M G Farquhar
Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.
A C Kirby, S Meghji, S P Nair, P White, K Reddi, T Nishihara, K Nakashima, A C Willis, R Sim, M Wilson
myo-inositol, a major compatible osmolyte in renal medulla, is accumulated in several kinds of cells under hypertonic conditions via Na+/myo-inositol cotransporter (SMIT). To investigate the physiological role of the SMIT, we sought to determine its localization by in situ hybridization and its acute regulation by NaCl and furosemide administration. Northern analysis demonstrated that SMIT is strongly expressed in the medulla and at low levels in the cortex of kidney. Intraperitoneal injection of NaCl rapidly induced SMIT mRNA in both the cortex and medulla, and furosemide completely abolished this induction. In situ hybridization revealed that SMIT it predominantly present in the medullary and cortical thick ascending limbs of Henle's loop (TALH) and macula densa cells. Less intense signals were seen in the inner medullary collecting ducts (IMCD). NaCl loading increased the signals throughout the TALH, and furosemide reduced the signals. SMIT in the IMCD is less sensitive to these kinds of acute regulation. Thus, the distribution pattern of SMIT does not correspond to the corticomedullary osmotic gradient, and SMIT in the TALH and macula densa cells is regulated very rapidly. These results suggest that SMIT expression in TALH may be regulated by intracellular and/or peritubular tonicity close to the basolateral membrane, which is supposed to be proportional to the magnitude of NaCl reabsorption.
A Yamauchi, A Miyai, S Shimada, Y Minami, M Tohyama, E Imai, T Kamada, N Ueda
Homozygous adhalin gene mutations were found in three patients from two consanguineous families with autosomal recessive childhood onset muscular dystrophy. Muscle biopsies from patients in each family showed complete absence of adhalin. Sequencing of adhalin cDNA prepared from skeletal muscle by reverse transcription PCR demonstrated a cytosine to thymidine substitution at nt 229 in the patient in family 1 and an adenine to guanine substitution at nt 410 and a 15-base insertion between nt 408 and 409 in the two patients in family 2. Sequencing of genomic DNA prepared from peripheral blood leukocytes by PCR confirmed these mutations. The parents in each family were found to be heterozygous for the respective mutations. These adhalin gene mutations are presumed to be responsible for the absence of adhalin in the skeletal muscle. Adhalin deficiency likely causes disruption of the muscle cell membrane, resulting in dystrophic changes in the skeletal muscle similar to dystrophin deficiency in Duchenne muscular dystrophy.
H Kawai, M Akaike, T Endo, K Adachi, T Inui, T Mitsui, S Kashiwagi, T Fujiwara, S Okuno, S Shin
Steady state levels of hypothalamic expression of the genes encoding corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), arginine vasopressin (AVP), and oxytocin (OT) were studied in rats to investigate the mechanisms underlying the transitions between hypercorticalism during lactation and normocorticalism upon weaning. During lactation, CRH mRNA levels and blood titers of adrenocorticotropin (ACTH) were found to be significantly reduced, although POMC mRNA levels in the anterior pituitary were not significantly different from those found in cycling virgin (control) rats; during all phases of lactation, an inverse relationship was observed between the blood levels of ACTH and corticosterone (CORT). Plasma prolactin (PRL) concentrations were elevated approximately 30-fold during lactation. Whereas steady state levels of OT mRNA were markedly increased throughout lactation, those of AVP mRNA were only transiently (initially) elevated, and the blood levels of these hormones were not significantly altered in lactating as compared with cycling virgin and postlactating rats. CRH and POMC gene expression and blood levels of ACTH, CORT, and PRL were normalized within 1-3 d of removal of suckling pups. The temporal relationships between the biosynthetic profiles of the various peptide hormones and the patterns of ACTH and CORT secretion during the two physiological states suggest that lactation-associated hypercorticalism does not merely result from increased ACTH secretion; although still not well substantiated at this time, the evidence points to contributory roles of PRL, OT, and AVP in the hypercorticalismic state found during lactation.
D Fischer, V K Patchev, S Hellbach, A H Hassan, O F Almeida
We have mapped a disease locus for Wolff-Parkinson-White syndrome (WPW) and familial hypertrophic cardiomyopathy (FHC) segregating in a large kindred to chromosome 7 band q3. Although WPW syndrome and FHC have been observed in members of the same family in prior studies, the relationship between these two diseases has remained enigmatic. A large family with 25 surviving individuals who are affected by one or both of these conditions was studied. The disease locus is closely linked to loci D7S688, D7S505, and D7S483 (maximum two point LOD score at D7S505 was 7.80 at theta = 0). While four different FHC loci have been described this is the first locus that can be mutated to cause both WPW and/or FHC.
C A MacRae, N Ghaisas, S Kass, S Donnelly, C T Basson, H C Watkins, R Anan, L H Thierfelder, K McGarry, E Rowland
We examined the effect of bronchial blood flow (BBF) on hyperpnea-induced airway obstruction (HIAO) in dogs. HIAO in in situ isolated pulmonary lobes with or without BBF was monitored via a bronchoscope. An intravascular tracer in conjunction with morphometric analysis was used to document the efficacy of our occlusion technique. We found that (a) Occlusion of the bronchial artery abolished bronchovascular leakage, but did not alter HIAO; (b) HIAO occurred in postmortem dogs, and was attenuated by cooling; (c) absence of BBF did not cause mucosal damage, although hyperpnea-induced injury was enhanced in airways lacking BBF; (d) BBF did not affect either goblet/ ciliated cell ratios or hyperpnea-induced goblet cell degranulation; (e) ligation of the bronchial artery and hyperpnea each caused mast cell degranulation, and these effects were additive; (f) hyperpnea-induced leukocyte infiltration was reduced in the absence of BBF; and (g) ligation of the bronchial artery and hyperpnea with dry air each increased airway vessel diameter, and these effects were additive. We conclude that either impairment or absence of BBF abolishes bronchovascular leakage and increases hyperpnea-induced mucosal injury, but fails to affect HIAO. Based on these results we speculate that bronchovascular leakage protects the bronchial mucosa from excessive losses of heat and water, and inhibits mucosal damage.
A N Freed, C Omori, B H Schofield
DBA/2J mouse contains two renin gene loci (Ren1d and Ren2d). Ren2d but not Ren1d is expressed in submandibular gland (SMG) while both are expressed in the kidney. Based on vitro studies, we have postulated that a negative regulatory element (NRE) in the renin gene promoter is involved in its tissue-specific expression. In this study, we examined the molecular mechanism at the in vivo level using direct gene transfer. Fragments of the Ren1d or Ren2d promoter were fused to a chloramphenicol acetyltransferase (CAT) gene expression vector. These constructs complexed in fusogenic liposomes were injected directly into the mouse SMG or intraarterially into the mouse kidney via the renal artery. The vector containing the CAT exhibited readily detectable in vivo expressions in both SMG and kidney. In the SMG, Ren1d fragment containing the NRE abolished CAT expression while deletion of the NRE restored CAT expression. The homologous fragment from the Ren2d promoter did not inhibit CAT expression while deletion of the 150-bp insertion resulted in the inhibition. Cotransfection of Ren1d construct with Ren1d-NRE oligonucleotides as transcriptional factor decoy restored CAT expression. Contrary to the SMG, transfection with Ren1d fragment-CAT construct or Ren2d fragment-CAT construct into the kidney resulted in similar levels of CAT expression. Interestingly, human c-myc NRE oligonucleotides which share homology with Ren1d-NRE competed effectively with these oligonucleotides for the regulation of Ren1d gene expression in vivo. This NRE sequence is also homologous to silencer elements found in multiple mammalian genes, suggesting the presence of a family of NRE/NRE binding proteins regulating expression of diverse genes.
T Yamada, M Horiuchi, R Morishita, L Zhang, R E Pratt, V J Dzau
Parathyroid hormone and other bone resorptive agents function, at least in part, by inducing osteoblasts to secrete cytokines that stimulate both differentiation and resorptive activity of osteoclasts. We previously identified two potentially important cytokines by demonstrating that parathyroid hormone induces expression by osteoblasts of IL-6 and leukemia inhibitory factor without affecting levels of 14 other cytokines. Although parathyroid hormone activates multiple signal transduction pathways, induction of IL-6 and leukemia inhibitory factor is dependent on activation of adenyl cyclase. This study demonstrates that adenyl cyclase is also required for stimulation of osteoclast activity in cultures containing osteoclasts from rat long bones and UMR106-01 rat osteoblast-like osteosarcoma cells. Since the stimulation by parathyroid hormone of both cytokine production and bone resorption depends on the same signal transduction pathway, we hypothesized that IL-6 might be a downstream effector of parathyroid hormone. We found that addition of exogenous IL-6 mimics the ability of parathyroid hormone to stimulate bone resorption. More importantly, an antibody directed against the IL-6 receptor blocks moderate stimulation of osteoclast activity induced by the hormone. Interestingly, strong stimulation of resorption overcomes this dependence on IL-6. Thus, parathyroid hormone likely induces multiple, redundant cytokines that can overcome the IL-6 requirement associated with moderate stimulation. Taken together with studies showing that many other bone resorptive agents also stimulate IL-6 production, our results suggest that IL-6 may be a downstream effector of these agents as well as of parathyroid hormone.
E M Greenfield, S M Shaw, S A Gornik, M A Banks
We investigated the effect of recombinant human transforming growth factor beta 2 (rhTGF-beta 2) administration on trabecular bone loss induced by unloading in rats. Hind limb suspension for 14 d inhibited bone formation and induced osteopenia as shown by decreased bone volume, calcium and protein contents in long bone metaphysis. Systemic infusion of rhTFG-beta 2 (2 micrograms/kg per day) maintained normal bone formation rate, and prevented the decrease in bone volume, bone mineral content, trabecular thickness and number induced by unloading. In vitro analysis of tibial marrow stromal cells showed that rhTGF-beta 2 infusion in unloaded rats increased the proliferation of osteoblast precursor cells, but did not affect alkaline phosphatase activity or osteocalcin production. Northern blot analysis of RNA extracted from the femoral metaphysis showed that rhTGF-beta 2 infusion in unloaded rats increased steady-state levels of type I collagen mRNA but not alkaline phosphatase mRNA levels. rhTGF-beta 2 infusion at the dose used had no effect on metaphyseal bone volume and formation, osteoblast proliferation or collagen expression in control rats. The results show that systemic administration of rhTGF-beta 2 enhances osteoblast precursor cell proliferation and type I collagen expression by osteoblasts, and prevents the impaired bone formation and osteopenia induced by unloading.
