LD78α and LD78β are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1α (MIP-1α). Two molecular forms of natural LD78β (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78α and LD78β only differ in 3 amino acids, both LD78β variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78α. On the contrary, LD78β was only 2-fold more efficient than LD78α in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor–transfected cells, both molecular forms of LD78β proved to be much more potent than LD78α in inducing an intracellular calcium rise through CCR5. Compared with LD78α and RANTES, this preferential binding of LD78β to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78β is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.
Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse — an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.
Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the nuclear hormone receptor superfamily originally shown to play a critical role in adipocyte differentiation and glucose homeostasis, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. Colonic epithelial cells, which express high levels of PPAR-γ protein, have the ability to produce inflammatory cytokines that may play a role in inflammatory bowel disease (IBD). We report here that PPAR-γ ligands dramatically attenuate cytokine gene expression in colon cancer cell lines by inhibiting the activation of nuclear factor-κB via an IκB-α–dependent mechanism. Moreover, thiazolidinedione ligands for PPAR-γ markedly reduce colonic inflammation in a mouse model of IBD. These results suggest that colonic PPAR-γ may be a therapeutic target in humans suffering from IBD.
Cardiac hypertrophy often presages the development of heart failure. Numerous cytosolic signaling pathways have been implicated in the hypertrophic response in cardiomyocytes in culture, but their roles in the hypertrophic response to physiologically relevant stimuli in vivo is unclear. We previously reported that adenovirus-mediated gene transfer of SEK-1(KR), a dominant inhibitory mutant of the immediate upstream activator of the stress-activated protein kinases (SAPKs), abrogates the hypertrophic response of neonatal rat cardiomyocytes to endothelin-1 in culture. We now report that gene transfer of SEK-1(KR) to the adult rat heart blocks SAPK activation by pressure overload, demonstrating that the activity of cytosolic signaling pathways can be inhibited by gene transfer of loss-of-function mutants in vivo. Furthermore, gene transfer of SEK-1(KR) inhibited pressure overload–induced cardiac hypertrophy, as determined by echocardiography and several postmortem measures including left ventricular (LV) wall thickness, the ratio of LV weight to body weight, cardiomyocyte diameter, and inhibition of atrial natriuretic factor expression. Our data suggest that the SAPKs are critical regulators of cardiac hypertrophy in vivo, and therefore may serve as novel drug targets in the treatment of hypertrophy and heart failure.
Parathyroid hormone (PTH) and parathyroid hormone–related peptide (PTHrP) bind to and activate the same PTH/PTHrP receptor. Deletion of either the PTHrP gene or the PTH/PTHrP receptor gene leads to acceleration of differentiation of growth plate chondrocytes. To explore further the functional relationships of PTHrP and the PTH/PTHrP receptor, bones of knockout mice were analyzed early in development, and the phenotypes of double-knockout mice were characterized.
Fcγ receptors on the surface of phagocytic cells bind the Fc region of IgG and mediate binding, phagocytosis, and destruction of particulate antigens opsonized by the antigen-specific IgG molecule. The present study evaluates the feasibility of converting lung epithelial cells into phagocytic cells using adenovirus (Ad) vector–mediated gene transfer of FcγRIIA cDNA to induce expression of the human FcγRIIA receptor. Binding and phagocytosis of opsonized sheep red blood cells (SRBCs) by the A549 human lung epithelial cell line after Ad-mediated FcγRIIA gene transfer was demonstrated using light and fluorescence microscopy and phagocytic assays with 51Cr-labeled SRBCs. When A549 cells were infected with an Ad vector expressing a FcγRIIA mutant in which 2 of 3 cytoplasmic tyrosines have been replaced with phenylalanine, only binding, but not phagocytosis, of opsonized SRBCs was observed. In vivo expression of FcγRIIA in the lung after intratracheal administration of the AdFcγRIIA enhanced clearance of opsonized Pseudomonas aeruginosa from the lung in normal rats and in mice deficient in Fcγ receptor expression. Similar results were observed with a chimeric FcγRIIA construct containing the extracellular domain of FcγRIIIA. Together, these data demonstrate that Ad-mediated FcγRIIA receptor cDNA expression can mediate the binding and phagocytosis of opsonized particulate antigens by normally nonphagocytic cells, suggesting that gene-transfer strategies might be used to utilize nonphagocytic cells to clear bacteria or other opsonized particulate antigens from the respiratory tract.
