Evaluation of Neutrophil Activation Status According to the Phenotypes of Adult Asthma

PURPOSE: Neutrophils are considered key effector cells in the pathogenic mechanisms of airway inflammation in asthma. This study assessed the activation status of neutrophils in adult asthmatics, and the therapeutic potential of FTY720, a synthetic sphingosine-1-phosphate analog, on activated neutrophils using an in vitro stimulation model. METHODS: We isolated peripheral blood neutrophils (PBNs) from 59 asthmatic patients (including 20 aspirin-exacerbated respiratory disease [AERD] and 39 aspirin-tolerant asthma [ATA] groups). PBNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharide (LPS) and their activation status was determined based on reactive oxygen species (ROS) production, cell surface expression of CD11b, interleukin (IL)-8 and matrix metallopeptidase (MMP)-9 release. PBNs were primed with FTY720 to evaluate its anti-inflammatory action. RESULTS: In vitro PBN stimulation with fMLP or LPS induced a significant increase in ROS/CD11b/IL-8/MMP-9 levels (P < 0.05 for all). In asthmatics, fMLP-induced ROS level was significantly correlated with values of forced expiratory volume in 1 second/forced vital capacity (r = −0.278; P = 0.036), maximal mid-expiratory flow (r = −0.309; P = 0.019) and PC20 methacholine (r = −0.302; P = 0.029). In addition, ROS levels were significantly higher in patients with AERD and in those with severe asthma than in those with ATA or non-severe asthma (P < 0.05 for all). FTY720 treatment could suppress ROS/CD11b levels, and LPS-induced IL-8 and MMP-9 levels (P < 0.05 for all). Responders to FTY720 treatment had significantly higher neutrophil counts in sputum (P = 0.004). CONCLUSIONS: Our findings suggest a useful in vitro PBN stimulation model for evaluating the neutrophil functional status and the therapeutic potentials of neutrophil-targeting candidates in asthmatics.

Simvastatin inhibits neutrophil degranulation induced by anti-neutrophil cytoplasm auto-antibodies and N-formyl-methionine-leucine-phenylalanine [fMLP] peptide

To test the hypothesis that simvastatin is capable of blocking human neutrophil degranulation induced by proteinase 3 [PR3]-anti-neutrophil cytoplasm auto-antibodies [ANCA] and myeloperoxidase [MPO]-ANCA, and by the chemotactic and inflammatory peptide N-formyl-methionine-leucine-phenylalanine [fMLP]. This study was conducted between March 2010 and September 2011 at the Renal Institute of Birmingham, University of Birmingham, Birmingham, United Kingdom. Immunoglobulin G [IgG] was purified from the plasma of 20 randomly selected patients with ANCA-associated vasculitis [10 PR3- and 10 MPO-ANCA], and their ability to induce neutrophil degranulation in the presence or absence of simvastatin [10 micro M] was tested. The ability of the same dose of simvastatin to block fMLP-induced neutrophil degranulation was also tested. In addition, the ability of serum obtained from rats that received simvastatin at a dose of 25 mg/kg/day to block neutrophil degranulation in vitro was tested. The addition of simvastatin significantly inhibited ANCA IgG-induced neutrophil degranulation by 48% [p=0.02]. There was no significant difference in response to simvastatin inhibition [p=0.73] between PR3- and MPO-ANCA. Simvastatin also inhibited neutrophil degranulation induced by 1 micro M fMLP [30%, p=0.04]. We further demonstrated that serum from rats that received simvastatin significantly inhibited neutrophil degranulation induced by ANCA [31.7%, p=0.01] and fMLP [23.5%, p=0.03] compared to serum from control animals. Simvastatin blocked both ANCA and fMLP-induced neutrophil degranulation. It is worth pursuing further therapeutic investigation of statins in vascular inflammatory diseases that involve neutrophil degranulation in their pathogenesis

Activation of macrophages with N-formyl-methionyl-leucyl-phenylalanine: involvement of protein kinase C and tyrosine kinase.

N-formyl-methionyl-leucyl-phenylalanine (fMLP) a potent chemotactic peptide stimulates immune responses by activating macrophages and other cells of the immune system. The present study reports inhibition of fMLP-induced activation of murine peritoneal and P388D-1 macrophage cell line by protein kinase C (PKC) inhibitors, H-7 and chelerythrine chloride. Similarly, tumoricidal activity was also downregulated by protein tyrosine kinase (PTK) inhibitors genestein and lavendustin A. Further, fMLP increased tyrosine phosphorylation of several proteins in murine macrophages, which were inhibited in presence of genestein and lavendustin A. These findings suggest the involvement of PKC and PTK in the activation of murine macrophages with fMLP.