M Machwate, E Zerath, X Holy, M Hott, D Godet, A Lomri, P J Marie
Despite increasing information about the mechanism of action of cyclosporine A (CsA), little is known about the way lymphocytes recover from CsA. Recovery is central to understanding the pharmacodynamics of CsA in vivo. We studied the recovery of calcineurin phosphatase (CN) activity in CsA-treated cells. Single dose kinetics in renal transplant patients showed that inhibition of CN activity in PBL increased and fell concomitant with CsA blood vessels. In vitro, control PBL treated with CsA 100 micrograms/l, washed, and resuspended in CsA-free medium showed little recovery (0-20%) after 24 h. Erythrocytes or anti-CsA Ab added to the recovery medium increased recovery to 50% within 4 h. Similar recovery was seen in the ability of cells to produce IFN-gamma after OKT3 stimulation. Recovery of CN activity was associated with the efflux of [3H]CsA, was not blocked by cycloheximide and was temperature sensitive. A cell line with high expression of surface P glycoprotein (PGP), showed rapid recovery. However, PGP blockade did not prevent recovery in PBL, indicating a different PGP-independent mechanism. In PBL, recovery from CsA is slow and limited in vitro, but rapid in vivo, where CsA equilibrates among a complex set of extralymphocytic binding sites.
T D Batiuk, F Pazderka, J Enns, L DeCastro, P F Halloran
It was the aim of this study to determine whether FFA inhibit insulin-stimulated whole body glucose uptake and utilization in patients with non-insulin-dependent diabetes. We performed five types of isoglycemic (approximately 11mM) clamps: (a) with insulin; (b) with insulin plus fat/heparin; (c) with insulin plus glycerol; (d) with saline; (e) with saline plus fat/heparin and two types of euglycemic (approximately 5mM) clamps: (a) with insulin; (b) with insulin plus fat/heparin. During these studies, we determined rates of glucose uptake, glycolysis (both with 3[3H] glucose), glycogen synthesis (determined as glucose uptake minus glycolysis), carbohydrate oxidation (by indirect calorimetry) and nonoxidative glycolysis (determined as glycolysis minus carbohydrate oxidation). Fat/heparin infusion did not affect basal glucose uptake, but inhibited total stimulated (insulin stimulated plus basal) glucose uptake by 40-50% in isoglycemic and in euglycemic patients at plasma FFA concentration of approximately 950 and approximately 550 microM, respectively. In isoglycemic patients, the 40-50% inhibition of total stimulated glucose uptake was due to near complete inhibition of the insulin-stimulated part of glucose uptake. Proportional inhibition of glucose uptake, glycogen synthesis, and glycolysis suggested a major FFA-mediated defect involving glucose transport and/or phosphorylation. In summary, fat produced proportional inhibitions of insulin-stimulated glucose uptake and of intracellular glucose utilization. We conclude, that physiologically elevated levels of FFa could potentially be responsible for a large part of the peripheral insulin resistance in patients with non-insulin-dependent diabetes mellitus.
G Boden, X Chen
Infection with the protozoan parasite Entamoeba histolytica results in a high mortality worldwide. To initiate infection, E. histolytica trophozoites in the bowel lumen penetrate the epithelium, and cause extensive lysis of host cells. The acute amebic lesions in animal models are characterized by infiltration with inflammatory cells, particularly neutrophils. The acute host response is likely important for determining whether the infection will spread systemically, but little is known regarding the signals which initiate an acute inflammatory response to E. histolytica. In the studies reported herein, we used an in vitro model system to define the proinflammatory signals produced by epithelial and other host cells in response to infection with E. histolytica trophozoites. Coculture of human epithelial and stromal cells and cell lines with trophozoites is shown to increase expression and secretion of an array of chemoattractant and proinflammatory cytokines, including IL-8, GRO alpha, GM-CSF, IL-1 alpha, and IL-6. Moreover, high-level secretion of those cytokines is regulated by the paracrine action of cytolytically released IL-1 alpha. A second mechanism for trophozoite-induced IL-8 production involves trophozoite-target cell contact via a galactose-inhibitable amebic adherence protein, and appears to be mediated through increased intracellular calcium levels. These studies define novel mechanisms through which acute inflammation can be initiated in the host in response to a cytolytic pathogen, such as E. histolytica.
L Eckmann, S L Reed, J R Smith, M F Kagnoff
We previously demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone mainly produced in the ventricle, while the major production site of atrial natriuretic peptide (ANP) is the atrium. To assess the pathophysiological role of BNP in ventricular overload, we have examined the gene expression of BNP, In comparison with that of ANP, in a model of cardiac hypertrophy using cultured neonatal rat ventricular cardiocytes. During cardiocyte hypertrophy evoked by endothelin-1, Phenylephrine, or PMA, the steady state level of BNP mRNA increased as rapidly as the "immediate-early" induction of the c-fos gene expression, and reached a maximal level within 1 h. Actinomycin D, a transcriptional inhibitor, completely diminished the response, while the translational blocked with cycloheximide did not inhibit it. In contrast, ANP mRNA began to increase 3 h after the stimulation, and accumulated during cardiocyte hypertrophy. The BNP secretion from ventricular cardiocytes was also stimulated, more rapidly than the ANP secretion. Furthermore, the turnover of BNP mRNA was significantly faster than that of ANP mRNA, being consistent with the existence of AUUUA motif in the 3'-untranslated region of BNP mRNA. These results demonstrate that the gene expression of BNP is distinctly regulated from that of ANP at transcriptional and posttranscriptional levels, and indicate that the characteristics of the BNP gene expression are suitable for its possible role as an " emergency" cardiac hormone against ventricular overload.
O Nakagawa, Y Ogawa, H Itoh, S Suga, Y Komatsu, I Kishimoto, K Nishino, T Yoshimasa, K Nakao
We recently demonstrated that chronic treatment with IL-1 beta induces coronary arteriosclerotic changes and vasospastic responses to autacoids in pigs in vivo and that those responses are importantly mediated by PDGF. The receptors for PDGF and other major growth factors are known to have tyrosine kinase activity. We therefore investigated the effects of a selective tyrosine kinase inhibitor, ST 638, on those responses induced by IL-1 beta in our swine model. Intimal thickening and coronary vasospastic responses to serotonin and histamine were induced at the site of the coronary artery where IL-1 beta was chronically and locally applied. These responses were significantly suppressed in a dose-dependent manner by cotreatment with ST 638. In addition, ST 494, which is an inactive form of ST 638, did not inhibit those responses. The treatment with ST 638 alone did not affect the coronary vasoconstricting responses to the autacoids. Immunoblotting using an antibody to phosphotyrosines confirmed the inhibitory effects of ST 638 on the tyrosine phosphorylations induced by IL-1 beta. These results thus suggest that tyrosine kinase activation may play an important role in mediating the effects of IL-1 beta, while also suggesting that ST 638 has an inhibitory effect on the arteriosclerotic changes and vasospastic responses to autacoids in our swine model in vivo.
A Ito, H Shimokawa, T Kadokami, Y Fukumoto, M K Owada, T Shiraishi, R Nakaike, T Takayanagi, K Egashira, A Takeshita
To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.
K Sato, K Yamazaki, K Shizume, Y Kanaji, T Obara, K Ohsumi, H Demura, S Yamaguchi, M Shibuya
Activated CD4+ helper T cells have been demonstrated in asthmatic airways and postulated to play a central role in eliciting allergic inflammation; direct evidence of their involvement seems to be lacking. We hypothesized that CD4+ T cells have the potential to induce allergic responses to antigen challenge, and tested this hypothesis in a model of allergic bronchoconstriction, the Brown Norway rat, using the approach of adoptive transfer. Animals were actively sensitized to either ovalbumin (OVA) or BSA and were used as donors of T cells. W3/25(CD4)+ or OX8(CD8)+ T cells were isolated from the cervical lymph nodes of sensitized donors and transferred to naive BN rats. 2 d after adoptive transfer recipient rats were challenged by OVA inhalation, and changes in lung resistance (RL), bronchoalveolar lavage (BAL) cells, and serum levels of antigen-specific IgE were studied. After OVA challenge recipients of OVA-primed W3/25+ T cells exhibited sustained increases in RL throughout the entire 8-h observation period and had significant bronchoalveolar lavage eosinophilia, which was detected by immunocytochemistry using an antimajor basic protein mAb. Recipients of BSA-primed W3/25+ T cells or OVA-primed OX8+ T cells failed to respond to inhaled OVA. OVA-specific immunoglobulin E was undetectable by ELISA or skin testing in any of the recipient rats after adoptive transfer. In conclusion, antigen-induced airway bronchoconstriction and eosinophilia were successfully transferred by antigen-specific W3/25+ T cells in Brown Norway rats. These responses were dependent on antigen-primed W3/25+ T cells and appeared to be independent of IgE-mediated mast cell activation. This study provides clear evidence for T cell mediated immune mechanisms in allergic airway responses in this experimental model.
A Watanabe, H Mishima, P M Renzi, L J Xu, Q Hamid, J G Martin
To determine whether similar or divergent pathways mediate atrial natriuretic factor (ANF) induction in neonatal and hypertrophied adult ventricular myocardium, and to assess whether studies using an in vitro model system of hypertrophy have fidelity to the in vivo context during pressure overload hypertrophy, we generated transgenic mice which harbor either 638 or 3,003 bp of the rat ANF 5' flanking region ligated upstream from a luciferase reporter. Luciferase activity in the ventricles of day 1 transgenic neonates was 8-24-fold higher than the levels expressed in the ventricles of adult mice. Adult mice expressed the luciferase reporter in an appropriate tissue-specific manner. Transverse aortic constriction of adult mice harboring ANF reporter transgenes demonstrated no significant increase in reporter activity in the ventricle. These findings demonstrate that distinct regions of the ANF 5'-flanking region are required for inducible expression of the ANF gene in the hypertrophic adult ventricle compared with those required for atrial-specific and developmentally appropriate expression in the intact neonatal heart. Furthermore, the cis regulatory elements necessary for induction of ANF expression in endothelin-1 or alpha 1-adrenergically stimulated cultured neonatal ventricular myocytes are not sufficient for induction in the in vivo context of pressure overload hypertrophy.
