Issue published April 1, 2001

I n agreement with evidence that estrogens slow the rate of bone remodeling by suppressing the production of both osteoclasts and osteoblasts, loss of estrogens leads to an increase in the number of osteoclast as well as early osteoblast progenitors (CFU-osteoblasts; CFU-OBs) in the murine bone marrow. Here we show that CFU-OBs are early transit-amplifying progenitors, i.e., dividing cells capable of limited self-renewal, and that 17β-estradiol acts in vivo and in vitro to attenuate their self-renewal by approximately 50%. Consistent with a direct receptor–mediated action of estrogens on early mesenchymal cell progenitors, anti–estrogen receptor-α (anti-ERα) Ab’s stain a small number of marrow cells that exhibit characteristics of primitive undifferentiated cells, including a high nucleus/cytoplasm ratio and lack of lineage-specific biochemical markers; the effect of 17β-estradiol on CFU-OB self-renewal is absent in mice lacking ERα. Because both osteoblasts and the stromal/osteoblastic cells that are required for osteoclast development are derived from CFU-OBs, suppression of the self-renewal of this common progenitor may represent a key mechanism of the anti-remodeling effects of estrogens.

I nherited and acquired cardiomyopathies are associated with marked intracellular lipid accumulation in the heart. To test the hypothesis that mismatch between myocardial fatty acid uptake and utilization leads to the accumulation of cardiotoxic lipid species, and to establish a mouse model of metabolic cardiomyopathy, we generated transgenic mouse lines that overexpress long-chain acyl-CoA synthetase in the heart (MHC-ACS). This protein plays an important role in vectorial fatty acid transport across the plasma membrane. MHC-ACS mice demonstrate cardiac-restricted expression of the transgene and marked cardiac myocyte triglyceride accumulation. Lipid accumulation is associated with initial cardiac hypertrophy, followed by the development of left-ventricular dysfunction and premature death. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining and cytochrome c release in transgenic hearts suggest that cardiac myocyte death occurs, in part, by lipid-induced programmed cell death. Taken together, our data demonstrate that fatty acid uptake/utilization mismatch in the heart leads to accumulation of lipid species toxic to cardiac myocytes. This novel mouse model will provide insight into the role of perturbations in myocardial lipid metabolism in the pathogenesis of inherited and acquired forms of heart failure.

E xpression of smooth muscle myosin heavy chain (SM-MHC) is tightly controlled depending on the differentiated state of smooth muscle cells (SMCs). To better understand the mechanisms that regulate transcription of the SM-MHC gene in vivo, we tested the function of several conserved CArG elements contained within the –4200 to +11600 region of this gene that we had previously shown to drive SMC-specific expression in transgenic mice. CArG1 in the 5′-flanking sequence was required for all SMCs, while CArG2 and a novel intronic CArG element were differentially required in SMC subtypes. Of particular note, mutation of the intronic CArG selectively abolished expression in large arteries. A promoter construct containing three repeats of a conserved 227-bp intronic CArG-containing region was sufficient to direct transcription in vascular SMCs in transgenic mice, although this construct was also expressed in skeletal and cardiac muscle. These results support a model in which transcriptional regulation of SM-MHC is controlled by multiple positive and negative modular control regions that differ between SMCs and non-SMCs and among SMC subtypes. We also demonstrated that the CArG elements of the endogenous SM-MHC gene were bound by SRF in chromatin.

T hree distinct dendritic cell (DC) subsets capable of stimulating allogeneic naive T cells were isolated from human thymus. The most abundant subset was represented by plasmacytoid DCs (pDCs), which secreted high amounts of IFN-α upon stimulation with inactivated influenza virus and thus likely correspond to the recently identified peripheral blood natural IFN-α/β–producing cells (IPCs). Like those latter cells, thymic pDCs had distinctive phenotypic features (i.e., Lin^–, HLA-DR^int, IL-3Rα^hi, CD45RA^hi, CD11c^–, CD13^–, and CD33^lo) and developed into mature DCs upon culture in IL-3 and CD40L. Of the two other DC subsets, one displayed a phenotype of immature myeloid DCs (imDCs) (HLA-DR^int, CD11c⁺, CD13⁺, CD33⁺), and the other represented HLA-DR^hi CD11c⁺ mature DCs (mDCs). Since they also expressed DC-LAMP, these mDCs appear to correspond to interdigitating dendritic cells (IDCs). Thymic pDCs, but not myeloid imDCs, strongly expressed lymphoid-specific transcripts such as pre-Tα, λ-like, and Spi-B, thereby suggesting a possible lymphoid origin. The detection of Spi-B mRNA, not only upon in vitro maturation of pDCs, but also in freshly purified IDCs, suggests that in vivo pDCs may differentiate into IDCs.

