Deficient iNOS in inflammatory bowel disease intestinal microvascular endothelial cells results in increased leukocyte adhesion

Microvascular endothelial cells play a key role in inflammation by undergoing activation and recruiting circulating immune cells into tissues and foci of inflammation, an early and rate-limiting step in the inflammatory process. We have previously [Binion et al., Gastroenterology112:1898-1907, 1997] shown that human intestinal microvascular endothelial cells (HIMEC) isolated from surgically resected inflammatory bowel disease (IBD) patient tissue demonstrate significantly increased leukocyte binding in vitro compared to normal HIMEC. Our studies [Binion et al., Am. J. Physiol.275 (Gastrointest. Liver Physiol. 38):G592-G603, 1998] have also demonstrated that nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) normally plays a key role in downregulating HIMEC activation and leukocyte adhesion. Using primary cultures of HIMEC derived from normal and IBD patient tissues, we sought to determine whether alterations in iNOS-derived NO production underlies leukocyte hyperadhesion in IBD. Both nonselective (N(G)-monomethyl-L-arginine) and specific (N-Iminoethyl-L-lysine) inhibitors of iNOS significantly increased leukocyte binding by normal HIMEC activated with cytokines and lipopolysaccharide (LPS), but had no effect on leukocyte adhesion by similarly activated IBD HIMEC. When compared to normal HIMEC, IBD endothelial cells had significantly decreased levels of iNOS mRNA, protein, and NO production following activation. Addition of exogenous NO by co-culture with normal HIMEC or by pharmacologic delivery with the long-acting NO donor detaNONOate restored a normal leukocyte binding pattern in the IBD HIMEC. These data suggest that loss of iNOS expression is a feature of chronically inflamed microvascular endothelial cells, which leads to enhanced leukocyte binding, potentially contributing to chronic, destructive inflammation in IBD.     

CD8+T淋巴细胞与高血压心肌纤维化的研究进展

血管生长因子增多,血管平滑肌细胞增殖和炎症在血管重塑方面起到了关键的作用。这种低级的炎症反应导致粘附分子表达,白细胞的侵入,细胞因子的产生,氧化应激的增加,从而激活免疫细胞和血管炎症信号通路,使T淋巴细胞及巨噬细胞等细胞活化,产生和释放多种活性因子,激活心肌的细胞外基质生成细胞,引起胶原形成及代谢异常,并可导致心肌实质细胞的变性、坏死或亚细胞结构变化等,从而引起心肌纤维化一系列病理生理变化。本文主要就CD8+T淋巴细胞在高血压心肌纤维化炎症反应中的细胞毒性作用、诱导细胞凋亡作用、分泌大量的炎症因子、增加MMPs的活性从而影响心肌纤维化的形成等方面做一综述!     

The protective role of localized nitric oxide production during inflammation may be mediated by the heme oxygenase-1/carbon monoxide pathway

Nitric oxide (NO) is an important part of the host defense mechanism; however, it displays both pro- and anti-inflammatory properties depending on its location and concentration. Importantly, excessive or inappropriate NO production can cause tissue damage. Systemic and local administration of NO synthase (NOS) inhibitors ameliorates and may exacerbate the inflammatory response, respectively. Here, we used a carrageenan-induced pleurisy model of acute inflammation in rats to confirm the location-dependent effects of NO and investigate the underlying mechanisms. As expected, localized suppression of NO production exacerbated inflammation, as evidenced by increased pleural exudate volumes and leukocyte counts and enhanced activity of enzymes related to oxidative stress. In contrast, local NO supplementation reduced leukocyte infiltration, vascular permeability, and the activity of oxidative stress-related enzymes. Interestingly, inhibition of heme oxygenase-1 (HO-1) reversed the anti-inflammatory effects of localized NO production, while the addition of hemin (HO-1 substrate) or carbon monoxide (CO; HO-1 metabolite) decreased leukocyte migration and exudation. Together, these findings confirm a protective role for NO at the inflammatory site, which appears to be mediated via NOS induction of the HO-1/CO pathway. Thus, NO supplementation may be a potential new treatment for oxidative stress-associated inflammatory diseases.     

Leukocyte entry into sites of inflammation requires overlapping interactions between the L-selectin and ICAM-1 pathways.