K U Knowlton, H A Rockman, M Itani, A Vovan, C E Seidman, K R Chien
Oncostatin M (OM), which shares functional similarity and structural homology to leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), functions as a potent growth factor for AIDS-associated Kaposi's sarcoma-derived cells (AIDS-KS cells). OM was also suggested to bind to the LIF receptor (LIF/OM receptor), which consists of a signal transducing subunit for LIF and IL-6 (gp130) and a LIF receptor alpha-subunit. Recent studies indicate that IL-6 has growth-stimulating activity for AIDS-KS cells. However, we find that AIDS-KS cell growth is exclusively induced by OM and not by LIF or IL-6. We also observed the lack of binding properties of AIDS-KS cells for LIF and IL-6. Scatchard plots revealed the existence of two affinity classes of OM receptor sites on AIDS-KS cells, with Kd values of 6-12 pM (high affinity) and 521-815 pM (low affinity). In competition binding studies, we find that the OM-specific receptor, but not the LIF/OM receptor, contributes to the OM-specific growth stimulation of AIDS-KS cells. We also noted that anti-gp130 antibodies can completely abolish OM-induced growth stimulation of AIDS-KS cells as well as OM binding to AIDS-KS cells. PCR amplification clearly revealed high levels of gp130 expression in AIDS-KS cells, while the transcript of LIF receptor alpha-subunit or IL-6 receptor alpha-subunit was not observed. Therefore, we conclude that (a) AIDS-KS cells express the OM-specific receptor with high and low affinity, but not the LIF/OM receptor; (b) gp130 on AIDS-KS cells plays a key role in OM binding and signaling on the OM-specific receptor; and (c) the lack of biological response of AIDS-KS cells to IL-6 and LIF can be explained by the absence of the IL-6 and LIF/OM receptors. All this evidence shows the correlation of OM-specific biological activity with expression of the OM-specific receptor and the involvement of gp130 on this receptor, as based on findings in in vitro growth assays and binding experiments for AIDS-KS cells.
K Murakami-Mori, T Taga, T Kishimoto, S Nakamura
Differentiated villus intestinal epithelial cells express globotriaosylceramide, the Shiga-like toxin 1 (SLT-1) receptor, and are sensitive to toxin-mediated cytotoxicity, whereas undifferentiated crypt cells neither express Gb3 nor respond to toxin. To investigate if SLT-1 receptors are maturationally regulated in human intestinal cells, we examined the effect of butyrate, a known transcriptional regulator of differentiation genes in many cell types, using cultured colonic cancer-derived epithelial cell lines. Exposure to butyrate increased villus cell marker enzymes such as alkaline phosphatase, sucrase, and lactase, expression of toxin receptors, and sensitivity to SLT-1 in villus-like CaCo-2A and HT-29 cells. These effects were reversibly inhibited by preincubation of CaCo-2A cells with actinomycin D or cycloheximide. Butyrate-treated CaCo-2A cells unable to bind fluoresceinated SLT-1 B subunit were undifferentiated as assessed by alkaline phosphatase activity. HT-29 cells induced to differentiate by another signal, glucose deprivation, upregulated receptor content and response to toxin. Crypt-like T-84 cells responded to butyrate with a modest increase in alkaline phosphatase and toxin binding, but no induction of sucrase or lactase, and no change in sensitivity to toxin. The results demonstrate that expression of SLT-1 toxin receptors and toxin sensitivity are coregulated with cellular differentiation in cultured intestinal cells.
M S Jacewicz, D W Acheson, M Mobassaleh, A Donohue-Rolfe, K A Balasubramanian, G T Keusch
Some human chronic dermal wounds treated with recombinant platelet-derived growth factor-BB (rPDGF-BB) show increased healing coupled with fibroblast activation and granulation tissue formation. To determine whether endogenous PDGF is associated with healing and nonhealing dermal ulcer phenotypes, we developed monoclonal antibodies capable of recognizing the three isoforms of PDGF, AA, AB, and BB dimers, and capable of discriminating between two alternatively spliced A chain transcripts. We detected little PDGF isoform expression in normal skin and in nonhealing dermal ulcers. In contrast, in surgically created acute wounds and chronic ulcers treated with rPDGF-BB, markedly upregulated levels of PDGF-AA (long form) were found. In both types of wounds, increased PDGF-AA was detected primarily in capillaries and fibroblasts, although in rPDGF-BB-treated chronic wounds, widespread expression of PDGF-AA was somewhat delayed. With continued treatment, the long form of PDGF-AA, which can preferentially bind extracellular matrix, was expressed only in capillaries, while fibroblasts began synthesizing the short form of PDGF-AA. Within capillaries, all endothelial cells and varying numbers of pericytes and smooth muscle cells contained PDGF-AA. In all wounds, macrophages and keratinocytes were not a major contributor. While PDGF-BB and PDGF-AB were present in a minority of healing wounds, they were usually present at lower levels than PDGF-AA. PDGF-beta receptors, which bind only PDGF-BB and not other isoforms, were found in normal skin and granulation tissue, providing a molecular basis for treating human chronic wounds with exogenous rPDGF-BB.
G F Pierce, J E Tarpley, J Tseng, J Bready, D Chang, W C Kenney, R Rudolph, M C Robson, J Vande Berg, P Reid
During the period of rapid cell growth which follows a two-thirds partial hepatectomy, the liver is able to compensate for the acute loss of two-thirds of its mass to maintain serum glucose levels and many of its differentiation-specific functions. However certain hepatic transcription factors, C/EBP alpha and beta, which are important for establishment and maintenance of the differentiated state, have been shown to be antagonistic to cellular proliferation. To study the interplay between differentiation and cell growth in the liver regeneration model of hepatocyte proliferation, we characterized the expression of C/EBP alpha and beta transcription factors throughout the temporal course of liver regeneration. As determined by immunoblot, the level of C/EBP alpha decreases more than twofold during the mid to late G1 and S phase (8-24 h after hepatectomy) coordinately with a threefold increase in expression of C/EBP beta. Renormalization of the levels of these proteins occurs after the major proliferative phase. This inverse regulation of C/EBP alpha and beta results in up to a sevenfold increase in the beta / alpha DNA binding ratio between 3 and 24 h after hepatectomy that may have an important impact on target gene regulation. However, total C/EBP binding activity in nuclear extracts remains relatively constant during the 7-d period after hepatectomy. By immunohistochemistry, both C/EBP alpha and beta are expressed in virtually all hepatocyte nuclei throughout the liver during the temporal course of liver regeneration, and there is no exclusion of expression from hepatocytes that are expressing immediate-early gene products or undergoing DNA synthesis. The persistent expression of C/EBP alpha and beta isoforms predicts that C/EBP proteins contribute to the function of hepatocytes during physiologic growth and that significant amounts of these proteins do not inhibit progression of hepatocytes into S phase of the cell cycle.
L E Greenbaum, D E Cressman, B A Haber, R Taub
Opioid peptides have been implicated in the regulation of tumor growth and biology; however, little attention has been given to the mechanisms that are involved. In this study we show that physiological concentrations of the endogenous opioid neuropeptide methionine-enkephalin (MET-ENK) and the synthetic enkephalins D-Ala2, Me-Phe4, Gly(ol)5 and D-Ala2, D-Leu5 are stimulants for the in vitro migration of pre-B acute lymphoblastoid leukemia (ALL) cells. Activation of the human pre-B ALL cell lines NALM 6 and LAZ 221 with MET-ENK resulted in both an increase in their migration and an augmentation in the surface expression of the leukemia cell marker CD9. The opiate receptor antagonist naloxone reversed these enkephalin-induced effects on the leukemia cells. When the pre-B ALL cells were preincubated with an anti-CD9 mAb before challenge with MET-ENK their migration to the enkephalin was markedly reduced. These studies show that endogenous and synthetic opioid peptides are stimulants for pre-B ALL cell migration and suggest that CD9 is important in the regulation of leukemia cell motility.
W Heagy, K Duca, R W Finberg
To address the question as to how zona glomerulosa (ZG) cell angiotensin II (Ang II) secretion is regulated, we developed an immuno-cell blot assay to measure its secretion from single cells. We compared these results with those obtained from population studies using a superfusion system. Modulation of Ang II secretion was investigated acutely (by administrating potassium [K+] or captopril) and chronically (by feeding the animals low or high sodium diets). The area of secretory cells, halo areas, and halo intensities varied widely but were highly significantly correlated (P < 0.001) with each other. A disproportionate amount of Ang II was secreted by a small number of large cells. When K+ concentration was increased from 3.6 to 0 mM, superfused ZG cells increased their Ang II secretion 2.32 +/- 0.59-fold. Administration of captopril reduced the K(+)-stimulated Ang II secretion 1.24 +/- 0.07 fold. These findings were reflected in the cell blot assay as a change in the frequency distribution of halo area by K+ and captopril in the same direction as in the population study. In both conditions, the percentage of secretory cells did not change significantly from control. Superfused ZG cells from rats on a low sodium diet secreted 1.85 +/- 0.58-fold more Ang II than cells from sodium-loaded rats (p < 0.05, n = 6). The cell blot assay confirmed these findings with sodium restriction significantly increasing (P < 0.001) both the halo area and its frequency distribution to a larger portion of high secreting cells. However, in contrast to acute treatment with K+ or captopril, the number of secretory cells also doubled. Thus, the individual ZG cell uses two mechanisms to modify Ang II production. In response to acute stimulation and suppression, the amount of Ang II secreted per cell is modified without changing the number of secretary cells. With chronic stimulation, both the amount of Ang II secreted per cell and the number of secretary cells increase.
C Y Chiou, G H Williams, I Kifor
High levels of CD36 expression are found in triglyceride storing and secreting cells such as differentiated adipocytes and mammary secretory epithelial cells and in some capillary endothelial cells. We have found high levels of CD36 in the capillary endothelium of murine adipose tissue and in cardiac and skeletal muscles. Muscle cells themselves were CD36 negative. No CD36 was found in brain endothelium. Cardiac and skeletal muscle tissues are highly oxidative and catabolize long-chain fatty acids as a source of energy while brain tissue does not use long-chain fatty acids for energy production. Since capillary endothelial cell CD36 expression appeared to correlate with parenchymal cell fatty acid utilization and since CD26 has been identified recently as a long-chain fatty acid-binding protein, we examined heart tissue CD36 expression in murine models of insulin-dependent (nonobese diabetic, NOD) and non-insulin-dependent diabetes mellitus (KKAY). Diabetic NOD and KKAY mice had serum triglyceride levels 2.6- and 4.2-fold higher, respectively, than normal mice and exhibited 7- and 3.5-fold higher levels of heart microsomal CD36, respectively, than control mice. Mice fed a 40% fat diet expressed heart tissue CD36 at a level 3.5-fold higher than those fed a 9% fat diet. These data suggest that endothelial cell CD36 expression is related to parenchymal cell lipid metabolism.
