Delta-opioid augments cardiac contraction through ß-adrenergic and CGRP-receptor co-signaling.

Cardiac epinephrine and calcitonin gene-related peptide (CGRP) are produced by intrinsic cardiac adrenergic cells (ICA cells) residing in human and animal hearts. ICA cells are neuroparicine cells expressing δ-opioid receptors (DOR). We hypothesized that δ-opioid stimulation of ICA cells enhances epinephrine and CGRP release, which results in the augmentation of heart contraction. Rats were injected with DOR-agonist DPDPE (100 µg/kg) with or without 10-min pretreatment with either ß-adrenergic receptor (ß-AR) blocker propranolol (2mg/kg) or CGRP-receptor (CGRPR) blocker CGRP(8-37) (300 µg/kg), or their combination. Hemodynamics were monitored with echocardiogram and systolic blood pressure (SBP) was monitored via a tail arterial catheter. Changes in left ventricular fraction-shortening (LVFS) and heart rate (HR) were observed at 5-min after DPDPE infusion. At 5-min DPDPE induced a 36 ± 18% (p<0.001) increase of the LVFS, which continues to increase to 51 ± 24% (p<0.0001) by 10 min, and 68 ± 19% (p<0.001) by 20 min. The increase in LVFS was accompanied by the decrease of HR by 9±5% (p<0.01) by 5 min and 11 ± 6% (p<0.001) by 15 min post DPDPE infusion. This magnitude of HR reduction was observed for the remainder of the 20 min. Despite the HR-reduction, cardiac output was increased by 17 ± 8% (p<0.05) and 28±5% (p<0.001) by 5- and 20-min post DPDPE administration, respectively. There was a modest (9 ± 9%, p=0.03) decrease in SBP that was not apparent until 20 min post DPDPE infusion. The positive inotropism of DPDPE was abrogated in animals pretreated with propranolol, CGRP(8-37), or combined propranolol+CGRP(8-37). Furthermore, in whole animal and cardiomyocyte cell culture preparations, DPDPE induced myocardial protein-kinase A (PKA) activation which was abrogated in the animals pretreated with propranolol+CGRP(8-37). DOR agonists augment myocardial contraction through enhanced ß-AR and CGRPR co-signaling.

Molecular regulation of CD40 gene expression in macrophages and microglia.

Inflammatory events in the central nervous system (CNS) contribute to the disease process in a variety of neuroinflammatory diseases such as multiple sclerosis (MS), Alzheimer's Disease (AD), and cerebral ischemia, and activated macrophages/microglia are central to this response. Immunological activation of these cells leads to the production of a wide array of cytokines, chemokines, matrix metalloproteinases and neurotoxins, and ultimately to glial/neuronal injury and death. The CD40 molecule has an important role in promoting inflammatory responses by macrophages/microglia, since interaction with its cognate ligand, CD154, leads to secretion of cytokines and neurotoxins. Aberrant CD40 expression by macrophages/microglia, induced by cytokines such as IFN-gamma and TNF-alpha, contributes to neuroimmunologic cascades in the CNS. Strategies to suppress CD40 expression may attenuate inflammation and neuronal damage within the CNS, which will ultimately be of benefit in neuroinflammatory diseases. The mediators that regulate expression of CD40 in macrophages/microglia (both induction and inhibition) function at the level of gene transcription. In this review, we present an overview of the molecular basis of CD40 expression in macrophages/microglia. The signal transduction pathways and transcription factors employed by IFN-gamma and TNF-alpha to induce CD40 expression are described, as are the cis-elements in the CD40 promoter that are critical for CD40 transcription. Information is provided on the mechanism(s) underlying suppression of CD40 in macrophages/microglia by immunomodulatory agents such as IL-4, TGF-beta, neuropeptides, neurotrophins, and statins. A comprehensive assessment of CD40 production and function in macrophages/microglia will establish the foundation for future therapeutic manipulation of this critical immunoregulatory protein.

Regulation and function of class II major histocompatibility complex, CD40, and B7 expression in macrophages and microglia: Implications in neurological diseases.