Leukocyte interactions with vascular endothelium during inflammation depend on cascades of adhesion molecule engagement, particularly during selectin-mediated leukocyte rolling. Leukocyte rolling is also facilitated by members of the integrin and Ig families. Specifically, leukocyte rolling velocities during inflammation are significantly increased in ICAM-1-deficient mice, with ICAM-1 expression required for optimal P- and L-selectin-mediated rolling. Elimination of ICAM-1 expression in L-selectin-deficient mice significantly reduces leukocyte rolling. Whether disrupted leukocyte rolling in L-selectin and ICAM-1 double-deficient (L-selectin/ICAM-1-/-) mice affects leukocyte entry into sites of inflammation in vivo was assessed in the current study by using experimental models of inflammation; thioglycollate-induced peritonitis, chemokine-induced neutrophil migration to the skin, delayed-type hypersensitivity responses, rejection of allogeneic skin grafts, and septic shock. In many cases, the loss of both L-selectin and ICAM-1 expression dramatically reduced leukocyte migration into sites of inflammation beyond what was observed with loss of either receptor alone. In fact, the effects from loss of both L-selectin and ICAM-1 effectively eliminated multiple chronic inflammatory responses in L-selectin/ICAM-1-/- mice. By contrast, the combined loss of L-selectin and ICAM-1 expression had minimal effects on the generation of Ag-specific T cell responses or humoral immunity. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rolling and entry into tissues, which is essential for the generation of effective inflammatory responses in vivo.     

Molecular signaling blockade as a new approach to inhibit leukocyte-endothelial interactions for Inflammatory bowel disease treatment

Mitogen-activated protein kinases (MAPK) are among the major widespread transduction pathways in humans. They are involved in several inflammatory disorders, including the pathogenesis of inflammatory bowel disease (IBD). A recent paper showed that activated MAPK are up-regulated on endothelium and fibroblasts from intestinal biopsies of active IBD patients. In vitro experiments demonstrated that MAPK activation on intestinal endothelial cells and fibroblasts are responsible for the production of certain chemokines, increased leukocyte adhesion and transmigration. Specific local inhibition of MAPK activity on endothelial cells and fibroblasts may provide a new mechanism to control mucosal inflammation and leukocyte recruitment into the intestine of active IBD patients.     

Bradykinin receptor 2 extends inflammatory cell recruitment in a model of acute gouty arthritis

The aim of this study was determine the effect of bradykinin receptor antagonism on MSU crystal-induced chemokine production and leukocyte recruitment. Mice were injected intraperitoneally with monosodium urate (MSU) crystals ± bradykinin B1- or B2 receptor antagonists, Des-Arg-HOE-140 and HOE-140, respectively. MSU crystal-induced chemokine production and leukocyte recruitment in the peritoneum were measured over 24h and B1 and B2 receptor expression on leukocytes and peritoneal membrane was determined by flow cytometry and fluorescence microscopy. Data analysis showed that only B2 receptor antagonism decreased monocyte and neutrophil infiltration 24 h post MSU crystal administration. Decreased leukocyte infiltration was associated with reduced monocyte (CCL2) chemokine levels. MSU crystal-induced damage to the surrounding visceral membrane was also attenuated in the presence of B2 receptor antagonism. Together, these data show that bradykinin receptor 2 plays a role in maintaining MSU crystal-induced leukocyte infiltration and membrane permeability and identify the B2 receptor as a potential therapeutic target for managing inflammation in gout.     

Epigallocatechin gallate inhibits endothelial exocytosis

Consumption of green tea is associated with a decrease in cardiovascular mortality. The beneficial health effects of green tea are attributed in part to polyphenols, organic compounds found in tea that lower blood pressure, reduce body fat, decrease LDL cholesterol, and inhibit inflammation. We hypothesized that epigallocatechin gallate (EGCG), the most abundant polyphenol in tea, inhibits endothelial exocytosis, the initial step in leukocyte trafficking and vascular inflammation. To test this hypothesis, we treated human umbilical-vein endothelial cells with EGCG and other polyphenols, and then measured endothelial exocytosis. We found that EGCG decreases endothelial exocytosis in a concentration-dependent manner, with the effects most prominent after 4 h of treatment. Other catechin polyphenols had no effect on endothelial cells. By inhibiting endothelial exocytosis, EGCG decreases leukocyte adherence to endothelial cells. In searching for the mechanism by which EGCG affects endothelial cells, we found that EGCG increases Akt phosphorylation, eNOS phosphorylation, and nitric oxide (NO) production. NOS inhibition revealed that NO mediates the anti-inflammatory effects of EGCG. Our data suggest that polyphenols can decrease vascular inflammation by increasing the synthesis of NO, which blocks endothelial exocytosis.     