Effect of nordy on FPR function of malignant human glioma cell line U87 / 药学学报

Nordy is a synthesized chrial compound. To investigate the effects of nordy (25 - 100 micromol x L(-1)) on the function of formylpeptide receptor (FPR) of malignant human glioma cells, human glioblastoma cell line U87 was used to detect its proliferation, migration, calcium mobilization, vascular endothelial growth factor (VEGF) mRNA and protein levels after activation of FPR by its agonist N-formyl-methionyl-leucyl-phenylalanine (fMLF). Cell proliferation, migration ability, VEGF mRNA, VEGF protein and calcium mobilization were evaluated by cell counting, chemotaxis assay, RT-PCR, ELISA and spectrometry. Nordy (50 - 100 micromol x L(-1)) potently inhibited the proliferation, migration and calcium mobilization of U87 cells induced by fMLF (P < 0.05). Moreover, 100 micromol x L(-1) nordy showed a significantly impaired VEGF mRNA expression and protein secretion induced by fMLF (P < 0.05). Nordy could inhibit FPR functioning in glioma cell proliferation, migration and angiogenesis, which might be a possible mechanism of its anti-cancer effects.

Protective Effect of Defibrotide on Splanchnic Injury following Ischemia and Reperfusion in Rats

A splanchic artery occlusion for 90 min followed by reperfusion of the mesenteric circulation resulted in a severe form of circulatory shock, characterized by endothelial dysfunction, severe hypotension, marked intestinal tissue injury, and a high mortality rate. The effect of defibrotide, a complex of single-stranded polydeoxyribonucleotides having antithrombotic effect, was investigated in a model of splanchnic artery occlusion (SAO) shock in urethane anesthetized rats. Occlusion of the superior mesenteric artery for 90 min produced a severe shock state, resulting in a fatal outcome within 120 min of reperfusion in many rats. Defibrotide (10 mg/kg body weight) 10 min prior to reperfusion significantly improved mean arterial blood pressure in comparison to vehicle treated rats (p<0.05). Defibrotide treatment also significantly attenuated in the increase of plasma amino nitrogen concentration, intestinal myeloperoxidase activity, intestinal lipid peroxidation, infiltration of neutrophils in intestine and thrombin induced adherence of neutrophils to superior mesentric artery segments. Superoxide anion and hydrogen peroxide production in 1 micrometer formylmethionylleucylphenylalanine (fMLP)-activated PMNs was inhibited by defibrotide in a dose-dependent fashion. Defibrotide effectively scavenged hydrogen peroxide, but not hydroxyl radical. Treatment of SAO rats with defibrotide inhibited tumor necrosis factor-alpha, and interleukin-1beta productions in blood in comparison with untreated rats. These results suggest that defibrotide partly provides beneficial effects by preserving endothelial function, attenuating neutrophil accumulation, and antioxidant in the ischemic reperfused splanchnic circulation.

Hypotesis: an alternative pathway for the regulation of inflammation

Hipótesis: una vía alternativa de regulación de procesos inflamatorios. La regulación de mecanismos inflamatorios es un evento crucial debido a que una alteración de los mismos, como sucede por ejemplo, en la sepsis, en enfermedades autoinmunes crónicas (artritis reumatoidea, lupus eritematoso) o en enfermedades infecciosas (tuberculosis, lepra), genera daños tisulares severos. Aunque hay un consenso general de que la regulación de procesos inflamatorios resulta de un balance entre vías proinflamatorias y antiinflamatorias, nosotros arribamos a la conclusión de que moléculas quimioatractantes / proinflamatorias como, por ejemplo, péptidos formilados bacterianos o complejos inmunes (CI), pueden también inducir, paradójicamente, potentes efectos ntiinflamatorios. Así, demostramos que el péptido formilado prototipo N-formilmetionil- leucil-fenilalanina (FMLP), ejerce un drástico efecto antiinflamatorio, inhibiendo la secreción de factor de necrosis tumoral alfa (TNF-α) inducido por lipopolisacáridos, un potente inductor de la secreción de TNF-α. También determinamos que el FMLP y los CI inducen la disminución de la expresión de receptores para el fragmento Fc de IgG (FcγRII and FcγRIIIB) en neutrófilos humanos. Más aún, el FMLP inhibe la inducción de la expresión de los FcγRI por interferón gamma (IFN-γ) y los CI disminuyen la expresión de moléculas de clase II del complejo mayor de histocompatibilidad en monocitos humanos. Parte de esos efectos fueron mediados por la liberación de aspártico-, serino-, o metaloproteasas. Todos estos resultados nos permiten especular sobre un nuevo concepto en el cual la regulación de los procesos inflamatorios también puede llevarse a cabo por una vía alternativa, no convencional, en la cual un agente quimioatractante / proinflamatorio, bajo determinadas circunstancias, puede actuar como una molécula antiinflamatoria.