D E Greenwalt, S H Scheck, T Rhinehart-Jones
T lymphocytes are present in atherosclerotic lesions, but the role of this cell type in the disease process has not been determined. To determine whether cell-mediated immunity influences atherogenesis, New Zealand White rabbits fed a cholesterol-supplemented diet (0.5% wt/wt) were treated with cyclosporin A (n = 20) or vehicle alone (n = 16) for 12 wk. The dose of cyclosporin A was adjusted so that a blood concentration between 100 and 200 ng/ml was maintained to achieve a selective action T-lymphocytes. Effectiveness of immunosuppression in cyclosporin A-treated rabbits was confirmed by allogeneic skin graft survival. Cyclosporin A administration did not affect total plasma lipid concentrations, body weight, or renal function. Percentage of aortic intimal area covered with atherosclerotic lesions was increased significantly by immunosuppression in both the arch region (75 +/- 3% [mean +/- SEM] compared with 60 +/- 5% in controls; P < 0.01) and the thoracic region (47 +/- 7% vs 27 +/- 6%; P = 0.04). Enhanced atherogenesis was not associated with diminished numbers of T lymphocytes in lesions, changes in T lymphocyte subtype, or any discernible change in cellular composition. Humoral immune responses to oxidized LDL were similar in the two groups: serum titres of autoantibodies against malondialdehyde-modified LDL were equivalent. These data demonstrate that cyclosporin A-induced suppression of cell-mediated immunity increased the development of macrophage-rich atherosclerotic lesions in cholesterol-fed rabbits.
S E Roselaar, G Schonfeld, A Daugherty
Vascular cell adhesion molecule-1 (VCAM-1), an inducible cell-cell recognition protein on the endothelial cell surface (EC), has been associated with early stages of atherosclerosis. In view of the accelerated vascular disease observed in patients with diabetes, and the enhanced expression of VCAM-1 in diabetic rabbits, we examined whether irreversible advanced glycation endproducts (AGEs), could mediate VCAM-1 expression by interacting with their endothelial cell receptor (receptor for AGE, RAGE). Exposure of cultured human ECs to AGEs induced expression of VCAM-1, increased adhesivity of the monolayer for Molt-4 cells, and was associated with increased levels of VCAM-1 transcripts. The inhibitory effect of anti-RAGE IgG, a truncated form of the receptor (soluble RAGE) or N-acetylcysteine on VCAM-1 expression indicated that AGE-RAGE-induced oxidant stress was central to VCAM-1 induction. Electrophoretic mobility shift assays on nuclear extracts from AGE-treated ECs showed induction of specific DNA binding activity for NF-kB in the VCAM-1 promoter, which was blocked by anti-RAGE IgG or N-acetylcysteine. Soluble VCAM-1 antigen was elevated in human diabetic plasma. These data are consistent with the hypothesis that AGE-RAGE interaction induces expression of VCAM-1 which can prime diabetic vasculature for enhanced interaction with circulating monocytes.
A M Schmidt, O Hori, J X Chen, J F Li, J Crandall, J Zhang, R Cao, S D Yan, J Brett, D Stern
S K Moestrup, S Cui, H Vorum, C Bregengård, S E Bjørn, K Norris, J Gliemann, E I Christensen
The goal of this study was to determine to what extent aging affects the antioxidant defense system of the rat adrenal and to evaluate the impact of any change in this system on the recognized age-related decline in steroidogenic capacity of adrenocortical cells. The studies were conducted on young (2-5 mo) and aging (12-27 mo) Sprague-Dawley rats and involved procedures measuring steroidogenesis; oxidative damage to tissue; non enzymatic antioxidants such as vitamin C, E, and glutathione; and tissue antioxidant enzyme (Mn and CuZn superoxide dismutases, catalase, and glutathione peroxidase) activity and expression (mRNA, protein mass, and location). Some measurements were made also on rats maintained on vitamin E-deficient diets. The data show that adrenals from young animals are especially well protected against oxidative events; i.e., these adrenals show the least endogenous lipid peroxidation and the highest level of resistance to prooxidant-induced damage (of various tissues measured) and show exceedingly high levels of tissue antioxidants. Aging, on the other hand, results in oxidative changes in adrenal tissue that are generally linked in time to a reduction in efficiency of the normally protective antioxidant defense system and to the decline in corticosterone production. We speculate that these events are causally related, i.e., that the age-related reduction in oxidative mechanisms in adrenal tissues leads to oxidative damage of membrane or cytosolic factors important to cholesterol transport, and, as a consequence of this damage, cholesterol cannot reach appropriate mitochondrial cholesterol side chain cleavage sites, and corticosterone production fails.
S Azhar, L Cao, E Reaven
Biologically active peptide hormones are synthesized from larger precursor proteins by a variety of posttranslational processing reactions. Endoproteolytic cleavage at the Lys74-Lys75 dibasic processing site of progastrin is the major determinant for the relative distribution of gastrin heptadecapeptide and tetratriacontapeptide in tissues. Thus, we explored the ability of two prohormone convertases, PC1/PC3 and PC2, to cleave this important site within progastrin. We expressed wild-type human gastrin cDNA and mutant cDNAs in which the Lys74Lys75 site was changed to Lys74Arg75, Arg74Arg75, and Arg74Lys75 residues in AtT-20 cells. Because AtT-20 cells express Pc1/PC3 but not PC2, we also coexpressed a cDNA encoding PC2 in both wild-type and mutant gastrin-producing AtT-20 cells. Wild-type Lys74Lys75 and mutant Arg74Arg75 progastrin processing sites were efficiently cleaved in AtT-20 cells only after coexpression of PC2. Mutant Lys74Arg75 progastrin was readily processed in cells in the presence or absence of PC2 coexpression, but, in contrast, mutant Arg74Lys75 progastrin was inefficiently cleaved regardless of PC2 coexpression. Northern analysis revealed the presence of PC2 but not PC1/ PC3 in canine antral gastrin-producing G cells. These data suggest that PC2 but not PC1/PC3 is responsible for the cleavage of the Lys74Lys75 site in wild-type progastrin.
C J Dickinson, M Sawada, Y J Guo, S Finniss, T Yamada
The current study evaluates the capacity of recombinant rat stem cell factor (rrSCF) to regulate enzymes that control AA release and eicosanoid generation in mouse bone marrow-derived mast cells (BMMCs). Initial studies indicated that rrSCF provided for 24 h inhibited the release of AA into supernatant fluids of antigen- and ionophore A23187-stimulated BMMCs. Agonist-induced increases in cellular levels of AA were also inhibited, albeit to a lesser degree by rrSCF. To determine the inhibitory mechanism, several steps (e.g., mobilization of cytosolic calcium, release of BMMC granules, and regulation of phospholipase A2 [PLA2] activity) that could influence AA release were measured in rrSCF-treated cells. rrSCF did not alter the capacity of BMMCs to mobilize cytosolic calcium or release histamine in response to antigen and ionophore. BMMCs released large amounts of PLA2 with characteristics of the group II family in response to antigen and ionophore A23187. rrSCF treatment of BMMCs reduced the secretion of this PLA2 activity by BMMCs. Partial purification of acid-extractable PLA2 from rrSCF-treated and untreated BMMCs suggested that rrSCF decreased the quantity of acid-stable PLA2 within the cells. In contrast to group II PLA2, the quantity of cPLA2 (as determined by Western blot analysis) increased in response to rrSCF. To assess the ramifications of rrSCF-induced reductions in AA and group II PLA2, eicosanoid formation was measured in antigen- and ionophore-stimulated BMMCs, rrSCF-inhibited (100 ng/ml, 24 h) prostaglandin D2 (PGD2), thromboxane B2, and leukotriene B4 by 48.4 +/- 7.7%, 61.1 +/- 10.0% AND 38.1 +/- 3.6%, respectively, in antigen-stimulated cells. Similar patterns of inhibition were observed in ionophore-stimulated BMMCs. The addition of a group I PLA2 or exogenous AA to BMMCs reversed the inhibition of eicosanoid generation induced by rrSCF. Together, these data indicate that rrSCF differentially regulates group II and cytosolic PLA2 activities in BMMCs. The resultant reductions in eicosanoid generation suggest that group II PLA2 provides a portion of AA that is used for eicosanoid biosynthesis by BMMCs.
A N Fonteh, J M Samet, F H Chilton
Human (Hu) lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol. To assess the effects of increased plasma levels of LCAT, four lines of transgenic mice were created expressing a Hu LCAT gene driven by either its natural or the mouse albumin enhancer promoter. Plasma LCAT activity increased from 1.2- to 1.6-fold higher than that found in control mouse plasma. Lipid profiles, upon comparing Hu LCAT transgenics to control animals, revealed a 20 t0 60% increase in total and cholesteryl esters that were mainly present in HDL. The in vivo substrate specificity of Hu LCAT was assessed by creating animals expressing Hu apo AI + Hu LCAT (HuAI/ LCAT), Hu apo AI + Hu apo AII + Hu LCAT (HuAI/ AII/LCAT), and Hu apo AII + Hu LCAT (HuAII/LCAT). Plasma cholesterol was increased up to 4.2-fold in HuAI/ LCAT transgenic mice and twofold in the HuAI/AII/LCAT transgenic mice, compared with HuAI and HuAI/AII transgenic mice. HDL cholesteryl ester levels were increased more than twofold in both the HuAI/LCAT and HuAI/AII/LCAT mice compared with the HuAI, HuAI/AII, and HuLCAT animals. The HDL particles were predominantly larger in the HuAI/LCAT and the HuAI/AII/LCAT mice compared with those in HuAI, HuAII/LCAT, and HuLCAT animals. The increase in LCAT activity in the HuAI/LCAT and HuAI/AII/LCAT mice was associated with 62 and 27% reductions respectively, in the proportion of Hu apo AI in the pre beta-HDL fraction, when compared with HuAI and HuAI/AII transgenic mice. These data demonstrate that moderate increases in LCAT activity are associated with significant changes in lipoprotein cholesterol levels and that Hu LCAT has a significant preference for HDL containing Hu apo AI.
O L Francone, E L Gong, D S Ng, C J Fielding, E M Rubin
In vivo, endothelial cells (EC) are subjected to hemodynamic forces which may influence the production of nitric oxide. This study was designed to examine the effect of cyclic strain on the expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic EC. EC were grown on flexible membranes which were subjected to deformation at 60 cycles/min with -5 or -20 kPa of vacuum. This results in an average strain of 6 and 10%, respectively, which is transmitted to the attached cells. Northern blot analysis of total cytosolic RNA demonstrated an increase in eNOS gene expression with both strain regimens but the increase with 10% average strain was greater than that at 6%. Nuclear runoff transcription assays confirmed the induction of eNOS transcripts. Western blot analysis showed an increase in eNOS level after 24 h of cyclic 10% average strain compared with controls or 6% average strain. Immunohistochemical staining of EC for eNOS was increased in the high strain periphery (7-24% strain) of membranes deformed with -20 kPa vacuum. These results demonstrate that cyclic strain upregulates the expression of eNOS transcripts and protein levels in bovine aortic EC thus emphasizing the importance of hemodynamic forces in the regulation of eNOS in vivo.
