Mast cells: a pivotal role in pulmonary fibrosis.

Pulmonary fibrosis is characterized by an inflammatory response that includes macrophages, neutrophils, lymphocytes, and mast cells. The purpose of this study was to evaluate whether mast cells play a role in initiating pulmonary fibrosis. Pulmonary fibrosis was induced with bleomycin in mast-cell-deficient WBB6F1-W/W(v) (MCD) mice and their congenic controls (WBB6F1-(+)/(+)). Mast cell deficiency protected against bleomycin-induced pulmonary fibrosis, but protection was reversed with the re-introduction of mast cells to the lungs of MCD mice. Two mast cell mediators were identified as fibrogenic: histamine and renin, via angiotensin (ANG II). Both human and rat lung fibroblasts express the histamine H1 and ANG II AT1 receptor subtypes and when activated, they promote proliferation, transforming growth factor ß1 secretion, and collagen synthesis. Mast cells appear to be critical to pulmonary fibrosis. Therapeutic blockade of mast cell degranulation and/or histamine and ANG II receptors should attenuate pulmonary fibrosis.

Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model.

Mast cells are associated with inflammation and fibrosis. Whether they protect against or contribute to renal fibrosis is unclear. Based on our previous findings that mast cells can express and secrete active renin, and that angiotensin (ANG II) is profibrotic, we hypothesized that mast cells play a critical role in tubulointerstitial fibrosis. We tested this hypothesis in the 14-day unilateral ureteral obstruction (UUO) model in rats and mast cell-deficient (MCD) mice (WBB6F1-W/Wv) and their congenic controls (CC). In the 14-day UUO rat kidney, mast cell number is increased and they express active renin. Stabilizing mast cells in vivo with administration of cromolyn sodium attenuated the development of tubulointerstitial fibrosis, which was confirmed by measuring newly synthesized pepsin-soluble collagen and blind scoring of fixed trichrome-stained kidney sections accompanied by spectral analysis. Fibrosis was absent in UUO kidneys from MCD mice unlike that observed in the CC mice. Losartan treatment reduced the fibrosis in the CC UUO kidneys. The effects of mast cell degranulation and renin release were tested in the isolated, perfused kidney preparation. Mast cell degranulation led to renin-dependent protracted flow recovery. This demonstrates that mast cell renin is active in situ and the ensuing ANG II can modulate intrarenal vascular resistance in the UUO kidney. Collectively, the data demonstrate that mast cells are critical to the development of renal fibrosis in the 14-day UUO kidney. Since renin is present in human kidney mast cells, our work identifies potential targets in the treatment of renal fibrosis.

Targeting cardiac mast cells: pharmacological modulation of the local renin-angiotensin system.

Enhanced production of angiotensin II and excessive release of norepinephrine in the ischemic heart are major causes of arrhythmias and sudden cardiac death. Mast cell-dependent mechanisms are pivotal in the local formation of angiotensin II and modulation of norepinephrine release in cardiac pathophysiology. Cardiac mast cells increase in number in myocardial ischemia and are located in close proximity to sympathetic neurons expressing angiotensin AT1- and histamine H3-receptors. Once activated, cardiac mast cells release a host of potent pro-inflammatory and pro-fibrotic cytokines, chemokines, preformed mediators (e.g., histamine) and proteases (e.g., renin). In myocardial ischemia, angiotensin II (formed locally from mast cell-derived renin) and histamine (also released from local mast cells) respectively activate AT1- and H3-receptors on sympathetic nerve endings. Stimulation of angiotensin AT1-receptors is arrhythmogenic whereas H3-receptor activation is cardioprotective. It is likely that in ischemia/reperfusion the balance may be tipped toward the deleterious effects of mast cell renin, as demonstrated in mast cell-deficient mice, lacking mast cell renin and histamine in the heart. In these mice, no ventricular fibrillation occurs at reperfusion following ischemia, as opposed to wild-type hearts which all fibrillate. Preventing mast cell degranulation in the heart and inhibiting the activation of a local renin-angiotensin system, hence abolishing its detrimental effects on cardiac rhythmicity, appears to be more significant than the loss of histamine-induced cardioprotection. This suggests that therapeutic targets in the treatment of myocardial ischemia, and potentially congestive heart failure and hypertension, should include prevention of mast cell degranulation, mast cell renin inhibition, local ACE inhibition, ANG II antagonism and H3-receptor activation.