M icrobial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. Borrelia burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn^–/–) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn^–/– mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn^–/– mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn^+/+ or Dcn^+/– mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borrelia-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn^–/– mice had diminished Borrelia numbers compared with issues harvested from Dcn^+/+ controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn^–/– mice. Conversely, no differences in the numbers of Borrelia-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

R ecent evidence argues strongly that the marked increase in risk for atherosclerotic heart disease seen in diabetics cannot be explained by a generalized increase in oxidative stress. Here, we used streptozotocin to induce hyperglycemia in cynomolgus monkeys for 6 months and tested whether high glucose levels promote localized oxidative damage to artery wall proteins. We focused on three potential agents of oxidative damage: hydroxyl radical, tyrosyl radical, and reactive nitrogen species. To determine which pathways operate in vivo, we quantified four stable end products of these reactants — ortho-tyrosine, meta-tyrosine, o,o’-dityrosine, and 3-nitrotyrosine — in aortic proteins. Levels of ortho-tyrosine, meta-tyrosine, and o,o’-dityrosine, but not of 3-nitrotyrosine, were significantly higher in aortic tissue of hyperglycemic animals. Of the oxidative agents we tested, only hydroxyl radical mimicked this pattern of oxidized amino acids. Moreover, tissue levels of ortho-tyrosine and meta-tyrosine correlated strongly with serum levels of glycated hemoglobin, a measure of glycemic control. We conclude that short-term hyperglycemia in primates promotes oxidation of artery wall proteins by a species that resembles hydroxyl radical. Our observations suggest that glycoxidation reactions in the arterial microenvironment contribute to early diabetic vascular disease, raising the possibility that antioxidant therapies might interrupt this process.

A denosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5′ AMP. Using intestinal epithelial cell line T84, we studied the effect of adenosine on the secretion of IL-6, a proinflammatory cytokine involved in neutrophil degranulation and lymphocyte differentiation. Stimulation of T84 monolayers with either apical or basolateral adenosine induces A2b receptor–mediated increase in IL-6 secretion, which is polarized to the apical (luminal) compartment. In addition, Salmonella typhimurium, TNF-α, and forskolin, known inducers of IL-6 secretion in intestinal epithelial cells, also stimulate IL-6 secretion into the apical compartment. We show that IL6 promoter induction by adenosine occurs through cAMP-mediated activation of nuclear cAMP-responsive element-binding protein (CREB). We also show that IL-6 released in the luminal (apical) compartment achieves a sufficient concentration to activate neutrophils (from which the adenosine signal originates), since such IL-6 is found to induce an intracellular [Ca⁺⁺] flux in neutrophils. We conclude that adenosine released in the intestinal lumen during active inflammation may induce IL-6 secretion, which is mediated by cAMP/CREB activation and occurs in an apically polarized fashion. This would allow sequential activation of neutrophil degranulation in the lumen — a flow of events that would, in an epithelium-dependent fashion, enhance microbicidal activity of neutrophils as they arrive in the intestinal lumen.

M HC class II molecules are critical determinants of genetic susceptibility to human type 1 diabetes. In patients, the most common haplotype contains the DRA1*0101-DRB1*0401 (DR4) and DQA1*0301-DQB1*0302 (DQ8) loci. To assess directly the relative roles of HLA-DQ8 and DR4 for diabetes development in vivo, we generated C57BL/6 transgenic mice that lack endogenous mouse MHC class II molecules but express HLA-DQ8 and/or DR4. Neither HLA-DQ nor HLA-DR transgenic mice developed insulitis or spontaneous diabetes. However, when they were crossed to transgenic mice (C57BL/6) expressing the B7.1 costimulatory molecules on pancreatic β cells that do not normally develop diabetes, T cells from these double transgenic mice were no longer tolerant to islet autoantigens. The majority of DQ8/RIP-B7 mice developed spontaneous diabetes, whereas only 25% of DR4/RIP-B7 mice did so. Interestingly, when DQ8 and DR4 were coexpressed (DQ8DR4/RIP-B7), only 23% of these mice developed diabetes, an incidence indistinguishable from the DR4/RIP-B7 mice. T cells from both DR4/RIP-B7 and DQ8DR4/RIP-B7 mice, unlike those from DQ8/RIP-B7 mice, exhibited a Th2-like phenotype. Thus, the expression of DR4 appeared to downregulate DQ8-restricted autoreactive T cells in DQ8DR4/RIP-B7 mice. Our data suggest that although both DQ8 and DR4 can promote spontaneous diabetes in mice with a non–autoimmune-prone genetic background, the diabetogenic effect of the DQ8 allele is much greater, whereas DR4 expression downregulates the diabetogenic effect of DQ8, perhaps by enhancing Th2-like immune responses.

T o examine whether B7 costimulation can be mediated by a molecule on T cells that is neither CD28 nor CTLA4, we generated mice lacking both of these receptors. CD28/CTLA4^–/– mice resemble CD28^–/– mice in having decreased expression of T-cell activation markers in vivo and decreased T-cell proliferation in vitro, as compared with wild-type mice. Using multiple approaches, we find B7-dependent costimulation in CD28/CTLA4^–/– mice. The proliferation of CD28/CTLA4^–/– T cells is inhibited by CTLA4-Ig and by the use of antigen-presenting cells lacking both B7-1 and B7-2. CD28/CTLA4^–/– T-cell proliferation is increased by exposure to Chinese hamster ovary cells transfected with B7-1 or B7-2. Finally, administration of CTLA4-Ig to CD28/CTLA4^–/– cardiac allograft recipients significantly prolongs graft survival. These data support the existence of an additional receptor for B7 molecules that is neither CD28 nor CTLA4.

