IL-1ß reduces tonic contraction of mesenteric lymphatic muscle cells, with the involvement of cycloxygenase-2 and prostaglandin E2.

BACKGROUND AND PURPOSE: The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, 'lymphangions'. Here we have studied the effects of the inflammatory cytokine IL-1ß on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC). EXPERIMENTAL APPROACH: We measured IL-1ß in colon-conditioned media (CM) from acute (AC-CM, dextran sodium sulfate) and chronic (CC-CM, T-cell transfer) colitis-induced mice and corresponding controls (Con-AC/CC-CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h-IL-1ß or h-TNF-α (5, 10, 20 ng·mL(-1) ), with or without COX inhibitors [TFAP (10(-5) M), diclofenac (0.2 × 10(-5) M)], PGE2 (10(-5) M)], IL-1-receptor antagonist, Anakinra (5 µg·mL(-1) ), or a selective prostanoid EP4 receptor antagonist, GW627368X (10(-6) and 10(-7) M). KEY RESULTS: Tonic contractility of RMLMC was reduced by AC- and CC-CM compared with corresponding control culture media, Con-AC/CC-CM. IL-1ß or TNF-α was not found in Con-AC/CC-CM, but detected in AC- and CC-CM. h-IL-1ß concentration-dependently decreased RMLMC contractility, whereas h-TNF-α showed no effect. Anakinra blocked h-IL-1ß-induced RMLMC relaxation, and with AC-CM, restored contractility to RMLMC. IL-1ß increased COX-2 protein and PGE2 production in RMLMC.. PGE2 induced relaxations in RMLMC, comparable to h-IL-1ß. Conversely, COX-2 and EP4 receptor inhibition reversed relaxation induced by IL-1ß. CONCLUSIONS AND IMPLICATIONS: The IL-1ß-induced decrease in RMLMC tonic contraction was COX-2 dependent, and mediated by PGE2 . In experimental colitis, IL-1ß and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC-CM and pharmacological blockade of IL-1ß restored tonic contraction.

Lymphatic system: an active pathway for immune protection.

Lymphatic vessels are well known to participate in the immune response by providing the structural and functional support for the delivery of antigens and antigen presenting cells to draining lymph nodes. Recent advances have improved our understanding of how the lymphatic system works and how it participates to the development of immune responses. New findings suggest that the lymphatic system may control the ultimate immune response through a number of ways which may include guiding antigen/dendritic cells (DC) entry into initial lymphatics at the periphery; promoting antigen/DC trafficking through afferent lymphatic vessels by actively facilitating lymph and cell movement; enabling antigen presentation in lymph nodes via a network of lymphatic endothelial cells and lymph node stroma cell and finally by direct lymphocytes exit from lymph nodes. The same mechanisms are likely also important to maintain peripheral tolerance. In this review we will discuss how the morphology and gene expression profile of the lymphatic endothelial cells in lymphatic vessels and lymph nodes provides a highly efficient pathway to initiate immune responses. The fundamental understanding of how lymphatic system participates in immune regulation will guide the research on lymphatic function in various diseases.

Review article: lymphatic system and associated adipose tissue in the development of inflammatory bowel disease.

BACKGROUND: The lymphatic system plays critical roles in tissue fluid homoeostasis, immune defence and metabolic maintenance. Lymphatic vessels transport lymph, proteins, immune cells and digested lipids, allowing fluid and proteins to be returned to the blood stream, lipids to be stored and metabolized and antigens to be sampled in lymph nodes. Lymphatic drainage is mainly driven by rhythmic constrictions intrinsic to the vessels and critically modulated by fluid pressure and inflammatory mediators. AIM: To collect and discuss the compelling available information linking the lymphatic system, adiposity and inflammation. METHODS: A literature search was performed through PubMed focusing on lymphatic system, inflammation, immune cells and fat transport and function in the context of IBD. RESULTS: Evidence collected allows us to propose the following working model. Compromised lymph drainage, reported in IBD, leads to oedema, lymphangiogenesis, impaired immune cell trafficking and lymph leakage. Lymph factor(s) stimulate adipose tissue to proliferate and produce cytokines, which affect immune cell functions and exacerbate inflammation. CONCLUSIONS: Understanding the lymphatic system's role in immune cell trafficking and immune responses, contribution to fat transport, distribution, metabolism and implication in the pathogenesis of chronic intestinal inflammation may provide the basis for new therapeutic strategies and improved quality-of life.

Octreotide in intestinal lymphangiectasia: lack of a clinical response and failure to alter lymphatic function in a guinea pig model.