IL-10 suppresses calcium-mediated costimulation of receptor activator NF-kappa B signaling during human osteoclast differentiation by inhibiting TREM-2 expression.

Induction of effective osteoclastogenesis by RANK (receptor activator of NF-kappaB) requires costimulation by ITAM-coupled receptors. In humans, the TREM-2 (triggering receptor expressed on myeloid cells 2) ITAM-coupled receptor plays a key role in bone remodeling, as patients with TREM-2 mutations exhibit defective osteoclastogenesis and bone lesions. We have identified a new rapidly induced costimulatory pathway for RANK signaling that is dependent on TREM-2 and mediated by calcium signaling. TREM-2-dependent calcium signals are required for RANK-mediated activation of calcium/calmodulin-dependent protein kinase (CaMK)II and downstream MEK and ERK MAPKs that are important for osteoclastogenesis. IL-10 inhibited RANK-induced osteoclastogenesis and selectively inhibited calcium signaling downstream of RANK by inhibiting transcription of TREM-2. Down-regulation of TREM-2 expression resulted in diminished RANKL-induced activation of the CaMK-MEK-ERK pathway and decreased expression of the master regulator of osteoclastogenesis NFATc1. These findings provide a new mechanism of inhibition of human osteoclast differentiation. The results also yield insights into crosstalk between ITAM-coupled receptors and heterologous receptors such as RANK, and they identify a mechanism by which IL-10 can suppress cellular responses to TNFR family members.

Mast cell renin and a local renin-angiotensin system in the airway: role in bronchoconstriction.

We previously reported that mast cells express renin, the rate-limiting enzyme in the renin-angiotensin cascade. We have now assessed whether mast cell renin release triggers angiotensin formation in the airway. In isolated rat bronchial rings, mast cell degranulation released enzyme with angiotensin I-forming activity blocked by the selective renin inhibitor BILA2157. Local generation of angiotensin (ANG II) from mast cell renin elicited bronchial smooth muscle contraction mediated by ANG II type 1 receptors (AT(1)R). In a guinea pig model of immediate type hypersensitivity, anaphylactic mast cell degranulation in bronchial rings resulted in ANG II-mediated constriction. As in rat bronchial rings, bronchoconstriction (BC) was inhibited by a renin inhibitor, an AT(1)R blocker, and a mast cell stabilizer. Anaphylactic release of renin, histamine, and beta-hexosaminidase from mast cells was confirmed in the effluent from isolated, perfused guinea pig lung. To relate the significance of this finding to humans, mast cells were isolated from macroscopically normal human lung waste tissue specimens. Sequence analysis of human lung mast cell RNA showed 100% homology between human lung mast cell renin and kidney renin between exons 1 and 10. Furthermore, the renin protein expressed in lung mast cells was enzymatically active. Our results demonstrate the existence of an airway renin-angiotensin system triggered by release of mast-cell renin. The data show that locally produced ANG II is a critical factor governing BC, opening the possibility for novel therapeutic targets in the management of airway disease.

Immediate hypersensitivity elicits renin release from cardiac mast cells.

BACKGROUND: We recently reported that murine and cavian heart mast cells are a unique extrarenal source of renin. Ischemia/reperfusion releases this renin leading to local angiotensin formation and norepinephrine release. As mast cells are a primary target of hypersensitivity, we assessed whether anaphylactic mast cell degranulation also results in renin and norepinephrine release. METHODS: Hearts isolated from presensitized guinea pigs were challenged with antigen. RESULTS: Cardiac anaphylaxis was characterized by mast cell degranulation, evidenced by beta-hexosaminidase release and associated with renin and norepinephrine release. Mast cell stabilization with cromolyn or lodoxamide markedly attenuated the release of beta-hexosaminidase, renin and norepinephrine. Renin inhibition with BILA2157 did not affect mast cell degranulation, but attenuated norepinephrine release. CONCLUSIONS: Our findings disclose that immediate-type hypersensitivity elicits renin release from mast cells, activating a local renin-angiotensin system, thereby promoting norepinephrine release. As renin is stored in human heart mast cells, allergic reactions could initiate renin release, leading to local angiotensin formation and hyperadrenergic dysfunction.

