two orders of magnitude in response to small changes in input tetracycline-controlled transactivator DNA. Transactivation was repressed to background levels by subtherapeutic concentrations of tetracycline in a dose-dependent manner. Target gene expression could be rapidly and reversibly controlled by manipulating antibiotic administration. This system may be particularly useful for in vivo studies of gene function or gene therapies where the timing or extent of expression are critical variables.
G I Fishman, M L Kaplan, P M Buttrick