Intestinal lymphangiectasia, which can be classified as primary or secondary, is an unusual cause of protein-losing enteropathy. The main clinical features include edema, fat malabsorption, lymphopenia and hypoalbuminemia. Clinical management generally includes a low-fat diet and supplementation with medium chain triglycerides. A small number of recent reports advocate the use of octreotide in intestinal lymphangiectasia. It is unclear why octreotide was used in these studies; although octreotide can alter splanchnic blood flow and intestinal motility, its actions on lymphatic function has never been investigated. A case of a patient with intestinal lymphangiectasia who required a shunt procedure after failing medium chain triglycerides and octreotide therapy is presented. During the management of this case, all existing literature on intestinal lymphangiectasia and all the known actions of octreotide were reviewed. Because some of the case reports suggested that octreotide may improve the clinical course of intestinal lymphangiectasia by altering lymphatic function, a series of experiments were undertaken to assess this. In an established guinea pig model, the role of octreotide in lymphatic function was examined. In this model system, the mesenteric lymphatic vessels responded to 5-hydroxytryptamine with a decrease in constriction frequency, while histamine administration markedly increased lymphatic constriction frequency. Octreotide failed to produce any change in lymphatic function when a wide range of concentrations were applied to the mesenteric lymphatic vessel preparation. In conclusion, in this case, octreotide failed to induce a clinical response and laboratory studies showed that octreotide did not alter lymphatic function. Thus, the mechanisms by which octreotide induced clinical responses in the cases reported elsewhere in the literature remain unclear, but the present study suggests that it does not appear to act via increasing lymphatic pumping.

Review article: lymphatic vessel pumping and inflammation--the role of spontaneous constrictions and underlying electrical pacemaker potentials.

The lymphatic circulation is important in maintaining tissue fluid homeostasis. It removes fluid, proteins and other particles from tissue spaces and returns them to the blood stream. This function is achieved by rhythmical contractions of the collecting lymphatic vessels. The contractile mechanism is intrinsic to the smooth muscles present in the vessel wall and consequent to action potentials. The underlying electrical mechanism has been proposed to be due to rhythmic synchronization of Ca2+-dependent spontaneous transient depolarizations. The lymphatic pumping activity adapts to changes in fluid load and has been observed to augment during inflammatory reactions to help resolve the associated oedema. This beneficial action has been generally attributed to the increase in interstitial pressure consequent to the oedema. However, little attention has been paid to the possible role inflammatory mediators that are present in the lymphatic vessel environment, could play in directly affecting the lymphatic contractile mechanism. This review article discusses our current knowledge on the mechanism and initiation of lymphatic pumping and how these events are modulated during inflammatory conditions.

Nitric oxide decreases pacemaker activity in lymphatic vessels of guinea pig mesentery.

Intracellular microelectrode recordings were used to determine whether nitric oxide (NO), affects the pacemaker events that initiate vasomotion in lymphatic vessels of the guinea pig mesentery. This pacemaker activity is recorded as spontaneous transient depolarizations (STDs) and is likely to arise through synchronized Ca2+ release from intracellular stores. We show here that acetylcholine-induced endothelium-derived NO and exogenous NO released by sodium nitroprusside (SNP; 100 microM) and DEA-NONOate (500 microM) reduced the frequency and amplitude of STDs. This inhibition of STD frequency and amplitude was independent of the NO-induced hyperpolarization of the smooth muscle. The SNP-induced inhibition of STD frequency and amplitude was abolished during superfusion with the soluble guanylyl cyclase inhibitor ODQ (10 microM) and was diminished in the presence of cGMP and cAMP-dependent protein kinase inhibitors. The data are consistent with the hypothesis that NO inhibits vasomotion primarily by production of cGMP and activation of both cGMP- and cAMP-dependent protein kinases, which reduce the size and frequency of STDs, probably by acting on the underlying synchronized Ca2+ release from intracellular stores.

ATP-sensitive K+ channels in smooth muscle cells of guinea-pig mesenteric lymphatics: role in nitric oxide and beta-adrenoceptor agonist-induced hyperpolarizations.