'Tuning' of type I interferon-induced Jak-STAT1 signaling by calcium-dependent kinases in macrophages.

Immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors modulate the amplitude and nature of macrophage responses to Toll-like receptor and cytokine receptor stimulation. However, the molecular mechanisms enabling this receptor crosstalk are not known. Here we investigated the function of the calcium-dependent kinases CaMK and Pyk2 'downstream' of ITAM-associated receptors in the regulation of cytokine-induced activation of Jak kinases and STAT transcription factors. CaMK and Pyk2 relayed signals from integrins and the ITAM-containing adaptor DAP12 to augment interleukin 10- and interferon-alpha-induced Jak activation and STAT1-dependent gene expression. CaMK inhibition suppressed STAT1-mediated interferon-alpha signaling in a mouse model of systemic lupus erythematosus. Our results associate Pyk2 and Jak kinases with the linkage of signals emanating from cytokine and heterologous ITAM-dependent receptors.

Renin: at the heart of the mast cell.

Cardiac mast cells proliferate in cardiovascular diseases. In myocardial ischemia, mast cell mediators contribute to coronary vasoconstriction, arrhythmias, leukocyte recruitment, and tissue injury and repair. Arrhythmic dysfunction, coronary vasoconstriction, and contractile failure are also characteristic of cardiac anaphylaxis. In coronary atherosclerosis, mast cell mediators facilitate cholesterol accumulation and plaque destabilization. In cardiac failure, mast cell chymase causes myocyte apoptosis and fibroblast proliferation, leading to ventricular dysfunction. Chymase and tryptase also contribute to fibrosis in cardiomyopathies and myocarditis. In addition, mast cell tumor necrosis factor-alpha promotes myocardial remodeling. Cardiac remodeling and hypertrophy in end-stage hypertension are also induced by mast cell mediators and proteases. We recently discovered that cardiac mast cells contain and release renin, which initiates local angiotensin formation. Angiotensin causes coronary vasoconstriction, arrhythmias, fibrosis, apoptosis, and endothelin release, all demonstrated mechanisms of mast-cell-associated cardiac disease. The effects of angiotensin are further amplified by the release of norepinephrine from cardiac sympathetic nerves. Our discovery of renin in cardiac mast cells and its release in pathophysiological conditions uncovers an important new pathway in the development of mast-cell-associated heart diseases. Several steps in this novel pathway may constitute future therapeutic targets.

Cardiac mast cell-derived renin promotes local angiotensin formation, norepinephrine release, and arrhythmias in ischemia/reperfusion.

Having identified renin in cardiac mast cells, we assessed whether its release leads to cardiac dysfunction. In Langendorff-perfused guinea pig hearts, mast cell degranulation with compound 48/80 released Ang I-forming activity. This activity was blocked by the selective renin inhibitor BILA2157, indicating that renin was responsible for Ang I formation. Local generation of cardiac Ang II from mast cell-derived renin also elicited norepinephrine release from isolated sympathetic nerve terminals. This action was mediated by Ang II-type 1 (AT1) receptors. In 2 models of ischemia/reperfusion using Langendorff-perfused guinea pig and mouse hearts, a significant coronary spillover of renin and norepinephrine was observed. In both models, this was accompanied by ventricular fibrillation. Mast cell stabilization with cromolyn or lodoxamide markedly reduced active renin overflow and attenuated both norepinephrine release and arrhythmias. Similar cardioprotection was observed in guinea pig hearts treated with BILA2157 or the AT1 receptor antagonist EXP3174. Renin overflow and arrhythmias in ischemia/reperfusion were much less prominent in hearts of mast cell-deficient mice than in control hearts. Thus, mast cell-derived renin is pivotal for activating a cardiac renin-angiotensin system leading to excessive norepinephrine release in ischemia/reperfusion. Mast cell-derived renin may be a useful therapeutic target for hyperadrenergic dysfunctions, such as arrhythmias, sudden cardiac death, myocardial ischemia, and congestive heart failure.

Ectonucleoside triphosphate diphosphohydrolase 1/CD39, localized in neurons of human and porcine heart, modulates ATP-induced norepinephrine exocytosis.