Resolution of bronchial hyperresponsiveness and pulmonary inflammation is associated with IL-3 and tissue leukocyte apoptosis

We have used two models of murine pulmonary inflammation to investigate the signals responsible for the resolution of bronchial hyperresponsiveness (BHR). Both protocols involved two sensitizations with OVA followed by serial aerosolized challenge with OVA. We determined that administration of the second sensitization by aerosol (model A) was associated with a transient response, whereas administration by the i.p. route (model B) induced a sustained response, in the form of BHR and eosinophilia. This difference in kinetics was due solely to the route of the second Ag administration and was not associated with Ag dose or adjuvant. Differences in kinetics of lung eosinophilia/BHR were shown to be independent of IgE levels and IL-4 or IL-5. However, IL-3 levels in model A closely correlated with the rate of leukocyte clearance by apoptosis and were observed concomitant with a decline in BHR. Blockage of IL-3 in model B increased leukocyte apoptosis but reduced tissue eosinophilia and BHR. The use of mouse models in which a single different administration of allergen is associated with a failure/success to resolve inflammation and BHR by 72 h postchallenge indicates a link between IL-3 production, leukocyte apoptosis, and BHR responses.     

Chemokines and myeloid cell recruitment

Chemokine-chemokine receptor interactions mediate constitutive leukocyte trafficking and leukocyte recruitment to sites of infection and inflammation. We suggest that a multiplicity of leukocyte chemoattractants exists to increase the selectivity of leukocyte recruitment in a range of physiological and pathological settings.     

The P2Y<Subscript>1</Subscript> receptor-mediated leukocyte adhesion to endothelial cells is inhibited by melatonin

Extracellular ATP (released by endothelial and immune cells) and its metabolite ADP are important pro-inflammatory mediators via the activation of purinergic P2 receptors (P2Y and P2X), which represent potential new targets for anti-inflammatory therapy. Endothelial P2Y₁ receptor (P2Y₁R) induces endothelial cell activation triggering leukocyte adhesion. A number of data have implicated melatonin as a modulator of immunity, inflammation, and endothelial cell function, but to date no studies have investigated whether melatonin modulates endothelial P2YR signaling. Here, we evaluated the putative effect of melatonin on P2Y₁R-mediated leukocyte adhesion to endothelial cells and TNF-α production, using mesenteric endothelial cells and fresh peripheral blood mononuclear cells isolated from rats. Endothelial cells were treated with the P2Y₁R agonist 2MeSATP, alone or in combination with melatonin, and then exposed to mononuclear cells. 2MeSATP increased leukocyte adhesion to endothelial cells and TNF-α production in vitro, and melatonin inhibited both effects without altering P2Y₁R protein expression. In addition, assays with the Ca²⁺ chelator BAPTA-AM indicate that the effect of melatonin on 2MeSATP-stimulated leukocyte adhesion depends on intracellular Ca²⁺ modulation. P2Y₁R is considered a potential target to control chronic inflammation. Therefore, our data unveiled a new endothelial cell modulator of purinergic P2Y₁ receptor signaling.     

Secretory leucocyte protease inhibitor inhibits interferon-gamma-induced cathepsin S expression

We have demonstrated that bronchoalveolar lavage fluid from chronic obstructive pulmonary disease patients contains higher levels of interferon-gamma compared with controls. Interferon-gamma is a potent inducer of various cathepsins and matrix metalloproteases. Therefore, we postulated that interferon-gamma could induce protease expression by macrophages in acute and chronic lung disease. Chronic obstructive pulmonary disease patients had greater levels of cathepsin S and matrix metalloprotease-12 in their bronchoalveolar lavage fluid. Macrophages incubated with chronic obstructive pulmonary disease bronchoalveolar lavage fluid exhibited increased expression of cathepsin S and matrix metalloprotease-12, which was inhibited by the addition of interferon-gamma-neutralizing immunoglobulin. Human secretory leukocyte protease inhibitor is an 11.7-kDa cationic non-glycosylated antiprotease synthesized and secreted by cells at the site of inflammation. We have demonstrated that secretory leukocyte protease inhibitor can inhibit interferon-gamma-induced cathepsin S production by macrophages. Pretreatment of macrophages with secretory leukocyte protease inhibitor inhibited interferon-gamma-induced inhibitor kappaB beta degradation and activation of nuclear factor kappaB. Secretory leukocyte protease inhibitor may prove to be therapeutically important as a potential inhibitor of protease expression in chronic obstructive pulmonary disease.     