M A Awolesi, W C Sessa, B E Sumpio
Adrenoleukodystrophy (ALD) is an inherited disorder of fatty acid metabolism marked by accumulation of very long chain saturated fatty acids (VLCFA), especially the 26-carbon acid, hexacosanoic acid (HA), in membranes and tissues. We have studied interactions of 13C-enriched HA with model membranes (phospholipid bilayer vesicles) and bovine serum albumin (BSA) by 13C NMR spectroscopy to compare properties of HA with those of typical dietary fatty acids. In phospholipid bilayers the carboxyl group of HA is localized in the aqueous interface, with an apparent pKa (7.4) similar to other fatty acids; the acyl chain must then penetrate very deeply into the membrane. Desorption of HA from vesicles (t1+2 = 3 h) is orders of magnitude slower than shorter chain fatty acids. In mixtures of vesicles and BSA, HA partitions much more favorably to phospholipid bilayers than typical fatty acids. BSA binds a maximum of only 1 mole of HA at one binding site. Calorimetric experiments show strong perturbations of acyl chains of phospholipids by HA. We predict that disruptive effects of VLCFA on cell membrane structure and function may explain the neurological manifestations of ALD patients. These effects will be further amplified by slow desorption of VLCFA from membranes and by the ineffective binding to serum albumin.
J K Ho, H Moser, Y Kishimoto, J A Hamilton
One or more brief periods of ischemia, termed preconditioning, dramatically limits infarct size and reduces intracellular acidosis during subsequent ischemia, potentially via enhanced sarcolemmal proton efflux mechanisms. To test the hypothesis that preconditioning increases the functional activity of sodium-dependent proton efflux pathways, isolated rat hearts were subjected to 30 min of global ischemia with or without preconditioning. Intracellular sodium (Nai) was assessed using 23Na magnetic resonance spectroscopy, and the activity of the Na-H exchanger and Na-K-2Cl cotransporter was measured by transiently exposing the hearts to an acid load (NH4Cl washout). Creatine kinase release was reduced by greater than 60% in the preconditioned hearts (P < 0.05) and was associated with improved functional recovery on reperfusion. Preconditioning increased Nai by 6.24 +/- 2.04 U, resulting in a significantly higher level of Nai before ischemia than in the control hearts. Nai increased significantly at the onset of ischemia (8.48 +/- 1.21 vs. 2.57 +/- 0.81 U, preconditioned vs. control hearts; P < 0.01). Preconditioning did not reduce Nai accumulation during ischemia, but the decline in Nai during the first 5 min of reperfusion was significantly greater in the preconditioned than in the control hearts (13.48 +/- 1.73 vs. 2.54 +/- 0.41 U; P < 0.001). Exposure of preconditioned hearts to ethylisopropylamiloride or bumetanide in the last reperfusion period limited in the increase in Nai during ischemia and reduced the beneficial effects of preconditioning. After the NH4Cl prepulse, preconditioned hearts acidified significantly more than control hearts and had significantly more rapid recovery of pH (preconditioned, delta pH = 0.35 +/- 0.04 U over 5 min; control, delta pH = 0.15 +/- 0.02 U over 5 min). This rapid pH recovery was not affected by inhibition of the Na-K-2Cl cotransporter but was abolished by inhibition of the Na-H exchanger. These results demonstrate that preconditioning alters the kinetics of Nai accumulation during global ischemia as well as proton transport after NH4Cl washout. These observations are consistent with stimulation of the Na-K-2Cl cotransporter and Na-H exchanger by preconditioning.
R Ramasamy, H Liu, S Anderson, J Lundmark, S Schaefer
IGF-I stimulation of cell proliferation and c-Fos expression in skeletal muscle cells is markedly enhanced by dexamethasone. The effect of dexamethasone is not mediated by changes in IGF-binding proteins, as evidenced by similar effects of dexamethasone on the actions of insulin, PDGF-BB, and the IGF-I analogue long R3IGF-I. Dexamethasone also does not alter autocrine IGF-II secretion by muscle cells. To investigate the mechanism of the augmentation of IGF-I action, the effects of dexamethasone on intracellular IGF-I signaling pathways were determined. In dexamethasone-treated cells, the levels of IGF-I receptor tyrosine phosphorylation and receptor-associated phosphatidylinositol 3-kinase activity were increased. Dexamethasone-treated cells also showed increased and prolonged tyrosine phosphorylation of the Shc proteins. In contrast, dexamethasone decreased both tyrosine phosphorylation and expression of insulin receptor substrate 1 (IRS-1) and IRS-1-associated phosphatidylinositol 3-kinase activity. Thus, distinct signaling pathways activated by the IGF-I receptor in skeletal muscle cells are differentially regulated by dexamethasone. Potentiation of IGF-I action correlates with increased IGF-I receptor-associated phosphatidylinositol 3-kinase activity and tyrosine phosphorylation of Shc, but appears to be independent of activation of the IRS-1/phosphatidylinositol 3-kinase signaling pathway.
F Giorgino, R J Smith
Mercurials may induce immune manifestations in susceptible individuals. Mercuric chloride (HgCl2) induced autoimmunity in the Brown Norway (BN) strain but an immuno-suppression in the Lewis strain with, however, autoreactive anti-class II T cells present in both strains. In the present study we looked at modifications of cytokine production by PCR and cytofluorometric analyses in normal BN and Lewis rat splenocytes, cultured with or without HgCl2. Unfractionated BN rat splenocytes and purified T cells exposed to HgCl2 expressed high levels of IL-4 mRNA. Increase in class II and CD23 molecule expression on B cells was partly inhibited by anti-IL-4 mAb showing that IL-4 was produced. By contrast, no overexpression of IL-4 mRNA could be seen in Lewis rats. Although an increase in class II molecule expression was observed suggesting that other T helper cell 2 cytokines were produced, there was also a concomitant decrease in CD23 molecule expression that was abrogated after addition of an anti-IFN-gamma mAb to the culture. IFN-gamma mRNA production was induced in unfractionated spleen cells and T cells from both strains after HgCl2 exposure. Altogether these findings demonstrate that HgCl2 has very early direct effects on cytokine production and that these effects differ depending on the strain. The early effect on IL-4 production observed on BN rat spleen cells and T cells may explain that the autoreactive anti-class II T cells that are found in HgCl2-injected BN rats have a Th2 phenotype.
P Prigent, A Saoudi, C Pannetier, P Graber, J Y Bonnefoy, P Druet, F Hirsch
It has been proposed that the contribution of myocardial tissue angiotensin converting enzyme (ACE) to angiotensin II (Ang II) formation in the human heart is low compared with non-ACE pathways. However, little is known about the actual in vivo contribution of these pathways to Ang II formation in the human heart. To examine angiotensin II formation in the intact human heart, we administered intracoronary 123I-labeled angiotensin I (Ang I) with and without intracoronary enalaprilat to orthotopic heart transplant recipients. The fractional conversion of Ang I to Ang II, calculated after separation of angiotensin peptides by HPLC, was 0.415 +/- 0.104 (n = 5, mean +/- SD). Enalaprilat reduced fractional conversion by 89%, to a value of 0.044 +/- 0.053 (n = 4, P = 0.002). In a separate study of explanted hearts, a newly developed in vitro Ang II-forming assay was used to examine cardiac tissue ACE activity independent of circulating components. ACE activity in solubilized left ventricular membrane preparations from failing hearts was 49.6 +/- 5.3 fmol 125I-Ang II formed per minute per milligram of protein (n = 8, +/- SE), and 35.9 +/- 4.8 fmol/min/mg from nonfailing human hearts (n = 7, P = 0.08). In the presence of 1 microM enalaprilat, ACE activity was reduced by 85%, to 7.3 +/- 1.4 fmol/min/mg in the failing group and to 4.6 +/- 1.3 fmol/min/mg in the nonfailing group (P < 0.001). We conclude that the predominant pathway for angiotensin II formation in the human heart is through ACE.
L S Zisman, W T Abraham, G E Meixell, B N Vamvakias, R A Quaife, B D Lowes, R L Roden, S J Peacock, B M Groves, M V Raynolds
The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to O2- at the expense of NADPH. Dormant in resting cells, the oxidase is activated by exposing the cells to appropriate stimuli. During activation, p47phox, a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303 and S379. To determine whether this phosphorylation is necessary for oxidase activation, we examined phorbol-elicited oxidase activity in EBV-transformed B lymphoblasts deficient in p47phox after transfection with plasmids expressing various S-->A mutants of p47phox. The mutant containing S-->A mutations involving all serines between S303 and S379 [S(303-379)A] was not phosphorylated, did not translocate to plasma membrane during activation and was almost devoid of function. As to individual serines, S379 was of special interest because (a) p47 phox S379 was phosphorylated in phorbol-activated lymphoblasts expressing wild-type p47phox, and (b) p47phox S379A failed to translocate to the membrane, and was as functionless as p47phox S(303-379)A; other single S-->A mutations had little effect on oxidase activity. These findings suggest that the phosphorylation of S379 may be important for oxidase activation in whole cells.
L R Faust, J el Benna, B M Babior, S J Chanock
Sera from patients with insulin-dependent diabetes immunoprecipitate 64,000-M(r) proteins, distinct from glutamate decarboxylase, that are cleaved to 37,000- and 40,000-M(r) fragments by trypsin. We investigated possible relationships between 37,000- or 40,000-M(r) fragments of antigen and the tyrosine phosphatase-like protein, IA-2 (ICA512). Antibodies from nondiabetic relatives bound differentially to 37,000- and 40,000-M(r) fragments indicating presence of distinct epitopes. Precursors of these fragments could be separated on immobilized lectins, suggesting different carbohydrate content. Levels of antibodies to 40,000-M(r) fragments were strongly associated with those to the intracellular domain of IA-2. Recombinant intracellular domain of IA-2 blocked binding of antibodies to 40,000-M(r) fragments expressed by insulinoma cells and partially blocked binding to 37,000-M(r) fragments. Furthermore, trypsinization of recombinant intracellular domain of IA-2 generated proteolytic fragments of identical M(r) to the 40,000-M(r) fragments of insulinoma antigen; 37,000-M(r) fragments were not generated. Thus, 40,000-M(r) fragments of islet autoantigen are derived from a protein similar or identical to the tyrosine phosphatase-like molecule, IA-2. The 37,000-M(r) fragments are derived from a different, although related, protein.