Effect of Defibrotide on Rat Reflux Esophagitis

This study was aimed at evaluating the effect of defibrotide on the development of the surgically induced reflux esophagitis, on gastric secretion, lipid peroxidation, polymorphonuclear leukocytes (PMNs) accumulation, polymorphonuclear leukocytes adherence, superoxide anion and hydrogen peroxide production in PMNs, scavenge of hydroxyl radical and hydrogen peroxide, cytokine (interleukin-1beta, tumor necrosis factor-alpha) production in blood, and intracellular calcium mobilization in PMNs. Defibrotide did not inhibit the gastric secretion and not change the gastric pH. Treatment of esophagitis rats with defibrotide inhibited lipid peroxidation, and myeloperoxidase (MPO) in the esophagus in comparison with untreated rats. Defibrotide significantly decreased the PMN adherence to superior mesenteric artery endothelium in a dose-dependent manner. Superoxide anion and hydrogen peroxide production in 1microM formylmethionylleucylphenylalanine (fMLP) - or 0.1microgram/ml N-phorbol 12- myristate 13-acetate (PMA) -activated PMNs was inhibited by defibrotide in a dose-dependent fashion. Defibrotide effectively scavenged the hydrogen peroxide but did not scavenge the hydroxyl radical. Treatment of esophagitis rats with defibrotide inhibited interleukin-1beta production in the blood in comparison with untreated rats, but tumor necrosis factor-alpha production was not affected by defibrotide. The fMLP-induced elevation of intracellular calcium in PMNs was inhibited by defibrotide. The results of this study suggest that defibrotide may have partly beneficial protective effects against reflux esophagitis by the inhibition lipid peroxidation, PMNs accumulation, PMNs adherence to endothelium, reactive oxygen species production in PMNs, inflammatory cytokine production (i.e. interleukin-1beta), and intracellular calcium mobilization in PMNs in rats.

Role of phospholipase D in priming of rat peripheral leukocytes by lipopolysaccharide and antigen / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate whether or not lipopolysaccharide (LPS) and ovalbumin (OA) prime rat peripheral leukocytes, the effect of sensitization on priming and the role of phospholipase D in priming.</p><p><b>METHODS</b>The peripheral leukocytes were separated and purified from sensitized or unsensitized rats. LPS or OA was used as a priming agent and formylmethionylphenylalanine (fMLP) as an activating agent. Degradation of leukocyte was determined by measurement of elastase release and myeloperoxidase (MPO) activity. Phospholipase D (PLD) activity was assayed by the generation of choline,which was measured by choline-oxidase-catalyzed formation of H(2)O(2) and Trinder reaction.</p><p><b>RESULT</b>Compared with cells treated by fMLP alone,leukocytes from unsensitized rat challenged with fMLP after incubated with LPS released more elastase and MPO (P<0.05). But there was no significant difference between leukocytes challenged with fMLP after incubated with OA and fMLP treated alone. In sensitized rat,there was no difference between leukocytes challenged with fMLP after incubated with LPS and fMLP treated alone. But leukocytes challenged with fMLP after incubated with OA released significantly more elastase and MPO than fMLP treated alone (P<0.05). A significant correlation was obtained between the release of elastase and PLD activity (r(s)=0.51,P<0.01), and also between the release of MPO and PLD activity (r(s)=0.73,P<0.01) in unsensitized rat. In sensitized rat, it was 0.48 (P<0.01) and 0.37 (P<0.05) respectively.</p><p><b>CONCLUSION</b>(1) LPS primes peripheral leukocytes from unsensitized rats; (2) OA primes peripheral leukocytes from actively sensitized rats; (3) PLD plays a role in priming of rat peripheral leukocytes.</p>