Allium sativum (garlic) suppresses leukocyte inflammatory cytokine production in vitro: potential therapeutic use in the treatment of inflammatory bowel disease

BACKGROUND: Cytokines involved in inflammatory bowel disease (IBD) direct a predominantly cell-mediated T- helper-1 (Th1) immune response. The nonspecific anti-inflammatory treatment being used in the management of patients with IBD has not changed much since the 1970s and new therapeutic agents are keenly sought. Several compounds isolated from Allium sativum (garlic) modulate leukocyte cell proliferation and cytokine production. METHODS: To investigate the possible therapeutic effects of garlic in the treatment of patients with IBD, whole blood and peripheral blood mononuclear cells (PBMCs) were stimulated in the presence of various concentrations of garlic extract and the effect on leukocyte cytokine production was determined in vitro using multiparameter flow cytometry. RESULTS: Monocyte interleukin (IL)-12 production was inhibited significantly in the presence of low concentrations of garlic extract (>or=0.1 microg/ml total protein). Monocyte IL-10 production increased significantly and monocyte tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, IL-6, IL-8, T-cell interferon-gamma (IFN-gamma), IL-2, and TNF-alpha decreased significantly in the presence of >or=10 microg/ml garlic extract. Twenty to fifty percent of the immunomodulatory activity of garlic extract on cytokine production was acid labile. The inhibitory activity of methylprednisolone, a commonly used anti-inflammatory in IBD, with garlic on leukocyte cytokine production was additive. CONCLUSIONS: By inhibiting Th1 and inflammatory cytokines while upregulating IL-10 production, treatment with garlic extract may help to resolve inflammation associated with IBD. An in vivo animal model study needs to be undertaken to determine the significance of these in vitro findings.     

Rapid up-regulation of CXC chemokines in the airways after Ag-specific CD4+ T cell activation

Ag-specific activation of CD4(+) T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4(+) T cell activation is important in lung chemokine production is currently not clear. Using alphabeta-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4(+) T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11. 10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4(+) T cell activation was critical because CD4(+) but not CD8(+) T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4(+) T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4(+) T cells with APCs. A likely secondary mediator was TNF-alpha, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4(+) T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.     

ICAM-1 and CD11b/CD18 expression during acute pancreatitis induced by bile-pancreatic duct obstruction: effect of N-acetylcysteine

Different molecules are involved in the recruitment of leukocytes during inflammation. The aim was to investigate (i) the contribution of acinar cells to the overall production of ICAM-1 and (ii) the kinetics of leukocyte CD11b/CD18 expression during acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) to evaluate the contribution of both molecules to leukocyte homing. The role of reactive oxygen species (ROS) as mediators in the expression of ICAM-1 and CD11b/CD18 was examined by using N-acetylcysteine (NAC) as an antioxidant treatment. By mechanisms resistant to NAC treatment, acinar cells were able to produce ICAM-1 at first onset of AP; other cell sources contribute to maintaining increased ICAM-1 plasma levels during AP. By contrast, CD11b/CD18 was overexpressed in leukocytes in the course of AP by oxidant-dependent mechanisms. Since NAC treatment reduced neutrophil infiltration in the pancreas, we conclude that CD11b/CD18 over-expression is required for leukocyte recruitment; however, other adhesion molecules in addition to ICAM-1 seem to contribute to leukocyte homing during BPDO-induced AP.     

Correlated expression of adhesive properties for both white and red blood cells during inflammation

The state of leukocyte and erythrocyte adhesiveness/aggregation was determined in the peripheral blood of 382 patients with infection/inflammation as well as in 72 controls by using a simple slide test and image analysis. A highly significant correlation (r = 0.4, n = 455, p < 0.001) was found between the state of leukocyte and erythrocyte adhesiveness/aggregation. The extent of both leukocyte and erythrocyte aggregation correlated with the concentration of fibrinogen. Significant aggregation of leukocytes with erythrocytes was noted as well. We conclude that both leukocyte and erythrocyte aggregation occur in the peripheral blood of patients with infection/inflammation. Such cell aggregation, which might have detrimental rheological consequences, can be detected by using our novel technique.     

Phospholipase C Activator m-3M3FBS Protects against Morbidity and Mortality Associated with Sepsis

Although phospholipase C (PLC) is a crucial enzyme required for effective signal transduction and leukocyte activation, the role of PLC in polymicrobial sepsis remains unclear. In this study, we show that the direct PLC activator m-3M3FBS treatment significantly attenuates vital organ inflammation, widespread immune cell apoptosis, and mortality in a mouse sepsis model induced by lethal cecal ligation and puncture challenge. Mechanistically, m-3M3FBS-dependent protection was largely abolished by pretreatment of mice with the PLC-selective inhibitor U-73122, thus confirming PLC agonism by m-3M3FBS in vivo. PLC activation enhanced the bactericidal activity and hydrogen peroxide production of mouse neutrophils, and it also enhanced the production of IFN-γ and IL-12 while inhibiting proseptic TNF-α and IL-1β production in cecal ligation and puncture mice. In a second model of sepsis, PLC activation also inhibited the production of TNF-α and IL-1β following systemic LPS challenge. In conclusion, we show that agonizing the central signal transducing enzyme PLC by m-3M3FBS can reverse the progression of toxic shock by triggering multiple protective downstream signaling pathways to maintain organ function, leukocyte survival, and to enhance microbial killing.     