M A Payton, C J Hawkes, M R Christie
The effects of chronic stress on the renin-angiotensin-aldosterone system were studied by analysis of plasma hormone levels, kidney renin mRNA levels, adrenal angiotensin II receptors, and steroidogenesis in rats subjected to repeated immobilization (2 h daily) or intraperitoneal injections of 1.5 M NaCI for 14 d. 24 after the last stress in both stress models, plasma aldosterone levels were reduced in spite of significant increases in plasma renin activity. Repeatedly intraperitoneal hypertonic saline-injected rats showed plasma renin activity responses to acute immobilization similar to controls, but markedly reduced plasma aldosterone responses. Concomitant with the increases in plasma renin activity, renin mRNA levels in the kidney were significantly increased in intraperitoneal hypertonic saline-injected rats, and these increases were prevented by beta-adrenergic receptor blockade with propranolol. In isolated adrenal glomerulosa cells from chronically stressed rats, maximum aldosterone responses to angiotensin II, ACTH, and 8-Br-cAMP were significantly decreased, whereas pregnenolone responses were increased. P450-aldosterone synthetase mRNA levels and binding of 125I-[Sar1,Ile8] angiotensin II were significantly reduced in the adrenal zona glomerulosa of stressed rats. These studies show that chronic repeated stress leads to renin stimulation due to sympathetic activation, and inhibition of aldosterone secretion due to inhibition of the late steroidogenic pathway. The data provide evidence for a role of chronic stress in the development of hyperreninemic hypoaldosteronism.
G Aguilera, A Kiss, B Sunar-Akbasak
Various immune mechanisms have been reported to contribute to the progressive destruction of Th cells in HIV-1-infected patients. Among these, complement mediated lysis of infected cells has been suggested. An increased sensitivity of lymphocytes from HIV-1-infected patients to lysis by monoclonal antibodies directed to MHC class I antigen and complement has been directly correlated with a decreased expression of the decay accelerating factor (CD55). It also has been reported that the expression of the membrane inhibitor of reactive lysis (CD59) is decreased during HIV-1 infection. We examined the effect of antibodies in the serum of HIV-1-positive individuals and normal human serum (NHS) as source of complement on several HIV-1-infected cell lines differing in their expression of CD55 and CD59. When HIV-1-infected target cells without membrane expression of CD55 and CD59 were used, a highly significant cytotoxic effect was observed in the presence of heat inactivated anti-HIV-1-positive sera and NHS, while heat-inactivated anti-HIV-1-negative sera and NHS were unable to induce cytolysis. Similar results were obtained using purified IgG isolated from HIV-1-positive sera and either NHS or guinea pig serum as source of complement. Lysis of HIV-1-infected cells correlated with expression of viral antigens on the cell surface. HIV-1-infected CD55 and CD59 positive target cells showed specific lysis, when the function of these molecules was abrogated by blocking antibodies to CD55 and CD59. The finding of anti-HIV-1-specific cytotoxic antibodies in sera from HIV-1-infected patients should be considered in the pathogenesis of the HIV-1-infection.
J Schmitz, J P Zimmer, B Kluxen, S Aries, M Bögel, I Gigli, H Schmitz
It has been suggested that immune selection pressure exerted by the cytotoxic T lymphocyte (CTL) response could be responsible for viral persistence during chronic hepatitis B virus infection. To address this question, in the current study we compared the DNA and amino acid sequences of, and the CTL responses to, multiple HLA-A2-restricted CTL epitopes in the hepatitis B virus in several HLA-A2-positive patients with acute and chronic hepatitis. Our results indicate that the CTL response to these epitopes is barely detectable in the majority of patients with chronic hepatitis. Further, we show that the weak CTL response is not secondary in infection by mutant viruses lacking these epitopes, and we show that the CTL response did not select for escape mutants in any of these patients. We conclude that an ineffective hepatitis B virus specific CTL response is the primary determinant of viral persistence in chronic hepatitis and that immune selection of viral variants is not a common event in the majority of patients.
B Rehermann, C Pasquinelli, S M Mosier, F V Chisari
Although folate receptors (FRs) mediate folate uptake into cells, the independent role of FRs in cell proliferation remains unclear. We tested the hypothesis that transduction of FR cDNA in sense or antisense orientation using recombinant adeno-associated virus modulated FR expression and altered proliferation of cervical carcinoma cells (which constitutively overexpress FR genes). We determined that the integration of recombinant adeno-associated virions was not site specific. When compared with untransduced cells, sense and antisense FR cDNA-transduced cells exhibited an increase and decrease in FR mRNA and FR expression on the cell surface, respectively. However, when compared with antisense FR cDNA-transduced and untransduced cells, sense FR cDNA-transduced cells exhibited statistically significant (a) increased in total FRs, (b) smaller colonies, (c) lowered cell proliferation in vitro, and (d) less tumor volume with dramatic prolongation of tumor doubling times (225.6 h vs. 96 h) after transplantation into nude mice. Finally, (f) using single cell-derived transduced clones, an inverse relationship between cell proliferation and FR expression was established (r = 0.90, P < 0.001). Thus, transduction of sense/antisense FR cDNA into cervical carcinoma cells modulated expression of FRs and had an impact on cell proliferation in vitro and in vivo.
X L Sun, B R Murphy, Q J Li, S Gullapalli, J Mackins, H N Jayaram, A Srivastava, A C Antony
HTLV-1 infection causes an adult T cell leukemia in humans. The viral encoded protein tax, is thought to play an important role in oncogenesis. Our previous data obtained from a tax transgenic mouse model revealed that tax transforms mouse fibroblasts but not thymocytes, despite comparable levels of tax expression in both tissues. Constitutive tyrosine phosphorylation of a 130-kD protein(s) was observed in the tax transformed fibroblast B line and in HTLV-1 transformed human lymphoid lines, but not in thymocytes from Thy-tax transgenic mice. Phosphotyrosine immunoprecipitation followed by Western blot analysis with a set of Jak kinase specific antibodies, identified p130 as Jak2 in the tax transformed mouse fibroblastic cell line and Jak3 in HTLV-1 transformed human T cell lines. Phosphorylation of Jak2 in tax transformed cells resulted from high expression of IL-6. Tyrosine phosphorylation of this protein could also be induced in Balb/c3T3 cells using a supernatant from the B line, which was associated with induction of cell proliferation. Both phosphorylation and proliferation were inhibited by IL-6 neutralizing antibodies. Constitutive phosphorylation of Jak kinases may facilitate tumor growth in both HTLV-1 infected human T cells and the transgenic mouse model.
X Xu, S H Kang, O Heidenreich, M Okerholm, J J O'Shea, M I Nerenberg
In mammals, urea is the predominant end-product of nitrogen metabolism and plays a central role in the urinary-concentrating mechanism. Urea accumulation in the renal medulla is critical to the ability of the kidney to concentrate urine to an osmolality greater than systemic plasma. Regulation of urea excretion and accumulation in the renal medulla depends on the functional state of specialized phloretin-sensitive urea transporters. To study these transporters and their regulation of expression we isolated a cDNA which encodes the rat homologue (rUT2) of rabbit UT2 (You, G., C.P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M.A. Hediger, et al. Nature (Lond.). 1993. 365:844-847). Rat UT2 has 88% amino acid sequence identity to rabbit UT2 and 64% identity to the recently cloned human erythrocyte urea transporter, HUT11 (Olives, B., P. Neav, P. Bailly, M.A. Hediger, G. Rousselet, J.P. Cartron, and P. Ripoch J. Biol. Chem. 1994. 269:31649-31652). Analysis of rat kidney mRNA revealed two transcripts of size 2.9 and 4.0 kb which had spatially distinct distributions. Northern analysis and in situ hybridization showed that the 4.0-kb transcript was primarily responsive to changes in the protein content of the diet whereas the 2.9-kb transcript was responsive to changes in the hydration state of the animal. These studies reveal that the expression levels of the two rUT2 transcripts are modulated by different pathways to allow fluid and nitrogen balance to be regulated independently. Our data provide important insights into the regulation of the renal urea transporter UT2 and provide a basis on which to refine our understanding of the urinary concentrating mechanism and its regulation.
C P Smith, W S Lee, S Martial, M A Knepper, G You, J M Sands, M A Hediger
Complement activation contributes to the systemic inflammatory response induced by cardiopulmonary bypass. At the cellular level, cardiopulmonary bypass activates leukocytes and platelets; however the contribution of early (3a) versus late (C5a, soluble C5b-9) complement components to this activation is unclear. We used a model of simulated extracorporeal circulation that activates complement (C3a, C5a, and C5b-9 formation), platelets (increased percentages of P-selectin-positive platelets and leukocyte-platelet conjugates), and neutrophils (upregulated CD11b expression). to specifically target complement activation in this model, we added a blocking mAb directed at the human C5 complement component and assessed its effect on complement and cellular activation. Compared with a control mAB, the anti-human C5 mAb profoundly inhibited C5a and soluble C5b-9 generation and serum complement hemolytic activity but had no effect on C3a generation. Additionally, the anti-human C5 mAb significantly inhibited neutrophil CD11b upregulation and abolished the increase in P-selectin-positive platelets and leukocyte-platelet conjugate formation compared to experiments performed with the control mAb. This suggests that the terminal components C5a and C5b-9, but not C3a, directly contribute to platelet and neutrophil activation during extracorporeal circulation. Furthermore, these data identify the C5 component as a site for therapeutic intervention in cardiopulmonary bypass.
C S Rinder, H M Rinder, B R Smith, J C Fitch, M J Smith, J B Tracey, L A Matis, S P Squinto, S A Rollins
The cholesteryl ester transfer protein (CETP) transfers lipids among lipoprotein particles and plays a central role in lipoprotein metabolism. Humans with genetic deficiency of CETP have both elevated HDL cholesterol and apolipoprotein A-I concentrations as well as decreased LDL cholesterol and apolipoprotein B levels. The present study was undertaken to elucidate the metabolic basis for the decreased LDL cholesterol and apo B levels in CETP deficiency. We conducted a series of in vivo apo B kinetic studies in tow unrelated homozygotes with CETP deficiency and in control subjects. A primed constant infusion of stable isotopically labeled phenylalanine was administered to the two CETP deficient subjects and control subjects and apo B kinetic parameters in VLDL, intermediate density lipoproteins, and LDL were obtained by using a multicompartmental model. The fractional catabolic rates (FCR) of LDL apo B were significantly increased in the CETP-deficient subjects (0.56 and 0.75/d) compared with the controls (mean FCR of 0.39/d). Furthermore, the production rates of apo B in VLDL and intermediate density lipoprotein were decreased by 55% and 81%, respectively, in CETP deficiency compared with the controls. In conclusion, CETP-deficient subjects were demonstrated to have substantially increased catabolic rates of LDL apo B as the primary metabolic basis for the low plasma levels of LDL apo B. This result indicates that the LDL receptor pathway may be up-regulated in CETP deficiency.
