Dehydration attenuates plasma prolactin response to suckling through a dopaminergic mechanism.

The tuberoinfundibular dopaminergic neurons projecting to the median eminence are well accepted as a major physiological regulator of adenohypophyseal PRL secretion. However, recent evidence has shown that dopamine (DA) in the neurointermediate lobe also has an inhibitory effect on PRL secretion by anterior pituitary. Since the neurointermediate is innervated by the tuberohypophyseal dopaminergic (THDA) neurons, which is known to be selectively activated by dehydration of the animal, the aim of this study was to investigate the physiological role of the THDA system in PRL release during lactation. On the day of the experiments, the litters were separated from the mothers for 4 h before initiation of the suckling stimulus. The suckling-induced PRL surge was detected on three consecutive days. On the first day the normal response was tested; then immediately after taking the last blood samples, drinking solutions were changed to the high salt (2.5% saline) containing bottles or were taken away. Suckling-induced PRL response was significantly decreased after 24 h and almost completely blocked 48 h later in dehydrated mothers. This effect could be prevented by haloperidol (a DA receptor antagonist) pretreatment (0.1 mg/kg BW sc), and it was only transient because rehydration of the mothers reestablished basal as well as suckling-induced PRL response. In addition, the effect of an acute osmotic stimulus on the plasma PRL levels (injecting 0.5 ml 10% saline solution iv) was also tested. There was a marked and immediate decrease in PRL concentration within 15 min of injection. Domperidone, another DA receptor blocker (20 micrograms/rat iv) completely abolished the depletion of plasma PRL in response to 10% saline injection. These results support our assumption that the dopaminergic regulation of PRL secretion during lactation involves the THDA system. Furthermore, these data underline the importance of an interaction between regulation of PRL secretion and water and sodium homeostasis.     

Neutrophil migration into indomethacin induced rat small intestinal injury is CD11a/CD18 and CD11b/CD18 co-dependent

BACKGROUND: Neutrophils may exacerbate intestinal inflammatory diseases through secretion of proteolytic enzymes and reactive oxygen and nitrogen intermediates. AIMS: To define the mechanisms involved in neutrophil infiltration into the non-steroidal anti-inflammatory disease inflamed intestine to develop strategies to regulate this process. METHODS: The small intestinal epithelium of (15 mg/kg) indomethacin treated rats was examined for cytokine mRNA. The kinetics of neutrophil accumulation into the gastrointestinal tract (including lumen contents) of inflamed rats was determined using radiolabelled (111In) neutrophils injected intravenously followed by a three hour migration period. To determine which adhesion molecules were critical for migration, rats were also injected with function blocking monoclonal antibodies to the beta2 (CD11/CD18) integrins. RESULTS: Interleukin 1beta, interleukin 1 receptor II, tumour necrosis factor alpha, and monocyte inflammatory peptide 2 but not monocyte chemoattractant protein 1 mRNA were detected in the epithelium within hours of indomethacin injection. Neutrophils were detectable in the small intestine and intestinal lumen by six hours and continued to accumulate until 48 hours post indomethacin injection. Neutrophil accumulation in the intestine was essentially blocked by anti-CD18, and partially blocked by either anti-CD11a or CD11b antibody treatment. Migration into the intestinal lumen was reduced by anti-CD11b. CONCLUSIONS: The small intestinal epithelium acts as one source of cytokines with properties important in the recruitment of neutrophils. In turn, neutrophil migration into the indomethacin inflamed small intestine is mediated by CD11a/CD18 and CD11b/CD18.     

