F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found that connexin (Cx) 26 is upregulated in BL6 cells. To examine gap junction formation, we devised a coculture system, in which an opened vein segment was placed at the bottom of a culture dish and then dye-labeled melanoma cells were seeded onto it. Immunohistochemistry indicated that the vein segment preserved the integrity of the endothelial monolayer. In this system, BL6 cells could transfer dye into endothelial cells but F10 cells could not. Transfection with wild-type Cx26 rendered F10 cells competent for coupling with endothelial cells and as spontaneously metastatic as BL6 cells. Conversely, transfection with a dominant-negative form of Cx26 rendered BL6 cells deficient in coupling and less metastatic. In human melanoma lesions, the level of Cx26 expression was low in melanoma cells residing in the basal layer, but significantly upregulated in melanoma cells invading the dermis. The results suggested that Cx26 plays a role in intravasation and extravasation of tumor cells through heterologous gap junction formation with endothelial cells.
Expression of Fas ligand (FasL) renders certain tissues immune privileged, but its expression in other tissues can result in severe neutrophil infiltration and tissue destruction. The consequences of enforced FasL expression in striated muscle is particularly controversial. To create a stable reproducible pattern of cardiomyocyte-specific FasL expression, transgenic (Tg) mice were generated that express murine FasL specifically in the heart, where it is not normally expressed. Tg animals are healthy and indistinguishable from nontransgenic littermates. FasL expression in the heart does result in mild leukocyte infiltration, but despite coexpression of Fas and FasL in Tg hearts, neither myocardial tissue apoptosis nor necrosis accompanies the leukocyte infiltration. Instead of tissue destruction, FasL Tg hearts develop mild interstitial fibrosis, functional changes, and cardiac hypertrophy, with corresponding molecular changes in gene expression. Induced expression of the cytokines TNF-α, IL-1β, IL-6, and TGF-β accompanies these proinflammatory changes. The histologic, functional, and molecular proinflammatory consequences of cardiac FasL expression are transgene-dose dependent. Thus, coexpression of Fas and FasL in the heart results in leukocyte infiltration and hypertrophy, but without the severe tissue destruction observed in other examples of FasL-directed proinflammation. The data suggest that the FasL expression level and other tissue-specific microenvironmental factors can modulate the proinflammatory consequences of FasL.
Hereditary hemochromatosis (HH) is a prevalent human disease caused by a mutation in HFE, which encodes an atypical HLA class I protein involved in regulation of intestinal iron absorption. To gain insight into the pathogenesis of hemochromatosis, we have bred Hfe knockout mice to strains carrying other mutations that impair normal iron metabolism. Compound mutant mice lacking both Hfe and its interacting protein, beta-2 microglobulin (B2m), deposit more tissue iron than mice lacking Hfe only, suggesting that another B2m-interacting protein may be involved in iron regulation. Hfe knockout mice carrying mutations in the iron transporter DMT1 fail to load iron, indicating that hemochromatosis involves iron flux through DMT1. Similarly, compound mutants deficient in both Hfe and hephaestin (Heph) show less iron loading than do Hfe knockout mice, indicating that iron absorption in hemochromatosis involves the function of Heph as well. Finally, compound mutants lacking Hfe and the transferrin receptor accumulate more tissue iron than do mice lacking Hfe alone, consistent with the idea that interaction between these two proteins contributes to the control of normal iron absorption. In addition to providing insight into the pathogenesis of HH, our results suggest that each of these genes might be a candidate modifier of the human hemochromatosis phenotype.
The kidneys are thought to be a major site of net de novo arginine synthesis, but the quantitative status of arginine metabolism and its substrate precursor relationship to nitric oxide (NO) synthesis in end stage renal disease (ESRD) patients have not been characterized. We have investigated kinetic aspects of whole body arginine metabolism in six patients with ESRD. They received two pre- and two post-hemodialysis intravenous tracer infusion studies with L-[guanidino-15N2]arginine and L-[13C]leucine during the first study, and L-[5-13C]arginine and L-[5-13C-ureido,5,5,2H2]citrulline during the second study. Arginine homeostasis in ESRD patients was found to be associated with a lower rate of arginine oxidation, and despite the decrease in renal function, the rate of de novo arginine synthesis appeared to be preserved. Plasma citrulline concentrations and flux were also elevated in these subjects compared with healthy adults. The rate of whole body NO synthesis was increased in the ESRD patients, but apparently not different pre- and post-hemodialysis therapy. The anatomic site(s) responsible for the maintenance of net de novo arginine synthesis and for the elevated NO synthesis and its pathophysiological importance in ESRD remain to be established.