K Ikewaki, M Nishiwaki, T Sakamoto, T Ishikawa, T Fairwell, L A Zech, M Nagano, H Nakamura, H B Brewer Jr, D J Rader
Ryanodine receptors (RyRs) reside in microsomal membranes where they gate Ca2+ release in response to changes in the cytosolic Ca2+ concentration. In the osteoclast, a divalent cation sensor, the Ca2+ receptor (CaR), located within the cell's plasma membrane, monitors changes in the extracellular Ca2+ concentration. Here we show that a RyR-like molecule is a functional component of this receptor. We have demonstrated that [3H] ryanodine specifically binds to freshly isolated rat osteoclasts. The binding was displaced by ryanodine itself, the CaR agonist Ni2+ and the RyR antagonist ruthenium red. The latter also inhibited cytosolic Ca2+ elevations induced by Ni2+. In contrast, the responses to Ni2+ were strongly potentiated by an antiserum Ab129 raised to an epitope located within the channel-forming domain of the type II RyR. The antiserum also stained the surface of intact, unfixed, trypan blue-negative osteoclasts. Serial confocal sections and immunogold scanning electron microscopy confirmed a plasma membrane localization of this staining. Antiserum Ab34 directed to a putatively intracellular RyR epitope expectedly did not stain live osteoclasts nor did it potentiate CaR activation. It did, however, stain fixed, permeabilized cells in a distinctive cytoplasmic pattern. We conclude that an RyR-like molecule resides within the osteoclast plasma membrane and plays in important role in extracellular Ca2+ sensing.
M Zaidi, V S Shankar, R Tunwell, O A Adebanjo, J Mackrill, M Pazianas, D O'Connell, B J Simon, B R Rifkin, A R Venkitaraman
The VH26 germline gene occupies two different loci, due to gene duplication, and is one of the most frequently expressed human immunoglobulin VH genes. This report identifies the alleles of each VH26 locus and describes distinct patterns of VH26 polymorphism in three ethnic groups. Oligonucleotide probes targeting VH26 were used in sequence-specific RFLP analysis of DNA from 72 Caucasians, 52 Asians, 35 American Blacks, and members of six families. The A locus, on a 7.0-kb TaqI band, was detected in 89% of Caucasians, 75% of Asians, and 26% of Blacks (chi2 = P < 0.0005). The B locus, detected on a 5.0-kb band in nearly all subjects, was found to have additional alleles occurring at 6.8 kb in 10% of Asians and 3% of Blacks (chi2 = 7.8, P < 0.02) and at 3.7 kb in 1.4% of Caucasians, 21% of Asians, and (9% of Blacks (chi2 = 13.8, P < 0.001). In Asians, only, the 3.7-kb hybridization band represented a multiple-duplication unit containing three or four gene copies. Duplications of other VH26 alleles, and mull alleles of the B locus, were also seen. An exact VH26 sequence was cloned from the 5.0-kb allele and likely exists in the 7.0- and 6.8-kb alleles. A novel sequence cloned from the 3.7-kb allele differed from VH26 by nine nucleotides and appears to have evolved by gene conversion in CDR2. The total diploid gene dose of the A and B loci ranged from one to as many as six copies of VH26-containing genes, and from zero to as many as six to eight copies of the 3.7-kb allele. We conclude that ethnic differences in polymorphism exist at both VH26 loci. These differences could influence VH26 expression because they involve variations in gene copy number and coding region sequence.
E H Sasso, J H Buckner, L A Suzuki
Human nasal polyps from non-CF and delta F 508 homozygous CF patients were used to compare the expression of CFTR and markers epithelial differentiation, such as cytokeratins (CK) and desmoplakins (DP), at the transcriptional and translational levels. mRNA expression was assessed by semiquantitative RT/PCR kinetic assays while the expression and distribution of proteins were evaluated by immunofluorescence analysis. In parallel, for each nasal tissue specimen, the importance of surface epithelium remodeling and inflammation was estimated after histological observations. Our results show that the steady-state levels of CFTR, CK13, CK18, CK18, CK14, or DP 1 mRNA transcripts in delta F 508 CF nasal polyps were not significantly different from those of non-CF tissues. A variability in the CFTR mRNA transcript level and in the pattern of CFTR immunolabeling has been observed between the different tissue samples. However, no relationship was found between the level of CFTR mRNA transcripts and the CFTR protein expression and distribution, either in the non-CF or in the CF group. The histological observations of non-CF and CF nasal polyp tissue indicated that the huge variations in the expression and distribution of the CFTR protein were associated with the variations in the degree of surface epithelium remodeling and inflammation in the lamina propria. A surface epithelium, showing a slight basal cell hyperplasia phenotype associated with diffuse inflammation, was mainly characterized by a CFTR protein distribution at the apex of ciliated cells in both non-CF and CF specimens. In contrast, in a remodeled surface epithelium associated with severe inflammation, CFTR protein presented either a diffuse distribution in the cytoplasm of ciliated cells, or was absent. These results suggest that abnormal expression and distribution of the CFTR protein of CF airways is not only caused by CFTR mutations. Airway surface epithelium remodeling and inflammation could play a critical role in the posttranscriptional and/or the posttranslational regulation of the CFTR protein expression in non-CF and CF airways.
F Dupuit, N Kälin, S Brézillon, J Hinnrasky, B Tümmler, E Puchelle
Apolipoprotein E (apoE)-deficient mice develop marked hyperlipidemia as well as atherosclerosis and thus are an excellent animal model for evaluating the potential for gene therapy in human genetic dyslipoproteinemias. Recombinant adenovirus containing either human apoE (rAdv.apoE) or the reporter gene luciferase (rAdv.luc) were generated and infused intravenously in apoE-deficient mice with preinfusion plasma total cholesterol of 644 +/- 149 mg/dl an cholesterol rich VLDL/IDL. After a single infusion of rAdv.apoE, plasma concentrations of human apoE ranging from 1.5 to 650 mg/dl were achieved. Adenovirus-mediated apoE replacement resulted in normalization of the lipid and lipoprotein profile with markedly decreased total cholesterol (103 +/- 18mg/dl), VLDL, IDL, and LDL, as well as increased HDL. Measurement of aortic atherosclerosis 1 mo after adenoviral infusion demonstrated a marked reduction in the mean lesion area of mice infused with rAdv.apoE (58 +/- 8 x 10(3) microns2) when compared with control mice infused with rAdv.luc (161 +/- 10 x 10(3) microns2; P < 0.0001). Thus, apoE expression for 4 wk was sufficient to markedly reduce atherosclerosis, demonstrating the feasibility of gene therapy for correction of genetic hyperlipidemias resulting in atherosclerosis. The combined use of adenovirus vectors and the apoE-deficient mouse represents a new in vivo approach that will permit rapid screening of candidate genes for the prevention of atherosclerosis.
V S Kashyap, S Santamarina-Fojo, D R Brown, C L Parrott, D Applebaum-Bowden, S Meyn, G Talley, B Paigen, N Maeda, H B Brewer Jr
Bleomycin-induced lung injury is an established murine model of human pulmonary fibrosis. Although procoagulant molecules (e.g., tissue factor [TF]) and fibrinolytic components (e.g., urokinase [u-PA] and type 1 plasminogen activator inhibitor [PAI-1]) have been detected in alveolar fluid from injured lungs, the origin of these molecules remains unknown. We therefore examined the expression of procoagulant and fibrinolytic components in relation to the distribution of parenchymal fibrin in bleomycin-injured lungs. Extravascular fibrin localized to the alveolar and extracellular matrix in injured lung tissue. Injured lung tissue extracts contained elevated levels of PAI-1 activity and decreased levels of u-PA activity. Whole lung PAI-1 and TF mRNAs were dramatically induced by lung injury. In situ hybridization of injured lungs revealed that PAI-1, u-PA, and TF mRNAs were induced within the fibrin-rich fibroproliferative lesions, primarily in fibroblast-like and macrophagelike cells, respectively, while TF mRNA was also induced in perilesional alveolar cells. Taken together, these observations suggest that the induction of PAI-1 and TF gene expression plays and important role in the formation and persistence of extracellular fibrin in bleomycin injured murine lungs.
M A Olman, N Mackman, C L Gladson, K M Moser, D J Loskutoff
RA synovial tissue (ST) was studied to determine if and where apoptosis occurs in situ. Genomic DNA was extracted from 5 RA and 1 osteoarthritis ST samples. Agarose gel electrophoresis demonstrated DNA ladders characteristic for apoptosis from each tissue. In situ and labeling (ISEL) was used to identify DNA strand breaks consistent with apoptosis in frozen sections. 12 RA and 4 osteoarthritis ST were studied by ISEL and all were positive, but only 2 of 4 normal tissues were positive. The primary location of apopotic cells was the synovial lining. Some sublining cells were also positive, but lymphoid aggregate staining was conspicuously absent. Immunohistochemistry and ISEL were combined and showed that the lining cells with DNA strand breaks were mainly macrophages, although some fibroblastlike cells were also labeled. Sublining cells with fragmented DNA included macrophages and fibroblasts, but T cells in lymphoid aggregates, which expressed large amounts of bcl-2, were spared. DNA strand breaks in cultured fibroblastlike synoviocytes was assessed using ISEL. Apoptosis could be induced by actinomycin D, anti-fas antibody, IL-1, and TNF-alpha but not by IFN-gamma. Fas expression was also detected on fibroblast-like synoviocytes using flow cytometry. Therefore, DNA strand breaks occur in synovium of patients with arthritis. Cytokines regulate this process, and the cytokine profile in RA (high IL-1/TNF; low IFN-gamma) along with local oxidant injury might favor induction of apoptosis.
G S Firestein, M Yeo, N J Zvaifler
The engineering of mice that express a human apoB transgene has resulted in animals with high levels of human-like LDL particles and through crosses with human apo(a) transgenics, high levels of human-like lipoprotein (a) (Lp[a]) particles. In this study, these animals have been used to compare the atherogenic properties of apo(a), LDL, and Lp(a). The presence of the high expressing apoB (apoBH) transgene was associated with a 2.5-fold increase in VLDL-LDL cholesterol (primarily in the LDL fraction) and a 15-fold increase in proximal lesions compared with non-transgenic mice (P < or = 0.0001), while the presence of the low expressing human apoB (apoBL) transgene was not associated with major changes in lipoprotein profiles or increases in aortic lesion size. Examination of aortas of apoBH mice demonstrated lesions along the entire length of the aorta and immunochemical analysis of the lesions revealed features characteristically seen in human lesions including the presence of oxidized lipoproteins, macrophages, and immunoglobulins. Unlike animals with the apoBL transgene, animals with the apo(a) transgene had significant increases in proximal aortic fatty streak lesions compared to nontransgenic control animals (threefold; P < 0.02), while animals with both transgenes, the apo(a)/apo BL double transgenics, had lesions 2.5 times greater than animals expressing the apo(a) transgene alone and eightfold (P < 0.0006) greater than nontransgenic animals. These murine studies demonstrate that marked increases in apoB and LDL resulted in atherosclerotic lesions extending down the aorta which resemble human lesions immunochemically and suggest that apo(a) associated with apoB and lipid may result in a more pro-atherogenic state than when apo(a) is free in plasma.