P rostaglandins (PGs) generated by the enzyme cyclooxygenase (COX) have been implicated in the pathological renal hemodynamics and structural alterations in diabetes mellitus, but the role of individual COX isoenzymes in diabetic nephropathy remains unknown. We explored COX-1 and COX-2 expression and hemodynamic responses to the COX-1 inhibitor valeryl salicylate (VS) or the COX-2 inhibitor NS398 in moderately hyperglycemic, streptozotocin-diabetic (D) and control (C) rats. Immunoreactive COX-2 was increased in D rats compared with C rats and normalized by improved glycemic control. Acute systemic administration of NS398 induced no significant changes in mean arterial pressure and renal plasma flow in either C or D rats but reduced glomerular filtration rate in D rats, resulting in a decrease in filtration fraction. VS had no effect on renal hemodynamics in D rats. Both inhibitors decreased urinary excretion of PGE₂. However, only NS398 reduced excretion of thromboxane A₂. In conclusion, we documented an increase in renal cortical COX-2 protein expression associated with a different renal hemodynamic response to selective systemic COX-2 inhibition in D as compared with C animals, indicating a role of COX-2–derived PG in pathological renal hemodynamic changes in diabetes.

O steoporosis is a common disease with a strong genetic component. We previously described a polymorphic Sp1 binding site in the COL1A1 gene that has been associated with osteoporosis in several populations. Here we explore the molecular mechanisms underlying this association. A meta-analysis showed significant associations between COL1A1 “s” alleles and bone mineral density (BMD), body mass index (BMI), and osteoporotic fractures. The association with fracture was stronger than expected on the basis of the observed differences in BMD and BMI, suggesting an additional effect on bone strength. Gel shift assays showed increased binding affinity of the “s” allele for Sp1 protein, and primary RNA transcripts derived from the “s” allele were approximately three times more abundant than “S” allele–derived transcripts in “Ss” heterozygotes. Collagen produced from osteoblasts cultured from “Ss” heterozygotes had an increased ratio of α1(I) protein relative to α2(I), and this was accompanied by an increased ratio of COL1A1 mRNA relative to COL1A2. Finally, the yield strength of bone derived from “Ss” individuals was reduced when compared with bone derived from “SS” subjects. We conclude that the COL1A1 Sp1 polymorphism is a functional genetic variant that predisposes to osteoporosis by complex mechanisms involving changes in bone mass and bone quality.

W e studied T-cell clones generated from grafts of rejecting and tolerant animals and investigated the regulatory function of Th2 clones in vitro and in vivo. To prevent allograft rejection, we treated LEW strain recipient rats of WF strain kidney grafts with CTLA4Ig to block CD28-B7 costimulation. We then isolated epitope-specific T-cell clones from the engrafted tissue, using a donor-derived immunodominant class II MHC allopeptide presented by recipient antigen-presenting cells. Acutely rejected tissue from untreated animals yielded self-restricted, allopeptide-specific T-cell clones that produced IFN-γ, whereas clones from tolerant animals produced IL-4 and IL-10. Adoptive transfer into naive recipients of Th1 clones, but not Th2 clones, induced alloantigen-specific delayed-type hypersensitivity (DTH) responses. In addition, Th2 clones suppressed DTH responses mediated by Th1 clones in vivo and blocked Th1 cell proliferation and IFN-γ production in vitro. A pilot human study showed that HLA-DR allopeptide-specific T-cell clones generated from patients with chronic rejection secrete Th1 cytokines, whereas those from patients with stable graft function produce Th2 cytokines in response to donor-specific HLA-DR allopeptides. We suggest that self-restricted alloantigen-specific Th2 clones may regulate the alloimmune responses and promote long-term allograft survival and tolerance.

T he inflammatory cytokine TNF-α stimulates several presumed pro-atherogenic signaling events in endothelial cells (ECs), including activation of c-Jun NH₂-terminal kinase (JNK) and induction of E-selectin. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a MAP kinase kinase kinase, is required for TNF-mediated JNK activation. TNF activates ASK1 in part by dissociating ASK1 from its inhibitor 14-3-3. Because the risk of atherosclerosis is decreased in regions of steady laminar flow, we hypothesized that laminar flow inhibits proinflammatory cytokine-mediated activation of JNK. Steady laminar flow inhibited both TNF activation of ASK1 and JNK. Inhibition of ASK1 by flow correlated with increased association of ASK1 with 14-3-3. A constitutively active form of ASK1 lacking the 14-3-3-binding site (ASK1-ΔNS967A) was not inhibited by flow. These data establish ASK1 as a target for flow-mediated inhibition of cytokine signaling and indicate a novel role for 14-3-3 as an anti-inflammatory mediator in ECs.