Using a guinea pig heart synaptosomal preparation, we previously observed that norepinephrine (NE) exocytosis was attenuated by a blockade of P2X purinoceptors, potentiated by inhibition of ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1)/CD39, and reduced by soluble CD39, a recombinant form of human E-NTPDase1/CD39. This suggests that norepinephrine and ATP are coreleased upon depolarization of cardiac sympathetic nerve endings and that ATP enhances norepinephrine exocytosis by an action modulated by E-NTPDase1/CD39 activity. Whether E-NTPDase1/CD39 is localized to cardiac neurons and modulates norepinephrine exocytosis in intact heart tissue remained untested. We report that E-NTPDase1/CD39 is selectively localized in human and porcine cardiac neurons and that depolarization of porcine heart tissue elicits omega-conotoxin-inhibitable release of both norepinephrine and ATP. Inhibition of E-NTPDase1/CD39 with ARL67156 markedly potentiated ATP release, demonstrating that E-NTPDase1/CD39 is a major determinant of ATP availability at sympathetic nerve terminals. Notably, inhibition of E-NTPDase1/CD39 enhanced both ATP and NE exocytosis, whereas administration of soluble CD39 reduced both ATP and NE exocytosis. The strong correlation between ATP and norepinephrine release was abolished in the presence of the purinergic P2X receptor (P2XR) antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). We conclude that released ATP governs norepinephrine exocytosis by activating presynaptic P2XR and that this action is controlled by neuronal E-NTPDase1/CD39. Clinically, excessive norepinephrine release is a major cause of arrhythmic and coronary vascular dysfunction during myocardial ischemia. By curtailing NE release, in addition to its effects as an antithrombotic agent, soluble CD39 may constitute a novel therapeutic approach to ischemic complications in the myocardium.

Histamine H3-receptor-induced attenuation of norepinephrine exocytosis: a decreased protein kinase a activity mediates a reduction in intracellular calcium.

We had reported that activation of presynaptic histamine H(3)-receptors inhibits norepinephrine exocytosis from depolarized cardiac sympathetic nerve endings, an action associated with a marked decrease in intraneuronal Ca(2+) that we ascribed to a decreased Ca(2+) influx. An H(3)-receptor-mediated inhibition of cAMP-dependent phosphorylation of Ca(2+) channels could cause a sequential attenuation of Ca(2+) influx, intraneuronal Ca(2+) and norepinephrine exocytosis. We tested this hypothesis in sympathetic nerve endings (cardiac synaptosomes) expressing native H(3)-receptors and in human neuroblastoma SH-SY5Y cells transfected with H(3)-receptors. Norepinephrine exocytosis was elicited by K(+) or by stimulation of adenylyl cyclase with forskolin. H(3)-receptor activation markedly attenuated the K(+)- and forskolin-induced norepinephrine exocytosis; pretreatment with pertussis toxin prevented this effect. Similar to forskolin, 8-bromo-cAMP elicited norepinephrine exocytosis but, unlike forskolin, it was unaffected by H(3)-receptor activation, demonstrating that inhibition of adenylyl cyclase is a pivotal step in the H(3)-receptor transductional cascade. Indeed, we found that H(3)-receptor activation attenuated norepinephrine exocytosis concomitantly with a decrease in intracellular cAMP and PKA activity in SH-SY5Y-H(3) cells. Moreover, pharmacological PKA inhibition acted synergistically with H(3)-receptor activation to reduce K(+)-induced peak intracellular Ca(2+) in SH-SY5Y-H(3) cells and norepinephrine exocytosis in cardiac synaptosomes. Furthermore, H(3)-receptor activation synergized with N- and L-type Ca(2+) channel blockers to reduce norepinephrine exocytosis in cardiac synaptosomes. Our findings suggest that the H(3)-receptor-mediated inhibition of norepinephrine exocytosis from cardiac sympathetic nerves results sequentially from H(3)-receptor-G(i)/G(o) coupling, inhibition of adenylyl cyclase activity, and decreased cAMP formation, leading to diminished PKA activity, and thus, decreased Ca(2+) influx through voltage-operated Ca(2+) channels.

Mast cells: a unique source of renin.