1. Intracellular microelectrode recordings were performed to investigate the membrane K+ conductances involved in smooth muscle hyperpolarization of lymphatic vessels in the guinea-pig mesentery. 2. Nitric oxide (NO), released either by the endothelium after acetylcholine (ACh; 10 microM) stimulation or by sodium nitroprusside (SNP; 50-100 microM), hyperpolarized lymphatic smooth muscle. These responses were inhibited with the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ, 10 microM). 3. ACh and SNP-induced hyperpolarizations were inhibited (by about 90%) upon application of the ATP-sensitive K+(K(ATP)) channel blocker, glibenclamide (10 microM), or with 4-aminopyridine (2.5 mM), but were not affected by the Ca2+-activated K+ channels blocker, penitrem A (100 nM). 4. Hyperpolarization caused by the K+ channel opener, cromakalim (0.1-10 microM), isoprenaline (0.1 microM) or forskolin (0.5 microM) were all significantly blocked by glibenclamide. 5. Hyperpolarization evoked by ACh and SNP were inhibited with N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dich loride (H89, 10 microM), suggesting the involvement of cyclic AMP dependent protein kinase (PKA). 6. These results suggest that K(ATP) channels play a central role in lymphatic smooth muscle hyperpolarization evoked by a NO-induced increase in cyclic GMP synthesis, as well as by beta-adrenoceptor-mediated production of cyclic AMP. Interestingly, both pathways lead to K(ATP) channels opening through the activation of PKA.

Functional electrical properties of the endothelium in lymphatic vessels of the guinea-pig mesentery.

1. The resting and agonist-stimulated properties of endothelial cells and electrical communication between the endothelium and smooth muscle were investigated in open segments of guinea-pig mesenteric lymphatic vessels using intracellular microelectrodes. 2. Endothelial cells had a mean resting membrane potential (RMP) of -71.5 +/- 0.5 mV (n = 100) which was significantly different from the value of -60.8 +/- 1.1 mV (n = 75) recorded in smooth muscle. 3. Acetylcholine (ACh, 5-10 microM) generally evoked an initial hyperpolarization followed by depolarization (mean 3.4 +/- 0.5 mV and 15.4 +/- 1.0 mV, respectively, n = 75). 4. Ca(2+)-activated K+ channels were likely to underlie the ACh-induced hyperpolarization as this response exhibited an increased in membrane conductance, was larger in 0.5 mM K+ solution and was blocked by charybdotoxin (50 nM). 5. The endothelium did not exhibit a response to nitric oxide (NO) as the NO-donor sodium nitroprusside did not alter the RMP and the electrical responses to ACh were not affected by the NO-synthase inhibitor N omega-nitro L-arginine at a concentration which markedly inhibited smooth muscle hyperpolarization. 6. Electrical coupling between the endothelium and smooth muscle was not functional as there was extremely limited electrical continuity (1 in 12, endothelial/smooth muscle cell simultaneous recordings) and bradykinin, noradrenaline and isoprenaline caused different electrical responses in the two cell types. 7. These results provide the first description of RMP and electrical responses to various agonists in the lymphatic endothelium and its lack of functional electrical coupling with the smooth muscle.

Co-ordination of contractile activity in guinea-pig mesenteric lymphatics.

1. Intraluminally perfused lymphatic vessels from the mesentery of the guinea-pig were examined in vitro to investigate their contractile activity and the co-ordination of this activity between adjacent lymphangions. 2. Lymphangions constricted at fairly regular intervals and exhibited 'refractory' periods of up to 3 s during which constrictions did not occur. 3. The contractile activity of adjacent lymphangions was highly co-ordinated. 4. The smooth muscle was found to be continuous between the adjacent lymphangions for the majority of valve regions examined morphologically (52 of 63 preparations). 5. Mechanical and electrical coupling between adjacent lymphangions was indicated, as some lymphangions underwent transient dilatations just prior to constriction, whereas direct electrophysiological measurements showed that the smooth muscle of most adjacent lymphangions was electrically coupled across the valve (15 out of 20 pairs of lymphangions). 6. It is concluded that perfused lymphangions of guinea-pig mesenteric lymphatic vessels rhythmically constrict, with the contractile activity of adjacent lymphangions highly co-ordinated. The findings also indicate that transmission of both mechanical and electrical signals between the adjacent lymphangions contribute to the co-ordination of their contractile activity.

Endothelium-dependent modulation of pacemaking in lymphatic vessels of the guinea-pig mesentery.