M J Callow, J Verstuyft, R Tangirala, W Palinski, E M Rubin
The obese (ob) gene has recently been isolated through a positional cloning approach, the mutation of which causes a marked hereditary obesity and diabetes mellitus in mice. In the present study, we isolated rat ob cDNA and examined the tissue distribution of the ob gene expression in rats. We also studied the gene expression in genetically obese Zucker fatty (fa/fa) rats. The rat ob gene product, a 167 amino acid protein with a putative signal sequence, was 96 and 83% homologous to the mouse and human ob proteins, respectively. Northern blot analysis using the rat ob cDNA probe identified a single mRNA species of 4.5 kb in size in the adipose tissue, while no significant amount of ob mRNA was present in other tissues in rats. The ob gene was expressed in the adipose tissue with region specificities. The rank order of the ob mRNA level in the adipose tissue was epididymal, retroperitoneal, and pericardial white adipose tissue > mesenteric and subcutaneous white adipose tissue > or = interscapular brown adipose tissue. The ob gene expression occurred in mature adipocytes rather than in stromalvascular cells isolated from the rat adipose tissue. Expression of the ob gene was markedly augmented in all the adipose tissue examined in Zucker fatty (fa/fa) rats at the stage of established obesity. The present study leads to the better understanding of the physiologic and pathophysiologic roles of the ob gene.
Y Ogawa, H Masuzaki, N Isse, T Okazaki, K Mori, M Shigemoto, N Satoh, N Tamura, K Hosoda, Y Yoshimasa
Electron spin resonance (ESR) spectroscopy has been used to investigate hydroxyl radical generation in rats with chronic dietary iron loading. A secondary radical spin-trapping technique was used where hydroxyl radical forms methyl radical upon reaction with DMSO. The methyl radical was then detected by ESR spectroscopy as its adduct with the spin trap alpha-phenyl-N-t-butylnitrone (PBN). This adduct was detected in the bile of rats 10 wk after being fed an iron-loading diet and 40 min after the i.p. injection of the spin trap PBN dissolved in DMSO. Bile samples were collected into a solution of the ferrous stabilizing chelator 2,2'-dipyridyl in order to prevent the generation of radical adducts ex vivo during bile collection. Identification of the ESR spectrum of the major radical adduct as that of PBN/.CH3 provides evidence for the generation of the hydroxyl radical during iron supplementation. Desferal completely inhibited in vivo hydroxyl radical generation stimulated by high dietary iron intake. No radical adducts were detected in rats which were fed the control diet for the same period of time. This is the first evidence of hydroxyl radical generation in chronic iron-loaded rats.
M B Kadiiska, M J Burkitt, Q H Xiang, R P Mason
The mutant gene responsible for obesity in the ob/ob mouse was recently identified by positional cloning (Zhang Y., R. Proenca, M. Maffel, M. Barone, L. Leopold, and J.M. Friedman. 1994. Nature (Lond.) 372:425). The encoded protein and to represent and "adipostat" signal reflecting the state of energy stores. We confirm that the adipocyte is the source of ob mRNA and that the predicted 16-kD ob protein is present in rodent serum as detected by Western blot. To evaluate the hypothesis that it might represent an adipostat, we assessed serum levels of ob protein and expression of ob mRNA in adipose cells and tissue of rodents in response to a variety of perturbations which effect body fat mass. Both ob protein and ob mRNA expression are markedly increased in obesity. The levels of ob protein are approximately 5-10-fold elevated in serum of db/db mice, in mice with hypothalamic lesions caused by neonatal administration of monosodium glutamate (MSG), and in mice with toxigene induced brown fat ablation, (UCP-DTA). Very parallel changes are observed in adipocyte ob mRNA expression in these models and in ob/ob mice. As predicted however, no serum ob protein could be detected in the ob/ob mice. By contrast to obesity, starvation of normal rats and mice for 1-3 d markedly suppresses ob mRNA abundance, and this is reversed with refeeding. Similarly, ob protein concentration in normal mice falls to undetectable levels with starvation. In the ob/ob, UCP-DTA and MSG models, overexpression of ob mRNA is reversed by caloric restriction. These data support the hypothesis that expression of ob mRNA and protein are regulated as a function of energy stores, and that ob serves as a circulating feedback signal to sites involved in regulation of energy homeostasis.
R C Frederich, B Löllmann, A Hamann, A Napolitano-Rosen, B B Kahn, B B Lowell, J S Flier
A polymorphism of the CETP gene (CETP/TaqIB) with two alleles B1 (60%) and B2 (40%) has been investigated in relation to lipid variables and the risk of myocardial infarction in a large case-control study (ECTIM) of men aged 25-64. No association was observed between the polymorphism and LDL or VLDL related lipid variables. Conversely, B2 carriers had reduced levels of plasma CETP (P < 0.0001) and increased levels of HDL cholesterol (P < 0.0001) and of other HDL related lipid variables. The effects of the polymorphism on plasma CETP and HDL cholesterol were independent, suggesting the presence of at least two functional variants linked to B2. A search for these variants on the coding sequence of the CETP gene failed to identify them. The effect of B2 on plasma HDL cholesterol was absent in subjects drinking < 25 grams/d of alcohol but increased commensurably, with higher values of alcohol consumption (interaction: P < 0.0001). A similar interaction was not observed for plasma CETP. The odds-ratio for myocardial infarction of B2 homozygotes decreased from 1.0 in nondrinkers to 0.34 in those drinking 75 grams/d or more. These results provide the first demonstration of a gene-environment interaction affecting HDL cholesterol levels and coronary heart disease risk.
F Fumeron, D Betoulle, G Luc, I Behague, S Ricard, O Poirier, R Jemaa, A Evans, D Arveiler, P Marques-Vidal
Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin, which are derived from proadrenomedullin, exhibit remarkable hypotensive action. We investigated the effect of PAMP and adrenomedullin on peripheral sympathetic neutral transmission. Using perfused rat mesenteric arteries, PAMP (0, 1, 5, and 10 pmol/ml) decreased norepinephrine overflow by periarterial electrical nerve stimulation in a dose-dependent fashion (0.244 +/- 0.043, 0.231 +/- 0.048, 0.195 +/- 0.061 and 0.168 +/- 0.051 ng/gram tissue weigh: NS, P < 0.05, and P < 0.02, respectively). In contrast to PAMP, adrenomedullin (1, 5, and 10 pmol/ml) did not change it. In contrast, vasoconstrictive response of mesenteric arteries to exogenous norepinephrine was significantly attenuated by 10 pmol/ml of adrenomedullin but not by the same dose of PAMP. Calcitonin gene-related peptide (8-37) [CGRP(8-37)], a CGRP receptor antagonist, inhibited the vasodilatory effect of adrenomedullin but could not suppress the sympathoinhibitory effect of PAMP. Neither a nicotinic antagonist, hexamethonium, nor a presynaptic alfa2 antagonist, yohimbine, blocked the sympathoinhibitory effect of PAMP. Thus, it suggests that PAMP and adrenomedullin, which are derived from the same gene, exhibit different hypotensive mechanisms: PAMP inhibits neural transmission at peripheral sympathetic nerve ending, although adrenomedullin directly dilates vascular smooth muscle, possibly through CGRP-like receptor.
T Shimosawa, Y Ito, K Ando, K Kitamura, K Kangawa, T Fujita
HIV-infected patients are defective in their ability to produce interleukin (IL)-12 in vitro in response to pathogenic bacteria and parasites. IL-12 enhances the patient's depressed natural killer cell cytotoxic activity, peripheral blood lymphocyte production of interferon-gamma (IFN-gamma), and proliferative T cell response in vitro to recall antigens, HIV antigens, alloantigens, and mitogens. However, these effects represent short-lived responses and imply the need for chronic IL-12 therapeutic administration in the clinical setting. To identify any long-term effects of IL-12 on T cell differentiation toward Th1 cells, peripheral blood T cells from 10 HIV-infected patients at different stages of disease were cloned by limiting dilution in the presence or absence of IL-12 and tested for cytokine production in response to stimulation with anti-CD3 antibodies and phorbol diesters IL-12 present during the first 2 wk of clonal expansion determined a stable severalfold enhancement on the ability of both CD4+ and CD8+ clones to produce IFN-gamma. Because priming for high IFN-gamma production is probably the most important mechanism by which IL-12 induces generation of efficient T helper type 1 (Th1) cells, these results suggest the possibility that IL-12 treatment in vivo of HIV-infected patients may stimulate a protective Th1 response against opportunistic pathogens and possibly HIV itself.
C Paganin, I Frank, G Trinchieri
Thrombopoietin (TPO), the ligand for the receptor protooncogene c-mpl, has been cloned and shown to be the critical regulator of platelet production. Several features of c-Mpl expression, including its presence on erythroid cell lines, and the panmyeloid transformation characteristic of myeloproliferative leukemia (MPL) viral disease led us to investigate whether this receptor-ligand system may play a role in erythropoiesis. We report that although TPO alone did not support the growth of either early or late erythroid progenitors, it acted in synergy with erythropoietin to expand these populations. Moreover, while the effects on erythropoiesis in normal animals were modest, TPO greatly expanded the number of erythroid progenitors and blood reticulocytes and was associated with accelerated red cell recovery in myelosuppressed mice. Together, these data strongly suggest that erythroid progenitors respond to TOP and that this newly cloned cytokine, critical for platelet production, can augment erythropoiesis in states of marrow failure.
K Kaushansky, V C Broudy, A Grossmann, J Humes, N Lin, H P Ren, M C Bailey, T Papayannopoulou, J W Forstrom, K H Sprugel
Lysophosphatidylcholine (lysoPC), a component of atherogenic lipoproteins and atherosclerotic lesions, has been recently suggested to play a role in atherogenesis. LysoPC is known to induce several endothelial genes involved in leukocyte recruitment, mitogenesis, and inflammation. Cyclooxygenases (prostaglandin H2 synthases) are rate-limiting enzymes involved in the endothelial synthesis of prostacyclin, an antiplatelet, vasorelaxant, and vasoprotective molecule. We investigated the effect of lysoPC on the endothelial expression of cyclooxygenases. Our results demonstrate that, in cultured human umbilical vein endothelial cells, lysoPC induces cyclooxygenase-2 mRNA and protein levels. Increased expression of cyclooxygenase-2 is accompanied by the enhancement of both basal- and calcium ionophore A23187-induced synthesis of prostacyclin. Nuclear runoff experiments demonstrated an increased rate of transcription of the cyclooxygenase-2 gene by lysoPC. In contrast, lysoPC did not affect the expression of constitutive cyclooxygenase-1. Our results suggest that the induction of endothelial cyclooxygenase-2 by lysoPC may be an important vasoprotective mechanism that limits progression of atherosclerotic lesions and promotes their regression.
A Zembowicz, S L Jones, K K Wu