To identify the mechanisms that cause monocyte localization in infarcted myocardium, we studied the impact of ischemia-reperfusion injury on the surface expression and function of the monocyte fibronectin (FN) receptor VLA-5 (α5β1 integrin, CD49e/CD29). Myocardial infarction was associated with the release of FN fragments into cardiac extracellular fluids. Incubating monocytes with postreperfusion cardiac lymph that contained these FN fragments selectively reduced expression of VLA-5, an effect suppressed by specific immunoadsorption of the fragments. Treating monocytes with purified, 120-kDa cell-binding FN fragments (FN120) likewise decreased VLA-5 expression, and did so by inducing a serine proteinase–dependent proteolysis of this β1 integrin. We postulated that changes in VLA-5 expression, which were induced by interactions with cell-binding FN fragments, may alter monocyte migration into tissue FN, a prominent component of the cardiac extracellular matrix. Support for this hypothesis came from experiments showing that FN120 treatment significantly reduced both spontaneous and MCP-1–induced monocyte migration on an FN-impregnated collagen matrix. In vivo, it is likely that contact with cell-binding FN fragments also modulates VLA-5/FN adhesive interactions, and this causes monocytes to accumulate at sites where the fragment concentration is sufficient to ensure proteolytic degradation of VLA-5.
Mannose-binding lectin (MBL) is a key factor in innate immunity, and lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Accordingly, we investigated whether MBL variant alleles, which are associated with recurrent infections, might be risk factors for CF patients. In 149 CF patients, different MBL genotypes were compared with respect to lung function, microbiology, and survival to end-stage CF (death or lung transplantation). The lung function was significantly reduced in carriers of MBL variant alleles when compared with normal homozygotes. The negative impact of variant alleles on lung function was especially confined to patients with chronic Pseudomonas aeruginosa infection. Burkholderia cepacia infection was significantly more frequent in carriers of variant alleles than in homozygotes. The risk of end-stage CF among carriers of variant alleles increased 3-fold, and the survival time decreased over a 10-year follow-up period. Moreover, by using a modified life table analysis, we estimated that the predicted age of survival was reduced by 8 years in variant allele carriers when compared with normal homozygotes. Presence of MBL variant alleles is therefore associated with poor prognosis and early death in patients with CF.
The mass of regenerating tissues, such as bone, is critically dependent on the number of executive cells, which in turn is determined by the rate of replication of progenitors and the life-span of mature cells, reflecting the timing of death by apoptosis. Bone mass can be increased by intermittent parathyroid hormone (PTH) administration, but the mechanism of this phenomenon has remained unknown. We report that daily PTH injections in mice with either normal bone mass or osteopenia due to defective osteoblastogenesis increased bone formation without affecting the generation of new osteoblasts. Instead, PTH increased the life-span of mature osteoblasts by preventing their apoptosis — the fate of the majority of these cells under normal conditions. The antiapoptotic effect of PTH was sufficient to account for the increase in bone mass, and was confirmed in vitro using rodent and human osteoblasts and osteocytes. This evidence provides proof of the basic principle that the work performed by a cell population can be increased by suppression of apoptosis. Moreover, it suggests novel pharmacotherapeutic strategies for osteoporosis and, perhaps, other pathologic conditions in which tissue mass diminution has compromised functional integrity.