In addition to the traditional renin-angiotensin system, a great deal of evidence favors the existence of numerous independent tissue-specific renin-angiotensin systems. We report that mast cells are an additional source of renin and constitute a unique extrarenal renin-angiotensin system. We use renin-specific antibodies to demonstrate that cardiac mast cells contain renin. Extending this observation to the human mast cell line HMC-1, we show that these mast cells also express renin. The HMC-1 renin RT-PCR product is 100% homologous to Homo sapiens renin. HMC-1 cells also contain renin protein, as demonstrated both by immunoblot and immunocytochemical analyses. Renin released from HMC-1 cells is active; furthermore, HMC-1 cells are able to synthesize renin. It is known that, in the heart, mast cells are found in the interstitium in close proximity to nerves and myocytes, which both express angiotensin II receptors. Inasmuch as myocardial interstitium contains angiotensinogen and angiotensin-converting enzyme, and because we were able to detect renin only in mast cells, we postulate that the release of renin from cardiac mast cells is the pivotal event triggering local formation of angiotensin II. Because of the ubiquity of mast cells, our results represent a unique paradigm for understanding local renin-angiotensin systems, not just in the heart, but in all tissues. Our findings provide a rationale for targeting mast cells in conjunction with renin-angiotensin system inhibitors in the management of angiotensin II-related dysfunctions.

Coupling of angiotensin II AT1 receptors to neuronal NHE activity and carrier-mediated norepinephrine release in myocardial ischemia.

In ischemia, cardiac sympathetic nerve endings (cSNE) release excessive amounts of norepinephrine (NE) via the nonexocytotic Na(+)-dependent NE transporter (NET). NET, normally responsible for NE reuptake into cSNE, reverses in myocardial ischemia, releasing pathological amounts of NE. This carrier-mediated NE release can be triggered by elevated intracellular Na(+) levels in the axoplasm. The fact that ischemia activates the intracellular pH regulatory Na(+)/H(+) exchanger (NHE) in cSNE is pivotal in increasing intraneuronal Na(+) and thus activating carrier-mediated NE release. Angiotensin (ANG) II levels are also significantly elevated in the ischemic heart. However, the effects of ANG II on cSNE, which express the ANG II receptor, AT(1)R, are poorly understood. We hypothesized that ANG II-induced AT(1)R activation in cSNE may be positively coupled to NHE activity and thereby facilitate the pathological release of NE associated with myocardial ischemia. We tested this hypothesis in a cSNE model, human neuroblastoma cells stably transfected with rat recombinant AT(1A) receptor (SH-SY5Y-AT(1A)). SH-SY5Y-AT(1A) constitutively expresses amiloride-sensitive NHE and the NET. NHE activity was assayed in BCECF-loaded SH-SY5Y-AT(1A) as the rate of the Na(+)-dependent alkalinization in response to an acute acidosis. ANG II activation of AT(1)R markedly increased NHE activity in SH-SY5Y-AT(1A) via a Ca(2+)-dependent pathway and promoted carrier-mediated NE release. In addition, in guinea pig cSNE expressing native AT(1)R, ANG II elicited carrier-mediated NE release. In SH-SY5Y-AT(1A) and cSNE, amiloride inhibited the ANG II-mediated release of NE. Our results provide a link between AT(1)R and NHE in cSNE, which can exacerbate carrier-mediated NE release during protracted myocardial ischemia.

In vivo trans-splicing of 5' and 3' segments of pre-mRNA directed by corresponding DNA sequences delivered by gene transfer.

We have developed a new paradigm of in vivo gene transfer termed "segmental trans-splicing" (STS), in which individual "donor" and "acceptor" DNA sequences, delivered in vitro or in vivo, generate pre-mRNAs with 5' and 3' splice signals, respectively, and complementary hybridization domains through which the two pre-mRNAs interact, facilitating trans-splicing of the two mRNA fragments. To demonstrate STS, we used alpha-cobratoxin, a neurotoxin that binds irreversibly to postsynaptic nicotinic acetylcholine receptors. Cells or animals receiving both donor and acceptor plasmids, but neither plasmid alone, yielded RT-PCR products with the correct sequence of mature alpha-cobratoxin mRNA, suggesting that trans-splicing had occurred. Mice receiving intravenous administration of > or = 7.5 microg donor + acceptor plasmids, but not either plasmid alone, died within 6 h. These data demonstrate that segmental trans-splicing occurs in vivo. This approach should permit the intracellular assembly of molecules hitherto too large to be accommodated within current gene transfer vectors.