Interferon-gamma receptor ligand-binding chain (IFN-gammaR1) or signaling chain (IFN-gammaR2) deficiency, like interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency, predispose to severe infections due to poorly virulent mycobacteria and salmonella. A child with bacille Calmette-Guérin and Salmonella enteritidis infection was investigated. Mutations in the genes for IFN-gammaR1, IFN-gammaR2, IL-12Rbeta1, and other molecules implicated in IL-12- or IFN-gamma-mediated immunity were sought. A large homozygous deletion within the IL-12 p40 subunit gene was found, precluding expression of functional IL-12 p70 cytokine by activated dendritic cells and phagocytes. As a result, IFN-gamma production by lymphocytes was markedly impaired. This is the first discovered human disease resulting from a cytokine gene defect. It suggests that IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella.
F Altare, D Lammas, P Revy, E Jouanguy, R Döffinger, S Lamhamedi, P Drysdale, D Scheel-Toellner, J Girdlestone, P Darbyshire, M Wadhwa, H Dockrell, M Salmon, A Fischer, A Durandy, Jean-Laurent Casanova, D S Kumararatne
HIV-infected patients suffer several renal syndromes, which can progress rapidly from renal insufficiency to end-stage renal disease. Histologically, HIV-induced nephropathy is characterized by prominent tubulopathy with apoptosis of tubular cells. Clinical and experimental evidence suggests that renal injury may be directly related to virus infection. Although HIV-1 is a polytropic and not solely lymphotropic pathogen, the susceptibility of renal cells to HIV-1 remains to be determined. This paper demonstrates in vitro the permissiveness of proximal tubular epithelial cells (PTEC) to HIV-1 and describes the effects of PTEC infection to explain the pathogenesis of tubular damage in vivo. The results indicate that PTEC express HIV-specific receptor and coreceptors and sustain virus replication. We observed that HIV-1 infection causes the death of tubular cells by triggering an apoptotic pathway involving caspase activation. Fas upregulation but not Fas ligand expression was found in the infected PTEC. However, after HIV-1 infection, tubular cells became susceptible to apoptosis induced through Fas stimulation. Caspase inhibition prevented the death of the infected PTEC in spite of persistent viral replication. These findings may explain the prominent histopathology of HIV-associated nephropathy and demonstrate that the apoptosis of nonlymphoid cells can be directly induced by HIV-1.
P G Conaldi, L Biancone, A Bottelli, A Wade-Evans, L C Racusen, M Boccellino, V Orlandi, C Serra, G Camussi, A Toniolo
Feedback regulation of cholesterol biosynthesis is mediated by membrane-bound transcription factors designated sterol regulatory element-binding proteins (SREBP)-1 and -2. In sterol-deprived cultured cells, SREBPs are released from membranes by a proteolytic process that is stimulated by SREBP cleavage-activating protein (SCAP), a membrane protein containing a sterol-sensing domain. Sterols suppress SREBP cleavage by blocking the action of SCAP, thereby decreasing cholesterol synthesis. A point mutation in SCAP(D443N) causes resistance to sterol suppression. In this article, we produced transgenic mice that express mutant SCAP(D443N) in liver. In these livers the nuclear content of SREBP-1 and -2 was increased, mRNAs encoding proteins involved in uptake and synthesis of cholesterol and fatty acids were elevated, and the livers were engorged with cholesteryl esters and triglycerides enriched in monounsaturated fatty acids. When the mice were challenged with a high cholesterol diet, cleavage of SREBP-1 and -2 was reduced in wild-type livers and less so in transgenic livers. We conclude that SCAP(D443N) stimulates proteolytic processing of native SREBPs in liver and decreases the normal sterol-mediated feedback regulation of SREBP cleavage, suggesting a central role for SCAP as a sterol sensor in liver.