Heparin impairs glycocalyx barrier properties and attenuates shear dependent vasodilation in mice

The endothelial glycocalyx is a hydrated mesh of polysaccharides and adsorbed plasma proteins that forms the true interface between the flowing blood and the endothelium. We hypothesized in the present study that competitive binding of heparin to glycocalyx-associated proteins would affect glycocalyx barrier properties and mechanotransduction of shear stress to the endothelium. In anesthetized mice, the clearance of 70-kDa dextrans from the circulation was increased (P<0.05 versus saline) 1 hour after heparin (1.25 U) and glycocalyx degradation with hyaluronidase (35 U; amount cleared in 30 minutes after saline: 11+/-5%; after heparin: 45+/-8%; after hyaluronidase: 30+/-3%). Clearance of 40-kDa dextrans increased (P<0.05 versus saline) to a lesser extent after both treatments (saline: 46+/-3%; heparin: 60+/-5%; hyaluronidase: 60+/-2%). The dilator response of second-order arterioles in cremaster muscle during reactive hyperemia was reduced for < or =90 minutes after heparin as reflected by a decrease (P=0.008) in t(50) of diameter recovery, and this effect was associated with a diminished NO bioavailability. Infusion of hyaluronidase resulted in reductions (P<0.05) in baseline and peak reactive hyperemic diameter, whereas, despite an increase in wall shear rate at the beginning of reactive hyperemia, t(50) of diameter recovery was not affected. In conclusion, our data in mice show that a heparin challenge is associated with increased vascular leakage of dextrans and impaired arteriolar vasodilation during reactive hyperemia. Our data suggest that protein-heparan sulfate interactions are important for a functional glycocalyx.     

Myeloperoxidase delays neutrophil apoptosis through CD11b/CD18 integrins and prolongs inflammation

Polymorphonuclear neutrophil granulocytes have a central role in innate immunity and their programmed cell death and removal are critical for efficient resolution of acute inflammation. Myeloperoxidase (MPO), a heme protein abundantly expressed in neutrophils, is generally associated with killing of bacteria and oxidative tissue injury. Because MPO also binds to neutrophils, we investigated whether MPO could affect the lifespan of neutrophils. Here, we report that MPO independent of its catalytic activity through signaling via the adhesion molecule CD11b/CD18 rescued human neutrophils from constitutive apoptosis and prolonged their life span. MPO evoked a transient concurrent activation of extracellular signal-regulated kinase and Akt, leading to phosphorylation of Bad at both Ser112 and Ser136, prevention of mitochondrial dysfunction, and subsequent activation of caspase-3. Consistently, pharmacological inhibition of extracellular signal-regulated kinase, Akt, or caspase-3 reversed the antiapoptosis action of MPO. Acute increases in plasma MPO delayed murine neutrophil apoptosis assayed ex vivo. In a mouse model of self-resolving inflammation, MPO also prolonged the duration of carrageenan-induced acute lung injury, as evidenced by enhanced alveolar permeability and accumulation of neutrophils parallel with suppression of neutrophil apoptosis. Our results indicate that MPO functions as a survival signal for neutrophils and thereby contribute to prolongation of inflammation.     

PyMIF enhances the inflammatory response in a rodent model by stimulating CD11b+Ly6C+ cells accumulation in spleen

Macrophage migration inhibitory factor (PMIF) expressed by Plasmodium parasites has been proved to be similar to the mammalian MIF in both structure and biological activity and is a critical upstream regulator in antimalaria innate immunity. In this work, using a genetically modified (MIF‐KO) strain of highly lethal rodent Plasmodium yoelii 17XL (Py17XL), we found that PyMIF could increase the secretion of pro‐inflammatory factors by eliciting the CD11b⁺Ly6C⁺ cells accumulated in the spleen of infected mouse. In addition, the chemotactic effect of PyMIF was demonstrated to associate with cell receptors CXCR2, CXCR4 and the cell surface markers ICAM‐1, LFA‐4.     

Alpha lipoic acid attenuates inflammatory response during extracorporeal circulation

Aim

Extracorporeal circulation (ECC) of blood during cardiopulmonary surgery has been shown to stimulate various pro-inflammatory molecules such as cytokines and chemokines. The biochemical oxidation/reduction pathways of a-lipoic acid suggest that it may have antioxidant properties.