Both insulin resistance and hyperinsulinemia have been reported to be independent risk factors for cardiovascular diseases. However, little is known regarding insulin signaling in the vascular tissues in insulin-resistant states. In this report, insulin signaling on the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways were compared in vascular tissues of lean and obese Zucker (fa/fa) rats in both ex vivo and in vivo studies. Ex vivo, insulin-stimulated tyrosine phosphorylation of insulin receptor β subunits (IRβ) in the aorta and microvessels of obese rats was significantly decreased compared with lean rats, although the protein levels of IRβ in the 2 groups were not different. Insulin-induced tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and their protein levels were decreased in the aorta of obese rats compared with lean rats. The association of p85 subunit to the IRS proteins and the IRS-associated PI 3-kinase activities stimulated by insulin in the aorta of obese rats were significantly decreased compared with the lean rats. In addition, insulin-stimulated serine phosphorylation of Akt, a downstream kinase of PI 3-kinase pathway, was also reduced significantly in isolated microvessels from obese rats compared with the lean rats. In euglycemic clamp studies, insulin infusion greatly increased tyrosine phosphorylation of IRβ- and IRS-2–associated PI 3-kinase activity in the aorta of lean rats, but only slight increases were observed in obese rats. In contrast, insulin stimulated tyrosine phosphorylation of MAP kinase (ERK-1/2) equally in isolated microvessels of lean and obese rats, although basal tyrosine phosphorylation of ERK-1/2 was higher in the obese rats. To our knowledge, these data provided the first direct measurements of insulin signaling in the vascular tissues, and documented a selective resistance to PI 3-kinase (but not to MAP kinase pathway) in the vascular tissues of obese Zucker rats.
We demonstrate an increase in graft-versus-host disease (GVHD) after experimental bone marrow transplant (BMT) when cyclophosphamide (Cy) is added to an otherwise well-tolerated dose (900 cGy) of total body irradiation (TBI). Donor T cell expansion on day +13 was increased after conditioning with Cy/TBI compared with Cy or TBI alone, although cytotoxic T lymphocyte (CTL) function was not altered. Histological analysis of the gastrointestinal tract demonstrated synergistic damage by Cy/TBI and allogeneic donor cells, which permitted increased translocation of LPS into the systemic circulation. TNF-α and IL-1 production in response to LPS was increased in BMT recipients after Cy/TBI conditioning. Neutralization of IL-1 significantly reduced serum LPS levels and GVHD mortality, but it did not affect donor CTL activity. By contrast, neutralization of TNF-α did not prevent GVHD mortality but did impair CTL activity after BMT. When P815 leukemia cells were added to the bone marrow inoculum, allogeneic BMT recipients given the TNF-α inhibitor relapsed at a significantly faster rate than those given the IL-1 inhibitor. To confirm that the role of TNF-α in graft versus leukemia (GVL) was due to effects on donor T cells, cohorts of animals were transplanted with T cells from either wild-type mice or p55 TNF-α receptor–deficient mice. Recipients of TNF-α p55 receptor–deficient T cells demonstrated a significant impairment in donor CTL activity after BMT and an increased rate of leukemic relapse compared with recipients of wild-type T cells. These data highlight the importance of conditioning in GVHD pathophysiology, and demonstrate that TNF-α is critical to GVL mediated by donor T cells, whereas IL-1 is not.
Multiple mutations in cardiac troponin T (cTnT) can cause familial hypertrophic cardiomyopathy (FHC). Patients with cTnT mutations generally exhibit mild or no ventricular hypertrophy, yet demonstrate a high frequency of early sudden death. To understand the functional basis of these phenotypes, we created transgenic mouse lines expressing 30%, 67%, and 92% of their total cTnT as a missense (R92Q) allele analogous to one found in FHC. Similar to a mouse FHC model expressing a truncated cTnT protein, the left ventricles of all R92Q lines are smaller than those of wild-type. In striking contrast to truncation mice, however, the R92Q hearts demonstrate significant induction of atrial natriuretic factor and β-myosin heavy chain transcripts, interstitial fibrosis, and mitochondrial pathology. Isolated cardiac myocytes from R92Q mice have increased basal sarcomeric activation, impaired relaxation, and shorter sarcomere lengths. Isolated working heart data are consistent, showing hypercontractility and diastolic dysfunction, both of which are common findings in patients with FHC. These mice represent the first disease model to exhibit hypercontractility, as well as a unique model system for exploring the cellular pathogenesis of FHC. The distinct phenotypes of mice with different TnT alleles suggest that the clinical heterogeneity of FHC is at least partially due to allele-specific mechanisms.