B S Korn, I Shimomura, Y Bashmakov, R E Hammer, J D Horton, J L Goldstein, M S Brown
Nitric oxide (NO), constitutively produced by endothelial nitric oxide synthase (eNOS), plays a major role in the regulation of blood pressure and vascular tone. We generated transgenic mice overexpressing bovine eNOS in the vascular wall using murine preproendothelin-1 promoter. In transgenic lineages with three to eight transgene copies, bovine eNOS-specific mRNA, protein expression in the particulate fractions, and calcium-dependent NOS activity were confirmed by RNase protection assay, immunoblotting, and L-arginine/citrulline conversion. Immunohistochemical studies revealed that eNOS protein was predominantly localized in the endothelial cells of aorta, heart, and lung. Blood pressure was significantly lower in eNOS-overexpressing mice than in control littermates. In the transgenic aorta, basal NO release (estimated by Nomega-nitro-L-arginine-induced facilitation of the contraction by prostaglandin F2alpha) and basal cGMP levels (measured by enzyme immunoassay) were significantly increased. In contrast, relaxations of transgenic aorta in response to acetylcholine and sodium nitroprusside were significantly attenuated, and the reduced vascular reactivity was associated with reduced response of cGMP elevation to these agents as compared with control aortas. Thus, our novel mouse model of chronic eNOS overexpression demonstrates that, in addition to the essential role of eNOS in blood pressure regulation, tonic NO release by eNOS in the endothelium induces the reduced vascular reactivity to NO-mediated vasodilators, providing several insights into the pathogenesis of nitrate tolerance.
Y Ohashi, S Kawashima, K i Hirata, T Yamashita, T Ishida, N Inoue, T Sakoda, H Kurihara, Y Yazaki, M Yokoyama
Although crucial to mucosal vaccine development, the mechanisms of defense against mucosal viral infection are still poorly understood. Protection, cytotoxic T lymphocytes (CTL), and neutralizing antibodies have all been observed, but cause and effect have been difficult to determine. The ability of CTL in the mucosa to mediate protection against mucosal viral transmission has never been proven. Here, we use an HIV peptide immunogen and an HIV-1 gp160-expressing recombinant vaccinia viral intrarectal murine challenge system, in which neutralizing antibodies do not play a role, to demonstrate for the first time that long-lasting immune resistance to mucosal viral transmission can be accomplished by CD8(+) CTL that must be present in the mucosal site of exposure. The resistance is ablated by depleting CD8(+) cells in vivo and requires CTL in the mucosa, whereas systemic (splenic) CTL are shown to be unable to protect against mucosal challenge. Furthermore, the resistance as well as the CTL response can be increased by local mucosal delivery of IL-12 with the vaccine. These results imply that induction of local mucosal CTL may be critical for success of a vaccine against viruses transmitted through a mucosal route, such as HIV.
I M Belyakov, J D Ahlers, B Y Brandwein, P Earl, B L Kelsall, B Moss, W Strober, J A Berzofsky
Antibodies against the extracellular domain of bullous pemphigoid antigen 2 (BPAG2) are thought to play a key role in the pathogenesis of bullous pemphigoid (BP), the most frequent autoimmune bullous disease of the skin. Autoreactive T cell responses to BPAG2 were investigated in 16 BP patients and 24 healthy controls by coculture of PBMC with two recombinant BPAG2 proteins (extracellular domain of BPAG2). Primary in vitro T cell responses to BPAG2 were observed in 10/12 BP patients expressing the BP-associated HLA-DQB1*0301 allele and 8/10 DQB1*0301 positive healthy individuals. DQB1*0301 also restricted three autoreactive T cell lines from two BP patients and a healthy donor. In contrast, PBMC from 14 normal patients carrying HLA class II alleles other than DQB1*0301 were not stimulated by BPAG2. Autoreactive BPAG2-specific CD4(+) T cell lines and clones from five BP patients produced both Th1 and Th2 cytokines, whereas three autoreactive T cell lines from three DQB1*0301 positive normal patients produced exclusively IFN-gamma. The absence of BPAG2-specific Th2 cells in healthy individuals strongly suggests that autoreactive Th2 responses to BPAG2 are restricted to BP patients and may thus be critical in the pathogenesis of BP.