1. Endothelial control of the rate of constrictions and the underlying pacemaker potentials has been studied in vitro in guinea-pig mesenteric lymphatic vessels. 2. ACh stimulated 60% of intraluminally perfused vessels to slow or abolish lymphatic constrictions. This action was inhibited by atropine and was likely to be due to the release of endothelium-derived nitric oxide (EDNO) as the effect was absent after endothelial lysis, mimicked by sodium nitroprusside (SNP), blocked by N omega-nitro L-arginine (NOLA) and partially inhibited by Methylene Blue (MB). 3. The remaining 40% of perfused vessels did not mechanically respond to ACh or SNP. In four of seven such vessels this appeared to be due to excessive perfusion-associated release of EDNO, as incubation with NOLA restored the response to SNP. 4. Application of NOLA or MB in perfused vessels significantly increased constriction frequency, further indicating perfusion-associated release of EDNO. 5. ACh induced a marked increase in endothelial [Ca2+]i of both mechanically responding and non-responding vessels. This ACh-induced increase could be repetitively induced when Ca2+ was present in the perfusate, but rapidly ran down when a Ca(2+)-free EGTA perfusate was used. 6. Intracellular recordings from the smooth muscle of non-perfused vessel segments demonstrated an ACh-induced hyperpolarization and decrease in membrane resistance, changes which were prevented by atropine, NOLA, MB and endothelial lysis and mimicked by SNP. 7. ACh directly reduced the size of the underlying pacemaker potentials termed spontaneous transient depolarizations (STDs). 8. NOLA and MB enhanced STDs in non-perfused vessel segments indicating an endogenous release of EDNO. 9. It is concluded that the lymphatic endothelium produces and releases EDNO endogenously, during perfusion or after stimulation with ACh, to decrease the efficacy of STDs to generate action potentials and resultant constrictions.

Beta-adrenoceptor-mediated hyperpolarization in lymphatic smooth muscle of guinea pig mesentery.

Intracellular microelectrode recordings were performed to investigate the consequences of beta-adrenoceptor activation in smooth muscle of guinea pig mesenteric lymphatic vessels. Isoproterenol (Iso) hyperpolarized the membrane with an associated increase in membrane conductance and decreased the amplitude of spontaneous transient depolarizations. Iso effects were mimicked by forskolin (FSK), 3-isobutyl-1-methylxanthine, and two adenosine 3',5'-cyclic monophosphate (cAMP) derivatives. Iso- and FSK-induced hyperpolarizations were inhibited by H89, an inhibitor of cAMP-dependent protein kinase A, increased in K+-free solution, but were not affected by ouabain or by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine. They were partially inhibited by 20 mM tetraethylammonium (approximately 40%) or by 2.5 mM 4-aminopyridine (approximately 55%). The-Iso-induced hyperpolarization was partially inhibited by iberiotoxin (20 nM) and charybdotoxin (40 nM), whereas the FSK-induced hyperpolarization was less affected. In cells where the Iso-induced hyperpolarization was decreased by 40 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, acetoxymethyl ester form, the FSK-induced hyperpolarization was little changed. Our results indicate that in guinea pig mesenteric lymphatic vessels, beta-adrenoceptor stimulation activates a protein kinase A-dependent K+ conductance, involving more than one channel type.

Simultaneous oscillations in the membrane potential of pig coronary artery endothelial and smooth muscle cells.

1. The effects of tetrabutylammonium (TBA) on the mechanical tension and on the electrical behaviour of endothelial and smooth muscle cells were studied in intact porcine coronary artery strips. 2. Superfusion of strips with TBA (2-20 mM) induced mechanical oscillations, leading to an increase in tonic isometric tension. 3. TBA-induced mechanical oscillations were correlated with fluctuations of the membrane potential of endothelial cells, which were identified by iontophoretic injection of Lucifer Yellow. 4. The endothelial cell membrane potential fluctuations appeared as action potentials or smaller amplitude slow waves, and were synchronized with electrical membrane potential fluctuations of the underlying coronary smooth muscle cells. 5. Oscillations induced by TBA in smooth muscle cells were not affected by removal of the endothelium, and depended on the presence of calcium in the external medium. 6. To our knowledge, this is the first description of action potential-like fluctuations in the endothelium. It is concluded that the oscillations were generated in the smooth muscle and that they propagate to the endothelium. The question of the mode of propagation of the signal is discussed.

Effects of ATP on cultured smooth muscle cells from rat aorta.

1. Membrane ionic currents provoked by externally applied ATP were studied by patch-clamp techniques in cultured aortic smooth muscle cells of the rat. 2. Using standard bath and pipette solutions and whole-cell voltage-clamp, ATP evoked an inward current when the cell membrane potential was held at -50 mV and an outward current when the potential was held at 30 mV, with a reversal potential near -10 mV. 3. Application of ATP gamma S gave results similar to those obtained with ATP, while adenosine, AMP and alpha,beta-methylene ATP were ineffective. The ATP-activated current was inhibited by suramin, 100 microM. 4. ATP also induced a biphasic rise in internal free Ca levels as shown directly by Fura-2 measurements and by the increase in Ca-dependent K single-channel activity in cell-attached patches. 5. With outward current through K channels blocked by internal Cs and TEA, modification of the ionic composition of bath and pipette solutions revealed that the reversal potential for the ATP-induced whole-cell current closely followed ECl, the chloride equilibrium potential, and was insensitive to manipulations of the monovalent cation gradient. 6. These results indicate that in rat cultured aortic smooth muscle cells, ATP binding to P2-purinoceptors produces increases of internal free Ca levels and subsequent activation of both Ca-dependent K and Cl currents.