Renal proximal tubule cells express in their apical brush border membrane (BBM) a Na/Pi cotransporter type IIa that is rapidly downregulated in response to parathyroid hormone (PTH). We used the rat renal Na/Pi cotransporter type IIa (NaPi-2) as an in vivo model to assess early cellular events in the rapid downregulation of this transporter. When rats were treated with PTH for 15 minutes, NaPi-2 abundance in the BBM was decreased. In parallel, transporter accumulated in intracellular vesicles. Concomitantly, microtubules (MTs) were found to form dense bundles of apical-to-basal orientation. After 60 minutes of PTH action, the cells were vastly depleted of NaPi-2, whereas their microtubular cytoskeleton had returned to its normal appearance. Prevention of MT rearrangement by taxol resulted in accumulation of NaPi-2 in the subapical cell portion after 15 minutes and a strong delay in depletion of intracellular transporter after 60 minutes of PTH action. Furthermore, the subapical accumulation of NaPi-2 was associated with the expansion of dense apical tubules of the subapical endocytic apparatus (SEA). Depolymerization of MTs by colchicine likewise caused a retardation of intracellular NaPi-2 depletion. These results suggest that NaPi-2 is downregulated in response to PTH through a rapid endocytic process in 2 separate steps: (a) internalization of the transporter into the SEA, and (b) its delivery to degradative organelles by a trafficking mechanism whose efficiency depends on a taxol-sensitive rearrangement of MTs.
Endothelial responses may contribute importantly to the pathology of high vascular pressure. In lung venular capillaries, we determined endothelial [Ca2+]i by the fura-2 ratioing method and fusion pore formation by quantifying the fluorescence of FM1-43. Pressure elevation increased endothelial [Ca2+]i. Concomitantly evoked exocytotic events were evident in a novel spatial-temporal pattern of fusion pore formation. Fusion pores formed predominantly at vascular branch points and colocalized with the expression of P-selectin. Blockade of mechanogated Ca2+ channels inhibited these responses, identifying entry of external Ca2+ as the critical triggering mechanism. These endothelial responses point to a proinflammatory effect of high vascular pressure that may be relevant in the pathogenesis of pressure-induced lung disease.
Central to the bone-sparing effect of estrogen (E2) is its ability to block the monocytic production of the osteoclastogenic cytokine TNF-α (TNF). However, the mechanism by which E2 downregulates TNF production is presently unknown. Transient transfection studies in HeLa cells, an E2 receptor–negative line, suggest that E2 inhibits TNF gene expression through an effect mediated by estrogen receptor β (ERβ). We also report that in RAW 264.7 cells, an E2 receptor–positive murine monocytic line, E2 downregulates cytokine-induced TNF gene expression by decreasing the activity of the Jun NH2-terminal kinase (JNK). The resulting diminished phosphorylation of c-Jun and JunD at their NH2-termini decreases the ability of these nuclear proteins to autostimulate the expression of the c-Jun and JunD genes, thus leading to lower production of c-Jun and JunD. The consequent decrease in the nuclear levels of c-Jun and JunD leads to diminished binding of c-Jun/c-Fos and JunD/c-Fos heterodimers to the AP-1 consensus sequence in the TNF promoter and, thus, to decreased transactivation of the TNF gene.
Prostaglandins (PGs) are lipid molecules that profoundly affect cellular processes including inflammation and immune response. Pathways contributing to PG output are highly regulated in antigen-presenting cells such as macrophages and monocytes, which produce large quantities of these molecules upon activation. In this report, we demonstrate aberrant constitutive expression of the normally inducible cyclooxygenase PG synthase 2 (PGS2/ COX-2) in nonactivated monocytes of humans with insulin-dependent diabetes mellitus (IDDM) and those with islet autoantibodies at increased risk of developing this disease. Constitutive PGS2 appears to characterize a high risk for diabetes as it correlates with and predicts a low first-phase insulin response in autoantibody-positive subjects. Abnormal PGS2 expression in at-risk subjects affected immune response in vitro, as the presence of a specific PGS2 inhibitor, NS398, significantly increased IL-2 receptor α-chain (CD25) expression on phytohemagglutinin-stimulated T cells. The effect of PGS2 on CD25 expression was most profound in subjects expressing both DR04 and DQβ0302 high-risk alleles, suggesting that this cyclooxygenase interacts with diabetes-associated MHC class II antigens to limit T-cell activation. These results indicate that constitutive PGS2 expression in monocytes defines an antigen-presenting cell defect affecting immune response, and that this expression is a novel cell-associated risk marker for IDDM.
Copyright © 2014 American Society for Clinical Investigation