L Büdinger, L Borradori, C Yee, R Eming, S Ferencik, H Grosse-Wilde, H F Merk, K Yancey, M Hertl
Defects of the mitochondrial genome are important causes of disease. Despite major advances in our investigation of patients, there is no effective therapy. Progress in this area is limited by the absence of any animal models in which we can evaluate treatment. To develop such a model we have injected human myoblasts into the tibialis anterior of SCID mice after inducing necrosis. After injection of normal human myoblasts, regenerating fibers expressed human beta-spectrin, confirming they were derived from fusion of human myoblasts. The stability of the muscle fibers was inferred by demonstrating the formation of motor end plates on the regenerating fibers. In addition, we show the presence of human cytochrome c oxidase subunit II, which is encoded by the mitochondrial genome, in the regenerated fibers. After injection of human myoblasts containing either the A8344G or the T8993C heteroplasmic mitochondrial DNA mutations, human beta-spectrin positive fibers were found to contain the mutation at a similar level to the injected myoblasts. These studies highlight the potential value of this model for the study of mitochondrial DNA defects.
K M Clark, D J Watt, R N Lightowlers, M A Johnson, J B Relvas, J W Taanman, D M Turnbull
Because monoclonal antibodies (mAbs) directed against alpha4-integrin and VCAM-1 inhibit the development of experimental autoimmune encephalomyelitis (EAE) in vivo, it has been concluded that the successful therapeutic effect is due to interference with alpha4beta1/VCAM-1-mediated interaction of autoaggressive T cells with the blood-brain barrier. A possible role for alpha4beta7-integrin, or interference with other T cell mediated events during the pathogenesis of EAE, has not been considered. We have compared the effects of mAb therapy on the development of EAE in the SJL/N mouse, using a large panel of mAbs directed against alpha4, beta7, the alpha4beta7-heterodimer, and against VCAM-1. Although encephalitogenic T cells express both alpha4-integrins, mAbs directed against the alpha4beta7-heterodimer or against the beta7-subunit did not interfere with the development of EAE. In contrast, mAbs directed against alpha4 and VCAM-1 inhibited or diminished clinical or histopathological signs of EAE. Our data demonstrate for the first time that alpha4beta7 is not essential for the development of EAE. Furthermore, our in vitro studies suggest that the therapeutic effect of anti-alpha4-treatment of EAE might also be caused by inhibition of antigen-specific T cell proliferation.
B Engelhardt, M Laschinger, M Schulz, U Samulowitz, D Vestweber, G Hoch
Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factors are regulated by binding proteins, but there is no information about binding proteins for BMPs in skeletal cells. Noggin specifically binds BMPs, but its expression by cells of the osteoblastic lineage has not been reported. We tested for the expression of noggin and its induction by BMP-2 in cultures of osteoblast-enriched cells from 22-d-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in noggin mRNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on noggin transcripts were dependent on protein, but independent of DNA synthesis. BMP-2 increased the rates of noggin transcription as determined by nuclear run-on assays. BMP-4, BMP-6, and TGF-beta1 increased noggin mRNA in Ob cells, but basic fibroblast growth factor, platelet- derived growth factor BB, and IGF-I did not. Noggin decreased the stimulatory effects of BMPs on DNA and collagen synthesis and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce noggin transcription in Ob cells, a probable mechanism to limit BMP action in osteoblasts.
E Gazzerro, V Gangji, E Canalis
There is evidence to suggest that the synthesis of type II collagen is increased in osteoarthritis (OA). Using an immunoassay, we show that the content of the C-propeptide of type II procollagen (CPII), released extracellularly from the newly synthesized molecule, is directly related to the synthesis of this molecule in healthy and osteoarthritic articular cartilages. In OA cartilage, CPII content is often markedly elevated (mean 7.6-fold), particularly in the mid and deep zones, reaching 29.6% of the content in newborn. Synthesis is also directly related to total collagen II content in OA, suggesting its importance in maintaining collagen content and cartilage structure. The release of CPII from cartilage is correlated directly with cartilage content. However, the increase in CPII in OA cartilage is not reflected in serum, where a significant reduction is observed. Together these studies provide evidence for alterations in procollagen II synthesis in vivo in patients with OA.