Effect of nitro-L-arginine on endothelium-dependent hyperpolarizations and relaxations of pig coronary arteries.

1. Endothelium-dependent relaxation is caused by an endothelium-derived relaxing factor (EDRF) identified as nitric oxide (NO). Our objective was to test whether one or several distinct endothelium-dependent relaxing factors exist. 2. In pig coronary arteries, a hyperpolarization accompanied by the relaxation caused by high concentrations of substance P (SP) and bradykinin (BK). 3. To examine the role played by nitric oxide and prostacyclin in the endothelium-dependent relaxations and hyperpolarizations caused by SP and BK on pig coronary arterial strips, the production of NO was inhibited by NG-nitro-L-arginine (L-NNA) and the production of prostacyclin was inhibited by indomethacin, while monitoring smooth muscle membrane potential and isometric tension. 4. Indomethacin had no effect on resting isometric tension nor on SP and BK relaxations of strips precontracted by prostaglandin F2 alpha. 5. L-NNA contracted arterial strips with intact endothelium, without changing the membrane potential of smooth muscles. 6. The inhibitor shifted to the right the concentration-response curve of kinins by 0.2 nM SP and 20 nM BK. It inhibited the maximal relaxations and hyperpolarizations by 30%. 7. The results show that, in pig coronary arteries, EDRF (NO) mainly controls the basal tension, whereas other factor(s) play(s) an important role in hyperpolarizations and relaxations caused by the kinins.

Effect of Ca2+ ionophores on membrane potential of pig coronary artery endothelial cells.

Ca2+ ionophores (A23187 and ionomycin) were used to determine whether an increase in cytosolic Ca2+ plays a direct role in pig coronary endothelial cell hyperpolarization. Ionophores induced concentration-dependent hyperpolarizations that were not altered by the presence of N omega-nitro-L-argnine (L-NNA), and inhibitor of nitric oxide synthesis. d-Tubocurarine decreased by 65-89% the A23187- and substance P (SP)-generated hyperpolarization of endothelial cells. To study the role of endothelial cell hyperpolarization in the endothelium-dependent relaxation of precontracted coronary artery strips, A23187 and SP concentration-response curves were built up in the presence of d-tubocurarine and/or L-NNA. A decrease in the maximal response was observed only when both d-tubocurarine and L-NNA were present. Our direct in situ approach gives results in agreement with a gating of Ca(2+)-activated K+ channels during A23187- and SP-induced hyperpolarizations of endothelial cells. We suggest that these hyperpolarizations play a role in the endothelial cell-dependent relaxation induced by A23187 and SP in the pig coronary artery.

Hydrogen peroxide: an endogenous smooth muscle cell hyperpolarizing factor.

Hydrogen peroxide can be released by different cells such as the nerves, the endothelial or phagocytotic white blood cells which can all interact with vascular smooth muscles. We show that hydrogen peroxide hyperpolarizes and relaxes pig coronary artery smooth muscle cells. The possibility that the endothelium derived hyperpolarizing factor released by the endothelium in response to bradykinin and substance P being hydrogen peroxide was tested using catalase, an enzyme which hydrolyses hydrogen peroxide. We find that this particular endothelial hyperpolarizing factor and hydrogen peroxide are two distinct molecules.

Identification of a 120-kD surface glycoprotein distinguishing cultured superior cervical ganglion from ciliary ganglion neurons.

Cell recognition during development of the nervous system involves specific interactions between neuronal cell surface molecules and their environment. Thus one type of neuron could carry on its surface a molecule which allows it to be distinguished from other types of neurons. We have tried to identify such specific components by comparing cell surfaces of cultured chick sympathetic (superior cervical ganglion) and parasympathetic (ciliary ganglion) neurons. Using metabolic labeling with (3H)-fucose or surface-labeling with the galactose oxidase-tritiated sodium borohydride method, we have identified a glycoprotein with an apparent molecular weight of 120 kD which is present on superior cervical ganglion neurons, but can barely be revealed on ciliary ganglion neurons. This molecule thus distinguishes two subsets of neurons and might therefore play a role in mediating specific interactions between the sympathetic neurons and their environment.