Methods

In this study we aimed to evaluate only patients with coronary heart disease and those planned for coronary artery bypass graft operation. Blood samples were obtained from the patients before the operation (P1) and one (P2), four (P3), 24 (P4) and 48 hours (P5) after administration of a-lipoic acid (LA). The patients were divided into two groups, control and LA treatment group. Levels of interleukin-6 (IL-6) and -8 (IL-8), complement 3 (C3) and 4 (C4), anti-streptolysin (ASO), C-reactive protein (CRP) and haptoglobin were assessed in the blood samples.

Results

Cytokine IL-6 and IL-8 levels were significantly higher after surgery. Compared with the control groups, LA significantly decreased IL-6 and IL-8 levels in a time-dependent manner. CRP levels did not show significant variation in the first three time periods. CRP levels were higher after surgery, especially in the later periods. These results demonstrate that CRP formation depends on cytokine release. C3 and C4 levels were significantly higher after surgery than in the pre-operative period. LA treatment decreased C3 and C4 levels. Therefore, LA administration may be useful for the treatment of diseases and processes where excessive cytokine release could cause oxidative damage.

Conclusions

Our findings suggest a possible benefit of using LA during cardiac surgery to reduce cytokine levels.     

Activation-dependent proteolytic degradation of polymorphonuclear CD11b

CD11b/CD18 is the principal integrin of polymorphonuclear (PMN) leucocytes and is involved in their adhesion, migration and phagocytosis. In quiescent cells, the receptor is stored in intracellular granules from where it is translocated to the cell surface in response to a variety of stimuli. In this study, we demonstrated that strong stimulation of PMNs not only leads to the upregulation of CD11b surface expression, but also to the subsequent time-dependent apparent loss of this receptor, as detected by fluorescence-activated cell sorting (FACS) using a monoclonal antibody (mAb) against an N-terminal CD11b epitope. This epitope loss was observed following either direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or after multiple receptor stimulation using a combination of the agonist N-formylmethionyl-leucyl-phenylalanine (FMLP) and the priming agents granulocyte macrophage-colony stimulating factor (GM-CSF) and platelet factor (PF) 4. However, upregulation following weak stimulation with FMLP alone was not followed by subsequent epitope loss of the receptor. The increases and subsequent decreases in CD11b expression induced by PMA were paralleled by an increase and a decrease in PMN adhesion to CD11b-specific ligands, fibrinogen and intercellular adhesion molecule (ICAM)-1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed that this epitope loss of PMN CD11b was the result of proteolytic degradation of the N-terminal region of the molecule. The use of a range of proteinase inhibitors indicated that this CD11b degradation involves a cell-associated serine proteinase. This is the first demonstration of the proteolytic alteration of CD11b in response to strong PMN stimulation. Given the central role of CD11b/CD18 in all aspects of PMN function, this alteration of the CD11b molecule and its effect on PMN adhesion are probably of considerable pathophysiological importance.     

Infliximab: mechanism of action beyond TNF-alpha neutralization in inflammatory bowel disease

Infliximab, a chimeric antibody to tumour necrosis factor-alpha (TNF-alpha), holds much promise for the treatment of patients with Crohn's disease. On the cellular level, infliximab affects survival and, as presented by Agnholt et al. in this issue of the journal, inhibits GM-CSF (granulocyte-macrophage colony-stimulating factor) production by intestinal T lymphocytes. Future studies will reveal whether the pro-apoptotic effect of infliximab is linked to its inhibition of endogenous GM-CSF expression in T cells. Treatment of Crohn's disease, a severe chronic intestinal disorder, may at times be challenging as it can be refractory to routine therapy. Among novel therapeutic strategies, agents that neutralize tumour necrosis factor-alpha (TNF-alpha) are of particular interest because of the crucial role of TNF-alpha in sustaining chronic mucosal inflammation. The exact mechanism of the anti-TNF action, apart from direct activity that neutralizes cytokines, is not fully understood. Cellular effects of TNF-alpha neutralizing treatment include an increased susceptibility to apoptosis of intestinal mucosal T cells. A novel pathway of anti-TNF-alpha interaction with T cells has been presented in the current issue of this journal. Agnholt et al. have found that in-vivo or in-vitro administration of infliximab, a chimeric antibody to TNF-alpha, resulted in a decreased production of GM-CSF (granulocyte-macrophage colony-stimulating factor) by T cells. Infliximab related down-regulation of TNF-alpha induced GM-CSF expression may be one of the mechanisms by which this drug increases the rate of apoptosis in T cells.     