F Nelson, L Dahlberg, S Laverty, A Reiner, I Pidoux, M Ionescu, G L Fraser, E Brooks, M Tanzer, L C Rosenberg, P Dieppe, A Robin Poole
We have demonstrated that a single injection of interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits acute graft-versus-host disease (GVHD) in mice. This effect of IL-12 can be diminished by anti-interferon (IFN)-gamma mAb. To determine the mechanism by which IFN-gamma affects IL-12-mediated GVHD protection, we have compared the effect of IL-12 on GVHD in C57BL/6 wild-type (WT) or IFN-gamma gene knockout (GKO) recipients of fully major histocompatibility complex plus minor antigen-mismatched allogeneic BMT from WT or GKO BALB/c mice. Lethal acute GVHD was readily induced in the absence of IFN-gamma. IL-12 inhibited GVHD mortality to a similar extent in WT and GKO recipients of WT allogeneic BMT. However, neither WT nor GKO recipients were protected by IL-12 from GVHD induced by GKO allogeneic BMT. Moreover, the effective inhibition of host-reactive donor T cell activation and expansion that is associated with IL-12-mediated GVHD protection was dependent on the ability of BALB/c donors to produce IFN-gamma. These results demonstrate that (a) acute GVHD can be induced in the absence of IFN-gamma, (b) host IFN-gamma does not play a critical role in IL-12-induced GVHD protection, and (c) the protective effect of IL-12 against GVHD is dependent on the ability of the donor to produce IFN-gamma.
Y G Yang, B R Dey, J J Sergio, D A Pearson, M Sykes
The new neuropeptide Y (NPY) Y5 receptor antagonist CGP 71683A displayed high affinity for the cloned rat NPY Y5 subtype, but > 1, 000-fold lower affinity for the cloned rat NPY Y1, Y2, and Y4 subtypes. In LMTK cells transfected with the human NPY Y5 receptor, CGP 71683A was without intrinsic activity and antagonized NPY-induced Ca2+ transients. CGP 71683A was given intraperitoneally (dose range 1-100 mg/kg) to a series of animal models of high hypothalamic NPY levels. In lean satiated rats CGP 71683A significantly antagonized the increase in food intake induced by intracerebroventricular injection of NPY. In 24-h fasted and streptozotocin diabetic rats CGP 71683A dose-dependently inhibited food intake. During the dark phase, CGP 71683A dose-dependently inhibited food intake in free-feeding lean rats without affecting the normal pattern of food intake or inducing taste aversion. In free-feeding lean rats, intraperitoneal administration of CGP 71683A for 28 d inhibited food intake dose-dependently with a maximum reduction observed on days 3 and 4. Despite the return of food intake to control levels, body weight and the peripheral fat mass remained significantly reduced. The data demonstrate that the NPY Y5 receptor subtype plays a role in NPY-induced food intake, but also suggest that, with chronic blockade, counterregulatory mechanisms are induced to restore appetite.
L Criscione, P Rigollier, C Batzl-Hartmann, H Rüeger, A Stricker-Krongrad, P Wyss, L Brunner, S Whitebread, Y Yamaguchi, C Gerald, R O Heurich, M W Walker, M Chiesi, W Schilling, K G Hofbauer, N Levens
Chronic granulomatous disease (CGD) is an inherited disorder of phagocyte function in which defective superoxide production results in deficient microbicidal activity. CGD patients suffer from recurrent, life-threatening infections, and nearly half develop chronic gastrointestinal (GI) complications (colitis, gastric outlet obstruction, or perirectal abscess) and/or autoimmune/rheumatologic disorders (AIDs). To identify genetic modifiers of disease severity, we studied a cohort of 129 CGD patients, in whom seven candidate genes (myeloperoxidase [MPO], mannose binding lectin [MBL], Fcgamma receptors IIa, IIIa, IIIb, TNF-alpha, and IL-1 receptor antagonist), each containing a physiologically relevant polymorphism predicted to influence the host inflammatory response, were selected for analysis. Genotypes of MPO (P = 0.003) and FcgammaRIIIb (P = 0.007) were strongly associated with an increased risk for GI complications, while an FcgammaRIIa (P = 0.05) genotype was suggestive for an association. Patients with all three associated genotypes had the highest risk for GI complications (P < 0.0001). The risk of AIDs was strongly associated with variant alleles of MBL (P = 0.01) and weakly associated with an FcgammaRIIa genotype (P = 0.04). Patients with variant forms of both MBL and FcgammaRIIa had the highest risk of developing an AID (P = 0.003).