Induction of myelin-specific CD4 T cells is a pivotal event in the development of experimental autoimmune encephalomyelitis (EAE). Other checkpoints in EAE pathogenesis have not been clearly defined, although multiple genetic loci are known to influence EAE development. We report here that targeted mutation of the heat-stable antigen (HSA) abrogates development of EAE despite a complete lack of effect on induction of autoimmune T cells. To test whether T-cell expression of HSA is sufficient, we created transgenic mice in which HSA is expressed exclusively in the T-cell lineage. We found that these mice remain resistant to EAE induction. Adoptive transfer studies demonstrate that both T cells and non–T cells must express HSA in order for the pathogenic T cells to execute their effector function. Moreover, HSAIg, a fusion protein consisting of the extracellular domain of the HSA and the Fc portion of immunoglobulin, drastically ameliorates the clinical sign of EAE even when administrated after self-reactive T cells had been expanded. Thus, identification of HSA as a novel checkpoint, even after activation and expansion of self-reactive T cells, provides a novel approach for immunotherapy of autoimmune neurologic diseases, such as multiple sclerosis.
We have found that EGF-R expression is associated with the development of the Schwann cell–derived tumors characteristic of neurofibromatosis type 1 (NF1) and in animal models of this disease. This is surprising, because Schwann cells normally lack EGF-R and respond to ligands other than EGF. Nevertheless, immunoblotting, Northern analysis, and immunohistochemistry revealed that each of 3 malignant peripheral nerve sheath tumor (MPNST) cell lines from NF1 patients expressed the EGF-R, as did 7 of 7 other primary MPNSTs, a non-NF1 MPNST cell line, and the S100+ cells from each of 9 benign neurofibromas. Furthermore, transformed derivatives of Schwann cells from NF1–/– mouse embryos also expressed the EGF-R. All of the cells or cell lines expressing EGF-R responded to EGF by activation of downstream signaling pathways. Thus, EGF-R expression may play an important role in NF1 tumorigenesis and Schwann cell transformation. Consistent with this hypothesis, growth of NF1 MPNST lines and the transformed NF1–/– mouse embryo Schwann cells was greatly stimulated by EGF in vitro and could be blocked by agents that antagonize EGF-R function.
To explore the pathophysiological role of leptin in obesity-related hypertension, we examined cardiovascular phenotypes of transgenic skinny mice whose elevated plasma leptin concentrations are comparable to those seen in obese subjects. We also studied genetically obese KKAy mice with hyperleptinemia, in which hypothalamic melanocortin system is antagonized by ectopic expression of the agouti protein. Systolic blood pressure (BP) and urinary catecholamine excretion are elevated in transgenic skinny mice relative to nontransgenic littermates. The BP elevation in transgenic skinny mice is abolished by α1-adrenergic, β-adrenergic, or ganglionic blockers at doses that do not affect BP in nontransgenic littermates. Central administration of an α-melanocyte–stimulating hormone antagonist causes a marked increase in cumulative food intake but no significant changes in BP. The obese KKAy mice develop BP elevation with increased urinary catecholamine excretion relative to control KK mice. After a 2-week caloric restriction, BP elevation is reversed in nontransgenic littermates with the Ay allele, in parallel with a reduction in plasma leptin concentrations, but is sustained in transgenic mice overexpressing leptin with the Ay allele, which remain hyperleptinemic. This study demonstrates BP elevation in transgenic skinny mice and obese KKAy mice that are both hyperleptinemic, thereby suggesting the pathophysiological role of leptin in some forms of obesity-related hypertension.
Chronic central administration of neuropeptide Y (NPY) causes hyperphagia, hyperinsulinemia, and obesity, a response that is prevented by prior adrenalectomy (ADX) in rats. The basis of NPY’s effect and how the acute responses to this peptide are affected by ADX remain unknown. This study investigates the role of glucocorticoids in acute NPY-stimulated food intake, acute NPY-induced insulin release, and hypothalamic NPY-receptor mRNA expression levels. NPY-induced food intake was similar in ADX and control rats after acute intracerebroventricular injection of NPY. Injection of NPY caused a significant increase in plasma insulin in control rats, but this effect was completely absent in ADX rats in which basal plasma insulin levels were also lower than controls. In addition, ADX significantly reduced the number of neurons expressing NPY receptor Y1 and Y5 mRNAs in the ventromedial hypothalamus (VMH), without affecting Y1- or Y5-mRNA expression in the paraventricular hypothalamus or the arcuate nucleus. These data indicate that glucocorticoids are necessary for acute NPY-mediated insulin release and suggest that the mechanisms involve glucocorticoid regulation of Y1 and Y5 receptors specifically within the VMH nucleus.