Peroxynitrite causes phosphorylation of vasodilator-stimulated phosphoprotein through a PKC dependent mechanism

Peroxynitrite is a potent nitrating and oxidizing agent that exerts differential effects on platelets. In the present study we investigated the influence of peroxynitrite on vasodilator-stimulated phosphoprotein (VASP), a protein that plays a key role in inhibition of platelet adhesion and spreading. In platelets, VASP is a substrate for protein kinase A (PKA), PKC and PKG and phosphorylation by these kinases is thought to block VASP-mediated actin cytoskeletal rearrangement. In the present study, we demonstrate that peroxynitrite phosphorylates VASP by a PKC-dependent mechanism. Peroxynitrite (0–100 µM) induced a concentration and time-dependent increase in phosphorylation of VASP at serine¹⁵⁷ (Ser¹⁵⁷) and Ser²³⁹. Inhibition of soluble guanylyl cyclase (sGC) did not significantly reduce peroxynitrite-mediated phosphorylation, indicating a cGMP-independent pathway for VASP phosphorylation. In contrast nitric oxide-mediated VASP phosphorylation was abolished under conditions of sGC inhibition. Further exploration of the mechanisms underlying VASP phosphorylation indicated a requirement for Ca²⁺ mobilization, but was independent of protein kinase A, Src kinases and protein nitration. Consistent with previous reports phorbol 12-myristate 13-acetate (PMA; 300 nM) induced phosphorylation of VASP at Ser¹⁵⁷, but not Ser²³⁹, which was blocked by general protein kinase C (PKC) inhibitors, Ro31-8220 and Bisindolylmaleimide I (BIM-1), and Gö6976, an inhibitor of conventional PKC isoforms. Interestingly, treatment of platelets with these PKC inhibitors significantly reduced peroxynitrite-mediated phosphorylation of both sites, indicating that phosphorylation occurred through PKC-dependent mechanism. Consistent with these findings peroxynitrite caused a small increase in PKC activity as evidenced by increased phosphorylation of PKC substrates. Together these data indicate that peroxynitrite may inhibit platelet function by inducing the phosphorylation of VASP through a mechanism that requires the activation of PKC.     

Interleukin 1 receptor blockade attenuates the host inflammatory response.

Cytokines, including interleukin 1 (IL-1), tumor necrosis factor alpha, and interleukin 6, are often produced in response to tissue injury and contribute to several host responses such as weight loss, anorexia, and acute-phase protein synthesis. However, the role of IL-1 in specific tissue responses is unclear. To test our hypothesis that specific in vivo blockade of IL-1's action might inhibit the catabolic host changes associated with inflammation, mice were passively immunized with a monoclonal antibody directed against the murine IL-1 receptor prior to initiation of a turpentine-induced sterile abscess. This antibody prevents IL-1-mediated proliferation of murine thymocytes in vitro by inhibiting IL-1 alpha and IL-1 beta by way of competition for a common receptor. Weight loss following turpentine challenge was prevented by daily injections of anti-IL-1 receptor monoclonal IgG. Body composition analysis confirmed that lean tissue and fat were preserved by passive immunization. Furthermore, pretreatment with an anti-IL-1 receptor monoclonal antibody significantly attenuated the plasma amyloid P and interleukin 6 responses but did not affect the decline in plasma albumin or the increase in circulating corticosterone. Passive immunization of similar mice with polyclonal antisera against another cytokine, tumor necrosis factor alpha, failed to prevent either the weight loss or hepatic acute-phase protein changes observed in this inflammatory model. These findings suggest that IL-1 orchestrates weight loss and body compositional changes during inflammation and contributes to the induction of interleukin 6 and acute-phase protein synthesis.     