C B Foster, T Lehrnbecher, F Mol, S M Steinberg, D J Venzon, T J Walsh, D Noack, J Rae, J A Winkelstein, J T Curnutte, S J Chanock
Unstimulated peripheral blood mononuclear cells (PBMCs) from corticosteroid-resistant (CR) but not corticosteroid-sensitive (CS) asthmatics demonstrate increased activating peptide-1 (AP-1)- and decreased glucocorticoid receptor (GR)-DNA binding. We test whether these abnormalities are associated with excessive generation of c-fos, the inducible component of AP-1. The c-fos transcription rate, mRNA and protein levels, and GR-DNA binding were quantitated in PBMCs, T cells, and monocytes from CS, CR, and nonasthmatic subjects. There was a 1.7-, 4.2-, and 2.3-fold greater increase in the baseline c-fos transcription rate, mRNA expression, and protein levels, respectively, in PBMCs derived from CR compared with CS patients. At optimal stimulation with PMA, there was a 5.7-, 3.4-, and 2-fold greater increase in the c-fos transcription rate, mRNA accumulation, and protein levels, respectively, in CR compared with CS PBMCs. These abnormalities were detected in both the T cell and monocyte subpopulations. PMA stimulation converted PBMCs from a CS to a CR phenotype and was associated with direct interaction between c-Fos and the GR. Pretreatment of PBMCs from CR patients with c-fos antisense oligonucleotides enhanced GR-DNA binding activity in CR PBMCs stimulated with dexamethasone. We suggest that increased c-fos synthesis provides a major mechanism for the increased AP-1- and decreased GR- DNA binding seen in CR asthma.
S J Lane, I M Adcock, D Richards, C Hawrylowicz, P J Barnes, T H Lee
We have shown previously that the 5-lipoxygenase product 5-oxo-6,8, 11,14-eicosatetraenoic acid (5-oxo-ETE) is a highly potent eosinophil chemoattractant in vitro. To determine whether this substance can induce pulmonary eosinophil infiltration in vivo, it was administered to Brown Norway rats by tracheal insufflation. Eosinophils were then counted in lung sections that had been immunostained with an antibody to eosinophil major basic protein. 5-Oxo-ETE induced a dramatic increase in the numbers of eosinophils (ED50, 2.5 microg) around the walls of the airways, which reached maximal levels (five times control levels) between 15 and 24 h after administration, and then declined. LTB4 also induced pulmonary eosinophil infiltration with a similar ED50 but appeared to be somewhat less effective. In contrast, LTD4 and LTE4 were inactive. 5-Oxo-ETE-induced eosinophilia was unaffected by the LTB4 and PAF antagonists LY255283 and WEB 2170, respectively. However, it was inhibited by approximately 75% by monoclonal antibodies to CD49d (VLA-4) or CD11a (LFA-1) but was not significantly affected by an antibody to CD11b (Mac-1). In conclusion, 5-oxo-ETE induces pulmonary eosinophilia in Brown Norway rats, raising the possibility that it may be a physiological mediator of inflammation in asthma.
P Stamatiou, Q Hamid, R Taha, W Yu, T B Issekutz, J Rokach, S P Khanapure, W S Powell
The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport.
V S Tanphaichitr, A Sumboonnanonda, H Ideguchi, C Shayakul, C Brugnara, M Takao, G Veerakul, S L Alper