The ability of microglia, the brain's resident macrophage, to present antigen through the class II major histocompatibility complex (MHC) to T cells allows these normally quiescent cells to play a critical role in shaping the outcome of many neurological diseases. The expression of class II MHC antigens and the costimulatory molecules CD40 and B7 on microglia and infiltrating macrophages is regulated through a complex network of cytokines in the inflamed brain. In this review, we describe the molecular mechanisms underlying class II MHC, CD40 and B7 regulation in microglia and macrophages. Our focus is on the cis-elements in the promoters of their genes and the transcription factors activated by cytokines that bind them. The functional implications of aberrant class II MHC, CD40 and B7 expression by microglia and macrophages as related to the diseases of Multiple Sclerosis and Alzheimer's Disease are discussed.

Suppressor of cytokine signaling 1 inhibits cytokine induction of CD40 expression in macrophages.

CD40 is a type I membrane-bound molecule belonging to the TNFR superfamily that is expressed on various immune cells including macrophages and microglia. The aberrant expression of CD40 is involved in the initiation and maintenance of various human diseases including multiple sclerosis, arthritis, atherosclerosis, and Alzheimer's disease. Inhibition of CD40 signaling has been shown to provide a significant beneficial effect in a number of animal models of human diseases including the aforementioned examples. We have previously shown that IFN-gamma induces CD40 expression in macrophages and microglia. IFN-gamma leads to STAT-1alpha activation directly and up-regulation of NF-kappaB activity due to the secretion and subsequent autocrine signaling of TNF-alpha. However, TNF-alpha alone is not capable of inducing CD40 expression in these cells. Suppressor of cytokine signaling 1 protein (SOCS-1) is a cytokine-inducible Src homology 2-containing protein that regulates cytokine receptor signaling by inhibiting STAT-1alpha activation via a specific interaction with activated Janus kinase 2. Given the important role of CD40 in inflammatory events in the CNS as well as other organ systems, it is imperative to understand the molecular mechanisms contributing to both CD40 induction and repression. We show that ectopic expression of SOCS-1 abrogates IFN-gamma-induced CD40 protein expression, mRNA levels, and promoter activity. Additionally, IFN-gamma-induced TNF-alpha secretion, as well as STAT-1alpha and NF-kappaB activation, are inhibited in the presence of SOCS-1. We conclude that SOCS-1 inhibits cytokine-induced CD40 expression by blocking IFN-gamma-mediated STAT-1alpha activation, which also then results in suppression of IFN-gamma-induced TNF-alpha secretion and subsequent NF-kappaB activation.

Critical role of tumor necrosis factor-alpha and NF-kappa B in interferon-gamma -induced CD40 expression in microglia/macrophages.

CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 expression on antigen-presenting cells (including macrophages and microglia) is crucial for T-cell activation. Aberrant expression of CD40 has been associated with autoimmune inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We have recently shown that the cytokine interferon (IFN)-gamma is the most potent inducer of CD40 expression in macrophages and microglia, and this induction is mediated by the IFN-gamma-activated transcription factor STAT-1alpha and constitutively expressed PU.1 and/or Spi-B. In this study, we have discovered that a major component of IFN-gamma-induced CD40 expression involves the endogenous production of the cytokine TNF-alpha. The inclusion of anti-TNF-alpha-neutralizing antibody significantly inhibits IFN-gamma-induced CD40 mRNA and CD40 promoter activity. IFN-gamma-induced CD40 protein expression is attenuated in TNF-alpha-deficient microglia and can be restored with exogenous TNF-alpha. Site-directed mutagenesis studies demonstrate that three of the four NF-kappaB elements in the CD40 promoter are required for IFN-gamma-induced CD40 promoter activity. IFN-gamma treatment leads to the activation of NF-kappaB in a time-dependent manner, which is inhibited in the presence of anti-TNF-alpha-neutralizing antibody. These results indicate that IFN-gamma-induced TNF-alpha production and subsequent NF-kappaB activation are integral parts of the mechanism of IFN-gamma-induced CD40 expression.