Chemotaxis of Blood Neutrophils from Patients with Primary Ciliary Dyskinesia

Primary ciliary dyskinesia is characterized by chronic upper and lower respiratory infections which are caused by the grossly impaired ciliary transport. Since the cilia and neutrophils both utilize microtubular system for their movement, it has been speculated that neutrophil motility such as chemotaxis might be impaired in patients with primary ciliary dyskinesia. Neutrophils were purified from whole blood from 16 patients with primary ciliary dyskinesia and from 15 healthy controls. Chemotactic responses of neutrophils to leukotriene B4 (LTB4), complement 5a (C5a), and formylmethion-ylleucylphenylalanine (fMLP) were examined using the under agarose method. The chemotactic differentials in response to LTB4, C5a, and fMLP in neutrophils from the patient group were significantly lower than the corresponding values in neutrophils from the control group (p<0.05 for all comparisons). The difference in chemotactic index between the two groups was statistically significant for LTB4 and fMLP (p<0.05 for both comparisons), but not for C5a (p=0.20). Neutrophils from patients with primary ciliary dyskinesia showed a decreased chemotactic response as compared with those from normal subjects. It is concluded that the increased frequency of respiratory tract infection in patients with primary ciliary dyskinesia is possibly due to the defective directional migration of neutrophils, as well as to the defective mucociliary clearance of the airways.

Polysaccharide sulfate 916 inhibits neutrophil-endothelial adhesion / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study the effect of polysaccharide sulfate 916 (PS916) on neutrophil-endothelial cell adhesion.</p><p><b>METHODS</b>Cell adhesion was evaluated by testing neutrophil myeloperoxidase activity. Expression of adhesion molecule in human umbilical vein endothelial cell (HUVEC) was measured by ELISA. The neutrophil activation rate induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was tested by nitroblue tetrazolium (NBT) reduction.</p><p><b>RESULTS</b>Tumor necrosis factor alpha (TNFalpha, 50 - 800 U/ml) increased the adherence of neutrophil to TNFalpha-stimulated HUVEC in a concentration and time dependent manner. PS916 (0.01 - 1.0 mg/ml) dose-dependently inhibited the adherence of neutrophils to TNFalpha-stimulated HUVEC. fMLP increased the activation rate of neutrophils independent of concentration. PS916 also inhibited the adherence of fMLP-activated neutrophils to HUVEC. Moreover, PS916 inhibited adhesion molecule expression in TNFalpha-stimulated HUVEC.</p><p><b>CONCLUSIONS</b>PS916 inhibited neutrophil-endothelial adhesion. The mechanism of its action was partially related to suppressing the expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1).</p>

Anti-inflammatory effects of IL-4 and IL-10 on Human Polymorphonuclear Leukocytes

Inflammatory responses are strictly regulated by coordination of pro-inflammatory and anti-inflammatory mediators. Interleukin-4 (IL-4) and interleukin-10 (IL-10) have typically the biologic anti-inflammatory effects on monocytes, but uncertain effects on polymorphonuclear leukocytes (PMNs). The PMNs are the first line of cellular response for host defense during acute inflammation. To modify hyper-inflammatory reaction with biologic anti-inflammatory mediators, we have determined the biologic anti-inflammatory activities of IL-4 and IL-10 on human PMNs. Human PMNs were pretreated with IL-4 or IL-10 and then stimulated with formyl methionyl leucyl phenylalanine (fMLP) for times indicated. The level of H2O2, interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) were determined in the each cell free supernatants. fMLP plays the role of a typical pro-inflammatory agent and, at least in determined conditions, down-regulated TNF release. IL-4 acts as an anti-inflammatory mediator but IL-10 did not show its anti-inflammatory activities on fMLP-stimulated human PMNs. IL-4 and IL-10 have different anti-inflammatory mechanisms. Perhaps, IL-10 needs co-factors to act as an anti-inflammatory mediator.

Molecular Mechanisms of Neutrophil Activation in Acute Lung Injury / 결핵

ABBREVIATIONS: Akt/PKB protein kinase B, ALI acute lung injury, ARDS acute respiratory distress syndrome, CREB C-AMP response element binding protein, ERK extracelluar signal-related kinase, fMLP fMet-Leu-Phe, G-CSF granulocyte colony-stimulating factor, IL interleukin, ILK integrin-linked kinase, JNK Jun N-terminal kinase, LPS lipopolysaccharide, MAP mitogen-activated protein, MEK MAP/ERK kinase, MIP-2 macrophage inflammatory protein-2, MMP matrix metalloproteinase, MPO myeloperoxidase, NADPH nicotinamide adenine dinucleotide phosphate, NE neutrophil elastase, NF-kappaB nuclear factor-kappa B, NOS nitric oxide synthase, p38 MAPK p38 mitogen activated protein kinase, PAF platelet activating factor, PAKs p21-activated kinases, PMN polymorphonuclear leukocytes, PI3-K phosphatidylinositol 3-kinase, PyK proline-rich tyrosine kinase, ROS reactive oxygen species, TNF-alpha tumor necrosis factor-alpha.