A γ-herpesvirus sneaks through a CD8+ T cell response primed to a lytic-phase epitope

To determine whether established CD8(+) T cell memory to an epitope prominent during the replicative phase of a gamma-herpesvirus infection protects against subsequent challenge, mice were primed with a recombinant vaccinia virus expressing the p56 peptide and then boosted by intranasal exposure to an influenza A virus incorporating p56 in the neuraminidase protein. Clonally expanded populations of functional, p56-specific CD8(+) T cells were present at high frequency in both the lung and the lymphoid tissue 1 month later, immediately before respiratory challenge with gammaHV-68. This prime-and-boost regime led to a massive reduction of productive gammaHV-68 infection in the respiratory tract and, initially, to much lower levels of latency in both the regional lymph nodes and the spleen. The CD8(+) T cell response to another epitope (p79) was diminished, there was less evidence of B cell activation, and the onset of the CD4(+) T cell-dependent splenomegaly was delayed. Within 3-4 weeks of the gammaHV-68 challenge, however, the extent of latent infection in the lymph nodes and spleen was equivalent, and both groups developed the prominent infectious mononucleosis-like syndrome that is characteristic of this infection. The reverse protocol (influenza then vaccinia) seemed to be slightly less effective. Even though immune CD8(+) T cells may be present at the time and site of virus challenge, establishing a high level of CD8(+) T cell memory to lytic-phase epitopes alone does not protect against the longer-term consequences of this gammaHV infection.     

Hypoxia-induced upregulation of CD11b expression in granulocytes

The aim of this study is to evaluate leukocyte-endothelial adhesion during ischemia/reperfusion injury. Polymorphonuclear neutrophils (PMN) were isolated from healthy volunteers and incubated in the presence of 2% O₂ (hypoxia) or 21% O₂ (normoxia) at 37°C for two hours. In some experiments, whole blood was subjected to hypoxia or normoxia prior to PMN isolation. Flowcytometric analysis and adhesion assays were carried out using these PMN. Moreover, adhesion assay was carried out using PMN, prepared from the patients who underwent infrarenal aortic aneurysmectomy with aortic clamping at various time points as an in vivo model of ischemial reperfusion injury. Flowcytometric analysis revealed an increased expression of CD11b in PMN subjected to hypoxia compared with those subjected to normoxia prior to isolation. Interestingly, these phenomena were not observed with PMN isolated prior to being subjected with hypoxia. Enzyme-linked immunosorbent assay (ELISA) using serum prepared from whole blood subjected to hypoxia revealed elevated levels of tumor necrosis factor (TNF)- compared with those subjected to normoxia. PMN obtained during aneurysmectomy exhibited an increased adhesion to activated human umbilical vein endothelial cells (HUVEC), compared with those taken from the same patients prior to the operation. In contrast, PMN prepared after aortic clamp, exhibited a significant reduced adhesion to activated HUVEC. Hypoxic condition, induced CD11b expression on PMN in vitro. PMN subjected to hypoxic condition in vivo exhibited an increased adhesion to activated endothelium. Elevated level of serum TNF- may be involved in this phenomenon.     

Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil capture and stable adhesion to intercellular adhesion molecule-1