The expression of several drug-resistance genes, including MRP and p53, increases with advancing stage of human prostate cancer. Altered transcription could account for the genotypic alterations associated with prostate cancer progression, and it was recently reported that the promoter of MRP1 is activated in the presence of mutant p53. To determine whether there is a relationship between p53 status and the expression of MRP1, a human, temperature-sensitive p53 mutant (tsp Val138) was transfected into LNCaP human prostate cancer cells. In the transfected cell line (LVCaP), the wild-type p53 produced growth arrest at the G1/S interface of the cell cycle, inhibited colony formation, and induced p21waf1/cip1. Temperature shifting to 38°C (p53 mutant) produced a time-dependent increase in expression of MRP1. This change in MRP1 expression was also seen in isogenic cell lines in which p53 was inactivated by human papilloma virus (HPV)16E6 protein or by a dominant-negative mutant. Functional assays revealed a decrease in drug accumulation and drug sensitivity associated with mutant p53 and increased MRP1 expression. These results provide the first mechanistic link between expression of MRP1 and mutation of p53 in human prostate cancer and support recent clinical associations. Furthermore, these data suggest a mechanism tying accumulation of p53 mutations to the multidrug resistance phenotype seen in this disease.
Corticotropin-releasing hormone (CRH)–deficient (KO) mice provide a unique system to define the role of CRH in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Despite several manifestations of chronic glucocorticoid insufficiency, basal pituitary proopiomelanocortin (POMC) mRNA, adrenocorticotrophic hormone (ACTH) peptide content within the pituitary, and plasma ACTH concentrations are not elevated in CRH KO mice. The normal POMC mRNA content in KO mice is dependent upon residual glucocorticoid secretion, as it increases in both KO and WT mice after adrenalectomy; this increase is reversed by glucocorticoid, but not aldosterone, replacement. However, the normal plasma levels of ACTH in CRH KO mice are not dependent upon residual glucocorticoid secretion, because, after adrenalectomy, these levels do not undergo the normal increase seen in KO mice despite the increase in POMC mRNA content. Administration of CRH restores ACTH secretion to its expected high level in adrenalectomized CRH KO mice. Thus, in adrenal insufficiency, loss of glucocorticoid feedback by itself can increase POMC gene expression in the pituitary; but CRH action is essential for this to result in increased secretion of ACTH. This may explain why, after withdrawal of chronic glucocorticoid treatment, reactivation of CRH secretion is a necessary prerequisite for recovery from suppression of the HPA axis.
Increased stromal cell production of M-CSF, an event caused by enhanced phosphorylation of the nuclear protein Egr-1, is central to the mechanism by which estrogen (E2) deficiency upregulates osteoclast (OC) formation. However, the contribution of enhanced M-CSF production to the bone loss induced by E2 deficiency remains to be determined. We found that treatment with an Ab that neutralizes M-CSF in vivo completely prevents the rise in OC number, the increase in bone resorption, and the resulting bone loss induced by ovariectomy (ovx). We also found that adult, intact Egr-1–deficient mice, a strain characterized by maximally stimulated stromal cell production of M-CSF, exhibit increased bone resorption and decreased bone mass. In these mice, treatment with anti–M-CSF Ab restored normal levels of bone resorption, thus confirming that increased M-CSF production accounts for the remodeling abnormalities of Egr-1–deficient mice. Consistent with the failure of ovx to further increase M-CSF production in Egr-1–deficient mice, ovx neither increased bone resorption further, nor caused bone loss in these animals. In summary, the data demonstrate that E2 deficiency induces M-CSF production via an Egr-1-dependent mechanism that is central to the pathogenesis of ovx-induced bone loss. Thus, Egr-1 and M-CSF are critical mediators of the bone sparing effects of E2 in vivo.