Mechanisms of the anti-inflammatory actions of the angiotensin type 1 receptor antagonist losartan in experimental models of arthritis

Angiotensin (Ang) II and its AT₁ receptors have been implicated in the pathogenesis of rheumatoid arthritis. Activation of the counter-regulatory Ang-(1–7)–Mas receptor axis may contribute to some of the effects of AT₁ receptor blockers (ARBs). In this study, we have used losartan, an ARB, to investigate the role of and the mechanisms by which AT₁ receptors participated in two experimental models of arthritis: antigen-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Treatment with losartan decreased neutrophil recruitment, hypernociception and the production of TNF-α, IL-1β and chemokine (C–X–C motif) ligand 1 in mice subjected to AIA. Histopathological analysis showed significant reduction of tissue injury and inflammation and decreased proteoglycan loss. In addition to decreasing cytokine production, losartan directly reduced leukocyte rolling and adhesion. Anti-inflammatory effects of losartan were not associated to Mas receptor activation and/or Ang-(1–7) production. Anti-inflammatory effects were reproduced in rats subjected to AdIA. This study shows that ARBs have potent anti-inflammatory effects in animal models of arthritis. Mechanistically, reduction of leukocyte accumulation and of joint damage was associated with local inhibition of cytokine production and direct inhibition of leukocyte–endothelium interactions. The anti-inflammatory actions of losartan were accompanied by functional improvement of the joint, as seen by reduced joint hypernociception. These findings support the use of ARBs for the treatment of human arthritis and provide potential mechanisms for the anti-inflammatory actions of these compounds.     

Direct in vivo monitoring of acute allergic reactions in human conjunctiva

Immediate allergic reactions are initiated by allergen-induced, specific IgE-mediated mast cell degranulation and involve leukocyte recruitment into the inflamed site. We compared conjunctival signs, symptoms, and in vivo leukocyte rolling and extravasation into sites of inflammation in five patients allergic to birch pollen and in 10 nonallergic controls who received a challenge to birch allergen or histamine. Both the specific allergen in allergic patients and histamine, both in patients and in healthy controls, induced symptoms and signs of an immediate allergic reaction together with leukocyte rolling within the conjunctival blood vessels. However, only allergen, not histamine, caused leukocyte extravasation into the site of inflammation in the allergic patients. Allergen also increased expression of endothelial P-selectin in conjunctival vessels and slowed the rolling of leukocytes which is required for their extravasation from blood circulation into the target tissue. Finally, i.v. heparin strongly reduced the number of slowly rolling cells during allergen- or histamine-induced reactions and this can probably hinder the leukocyte extravasation after allergen exposure. These findings suggest that slow rolling is required for leukocyte extravasation in acute allergic reactions, and it can be inhibited by heparin in vivo in therapeutically relevant conditions.     

Novel aspects of fibrin(ogen) fragments during inflammation

Coagulation is fundamental for the confinement of infection and/or the inflammatory response to a limited area. Under pathological inflammatory conditions such as arthritis, multiple sclerosis or sepsis, an uncontrolled activation of the coagulation system contributes to inflammation, microvascular failure and organ dysfunction. Coagulation is initiated by the activation of thrombin, which, in turn, triggers fibrin formation by the release of fibrinopeptides. Fibrin is cleaved by plasmin, resulting in clot lysis and an accompanied generation of fibrin fragments such as D and E fragments. Various coagulation factors, including fibrinogen and/or fibrin [fibrin(ogen)] and also fibrin degradation products, modulate the inflammatory response by affecting leukocyte migration and cytokine production. Fibrin fragments are mostly proinflammatory, however, Bβ15-42 in particular possesses potential antiinflammatory effects. Bβ15-42 inhibits Rho-kinase activation by dissociating Fyn from Rho and, hence prevents stress-induced loss of endothelial barrier function and also leukocyte migration. This article summarizes the state-of-the-art in inflammatory modulation by fibrin(ogen) and fibrin fragments. However, further research is required to gain better understanding of the entire role fibrin fragments play during inflammation and, possibly, disease development.