The relative contributions of CD11a/CD18 and CD11b/CD18 to the dynamics and strength of neutrophil adhesion to intercellular adhesion molecule (ICAM)-1-transfected cells were examined over the time course of chemotactic stimulation. Suspensions of neutrophils and transfectants were sheared in a cone-plate viscometer, and formation of heterotypic aggregates was measured by 2-color flow cytometry. The 2-body collision theory was used to compute adhesion efficiency, defined as the proportion of collisions between neutrophils and target cells that resulted in capture. ICAM-1 surface density and shear rate both regulated adhesion efficiency. Target cells expressing approximately 1000 ICAM-1 sites/microm(2) (I(low)) were captured with an efficiency of 0.15 at 100 s(-1), which decreased to zero at 300 s(-1). At 8-fold higher ICAM-1 expression (I(high)) corresponding to levels measured on interleukin-1-stimulated endothelium, efficiency was 0.3 at 100 s(-1) and remained above background to 900 s(-1). Shear alone was sufficient for CD11a/CD18-mediated adhesion to ICAM-1, and stimulation with formyl-methionyl-leucyl-phenylalanine boosted capture efficiency through CD11a/CD18 by 4-fold. In comparison, CD11b/CD18 supported one third of this efficiency, but was necessary for aggregate stability over several minutes of shear and at shear stresses exceeding 5 dyne/cm(2). Hydrodynamics influenced capture efficiency predominantly through the collisional contact duration, predicted to be approximately 9 milliseconds for successful capture of I(low) and 4 milliseconds for I(high). The implication is that an increase in ICAM-1 from resting levels to those on inflamed endothelium effectively increases the permissible shear in which capture through beta(2)-integrins may occur. Neutrophil adhesion to ICAM-1 appears to be a cooperative and sequential process of CD11a-dependent capture followed by CD11b-mediated stabilization.     

Effect of surgically-induced weight loss on inflammatory mediators and peripheral blood monocyte CD11b expression in morbid obesity

Obesity is characterized by a state of chronic mild inflammation, with raised circulating levels of inflammatory markers. Expression and release of inflammation-related adipokines, generally, rise as adipose tissue expands. In the present study we evaluated the level of serum mediators concerned in inflammation and monocyte activation (TNF-alpha, hs-CRP, MCP-1) together with percentage of CD11-b expression on monocytes in a group of morbidly obese individuals (n = 20) before and (3-6 months) after restrictive surgery, and in 15 healthy normal weight individuals. Serum MCP-1, TNF-alpha and hs-CRP were assayed by enzymatic immunoassay, while the percentage of CD11b expression on monocytes was assayed by flow cytometry. The total lipid profile and random blood glucose levels were also assessed. Morbidly obese individuals ( before surgical weight loss) had significantly increased levels of MCP-1, TNF-alpha, hs-CRP, CD11b expression on monocytes as compared to controls (P < 0.01). Levels of MCP-1, TNF-alpha, hs-CRP were significantly decreased 3 to 6 months after restrictive surgery than before the operation (P < 0.01). hs-CRP, MCP-1 and TNF-alpha were positively correlated versus each other. TNF-alpha and hs-CRP also showed positive correlation with the body mass index. Our data suggested that the studied serum and monocyte parameters may link obesity with systemic inflammation and metabolic disorders. The interactions of MCP-1, CD11b and other inflammatory parameters might provide the basis for development of new therapies for this syndrome.     

CD44 regulates vascular endothelial barrier integrity via a PECAM-1 dependent mechanism

Vascular integrity is a critical parameter in normal growth and development. Loss of appropriate vascular barrier function is present in various immune- and injury-mediated pathological conditions. CD44 is an adhesion molecule expressed by multiple cell types, including endothelial cells (EC). The goal of the present study was to examine how loss of CD44 affected vascular permeability. Using C57BL/6 WT and CD44-KO mice, we found no significant permeability to Evan’s Blue in either strain at baseline. However, there was significantly increased histamine-induced permeability in CD44-deficient mice compared to WT counterparts. Similar results were observed in vitro, where CD44-deficient endothelial monolayers were also impermeable to 40kD-FITC dextran in the absence of vasoactive challenge, but exhibited enhanced and prolonged permeability following histamine. However, CD44-KO monolayers have reduced baseline barrier strength by electrical resistance, which correlated with increased permeability, at baseline, to smaller molecular weight 4-kD FITC-dextran, suggesting weakly formed endothelial junctions. The CD44-KO EC displayed several characteristics consistent with impaired barrier function/dysfunctional EC junctions, including differential expression, phosphorylation, and localization of endothelial junction proteins, increased matrix metalloprotease expression, and altered cellular morphology. Reduced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression by CD44-KO EC in vivo and in vitro was also observed. Reconstitution of murine CD44 or PECAM-1 restored these defects to near WT status, suggesting CD44 regulates vascular permeability and integrity through a PECAM-1 dependent mechanism.