STAT4 and STAT6 are transcription factors that play crucial roles in responding to IL-12 and IL-4, respectively. STAT4 gene knockout (STAT4–/–) mice have markedly reduced Th1 responses and enhanced Th2 responses. STAT6–/– mice show the inverse phenotype. We compared the ability of bone marrow transplantation (BMT) with the inclusion of spleen cells from STAT6–/–, STAT4–/–, and wild-type (WT) mice to produce graft-versus-host disease (GVHD) in lethally irradiated MHC-mismatched recipients. Acute GVHD mortality was more rapid when induced by cells from STAT6–/– mice than when induced by STAT4–/– cells. However, cells from STAT4–/– and STAT6–/– donors both induced delayed GVHD mortality compared with WT controls, or compared with combined STAT4–/– and STAT6–/– cells, indicating a contribution of both Th1 cells and Th2 cells to acute GVHD. Recipients of STAT6–/– BMT showed evidence of acute GVHD with severe diarrhea and marked weight loss. Recipients of STAT4–/– BMT showed signs of GVHD with only initial transient weight loss and later development of severe skin GVHD. Histopathology showed that Th2 responses were required for the induction of both hepatic and severe skin GVHD. In contrast, both Th1 cells and Th2 cells were capable of causing intestinal pathology of GVHD. Our studies demonstrate an additive role for Th1 and Th2 cells in producing acute GVHD, and suggest a cytokine-directed approach to treating end-organ manifestations of GVHD.
Recruitment of macrophages to sites of cell death is critical for induction of an immunologic response. Calcium concentrations in extracellular fluids vary markedly, and are particularly high at sites of injury or infection. We hypothesized that extracellular calcium participates in modulating the immune response, perhaps acting via the seven-transmembrane calcium-sensing receptor (CaR) on mature monocytes/macrophages. We observed a dose-dependent increase in monocyte chemotaxis in response to extracellular calcium or the selective allosteric CaR activator NPS R-467. In contrast, monocytes derived from mice deficient in CaR lacked the normal chemotactic response to a calcium gradient. Notably, CaR activation of monocytes bearing the receptor synergistically augmented the transmigration response of monocytes to the chemokine MCP-1 in association with increased cell-surface expression of its cognate receptor, CCR2. Conversely, stimulation of monocytes with MCP-1 or SDF-1α reciprocally increased CaR expression, suggesting a dual-enhancing interaction of Ca2+ with chemokines in recruiting inflammatory cells. Subcutaneous administration in mice of Ca2+, MCP-1, or (more potently) the combination of Ca2+ and MCP-1, elicited an inflammatory infiltrate consisting of monocytes/macrophages. Thus extracellular calcium functions as an ionic chemokinetic agent capable of modulating the innate immune response in vivo and in vitro by direct and indirect actions on monocytic cells. Calcium deposition may be both consequence and cause of chronic inflammatory changes at sites of injury, infection, and atherosclerosis.
Pituitary adenylate cyclase–activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic β cells express two PACAP receptor subtypes, a PACAP-preferring (PAC1) and a VIP-shared (VPAC2) receptor. We have applied a gene targeting approach to create a mouse lacking the PAC1 receptor (PAC1–/–). These mice were viable and normoglycemic, but exhibited a slight feeding hyperinsulinemia. In vitro, in the isolated perfused pancreas, the insulin secretory response to PACAP was reduced by 50% in PAC1–/– mice, whereas the response to VIP was unaffected. In vivo, the insulinotropic action of PACAP was also acutely reduced, and the peptide induced impairment of glucose tolerance after an intravenous glucose injection. This demonstrates that PAC1 receptor is involved in the insulinotropic action of the peptide. Moreover, PAC1–/– mice exhibited reduced glucose-stimulated insulin secretion in vitro and in vivo, showing that the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose. The defective insulinotropic action of glucose was associated with marked glucose intolerance after both intravenous and gastric glucose administration. Thus, these results are consistent with a physiological role for the PAC1 receptor in glucose homeostasis, notably during food intake.
The lethality of Mycobacterium tuberculosis remains the highest among infectious organisms and is linked to inadequate immune response of the host. Containment and cure of tuberculosis requires an effective cell-mediated immune response, and the absence, during active tuberculosis infection, of delayed-type hypersensitivity (DTH) responses to mycobacterial antigens, defined as anergy, is associated with poor clinical outcome. To investigate the biochemical events associated with this anergy, we screened 206 patients with pulmonary tuberculosis and identified anergic patients by their lack of dermal reactivity to tuberculin purified protein derivative (PPD). In vitro stimulation of T cells with PPD induced production of IL-10, IFN-γ, and proliferation in PPD+ patients, whereas cells from anergic patients produced IL-10 but not IFN-γ and failed to proliferate in response to this treatment. Moreover, in anergic patients IL-10–producing T cells were constitutively present, and T-cell receptor–mediated (TCR-mediated) stimulation resulted in defective phosphorylation of TCRζ and defective activation of ZAP-70 and MAPK. These results show that T-cell anergy can be induced by antigen in vivo in the intact human host and provide new insights into mechanisms by which M. tuberculosis escapes immune surveillance.
Copyright © 2014 American Society for Clinical Investigation