Heparin and low molecular weight heparin decrease nitric oxide production by human polymorphonuclear cells

Background. Heparin and heparin derivatives with low anticoagulant activity exhibit a wide spectrum of biological functions affecting adhesion, activation and trafficking of luekocytes. Methods. We investigated the in vitro effect of heparin and low molecular weight heparin derivative (LMWH) on nitric oxide (NO) production by human polymorphonuclear leukocytes (PMN). Results. N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated NO production was significantly decreased by heparin at doses of 0.5 and 5 µg/mL, while LMWH was only effective at doses of 50 and 200 µg/mL by means of a mechanism not related to No synthase (NOS) activity. Conclusions. These results support the hypothesis that heparin and LMWH derivatives may offet therapeutic benefit for inflammatory diseases where No plays a protagonic role

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 ñ 0.19 and Dex = 0.35 ñ 0.13 vs saline (S) = 2.85 ñ 0.59; fMLP: Ptx = 0.43 ñ 0.09 and Dex 0.01 ñ 0.01 vs S = 1.08 ñ 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 ñ 1.4 and Dex = 3.06 ñ 0.76 vs S = 15.94 ñ 3.97; fMLP: Ptx = 3.85 ñ 0.56 and Dex = 0.40 ñ 0.16 vs S = 7.15 ñ 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 µm), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 ñ 0.38 vs S = 4.20 ñ 1.01; fMLP: Dex = 0.25 ñ 0.11 vs S = 2.20 ñ 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 ñ 2.25 vs S = 4.20 ñ 1.01; fMLP: Ptx = 4.66 ñ 1.24 vs S = 2.20 ñ 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.

Calyculin A modulates activation of the NADPH-oxidase in Me2SO-differentiated HL-60 cells

Human promyelocytic leukemia cells (HL-60) have been used as a model system in which to study the effects of protein phosphatase inhibitors on NADPH-oxidase activation. Since O2- is generated by NADPH-oxidase, we examined the effect of calyculin A pretreatment on oxidase activation in response to various agonists. When Me2SO-differentiated HL-60 cells were treated with calyculin A prior to the addition of phorbol 12-myristate 13-acetate (PMA), O2- production was inhibited; however, calyculin A enhanced O2- production by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The decreased O2- production seen with calyculin A pretreatment followed by PMA may be due to diminished translocation of the p47-phox and p67-phox, cytosolic components of the oxidase, and inhibition of arachidonic acid release. Interestingly calyculin A pretreatment followed by either agonist significantly enhanced mitogen-activated-protein kinase (MAPK) activity. The differential effects of pretreatment with calyculin A on subsequent oxidase stimulation elicited by FMLP or PMA provide further evidence for substantial heterogeneity in the activation of the respiratory burst.

Efecto de los péptidos formilados sobre funciones dependientes de los receptores para Fc de IgG (FcgR) en neutrófilos humanos / Effect of formyl peptides on FcgR- dependent functions in human neutrophils

Los neutrófilos polimorfonucleares humanos (PMN) participan en diferentes funciones celulares, como fagocitosis, citotoxicidad celular dependiente de anticuerpos (ADCC) y la liberación de reactivos intermediarios del oxígeno. Cada una de dichas funciones pueden generarse a través de la estimulación de los receptores para la porción Fc de moléculas IgG (FcgR). Los neutrófilos poseen dos clases de FcgR, FcgRII y FcgRIII. Dichas células también poseen receptores para péptidos formilados de naturaleza bacteriana, como el péptido formilado prototipo N-formil-methionil-leucil-phenilalanina (FMLP). En este trabajo, presentamos evidencia que la preincubación de PMN con FMLP inhibe diferentes funciones celulares dependientes de los FcgRs como lo son la ADCC, fagocitosis y la citotoxicidad inespecífica dependiente de CI. Estos efectos inhibitorios podrían ser explicados, al menos parcialmente, por una disminución de la expresión de ambos, FcgRII y FcgRIII en la superficie celular (down-regulation). Sin embargo, la preincubación de PMN con FMLP no es necesaria para la inhibición de la ADCC. Teniendo en cuenta que la down-regulation de los FcgRs inducida por FMLP no se observa hasta después de los 30 min de incubación, y que la ADCC es una función que se desarrolla rápidamente (en segundos), es muy factible que el FMLP pueda modificar dicha función a través de la inducción de eventos intracelulares.

Stimulation of inflammatory mediators secretion by chemotactic peptides in rat colitis model

Colonic inflammation was produced in rats by chemotactic peptides acting on polymorphonuclear leucocytes. Instillation during one hour of formylated tripeptide: formylmethionyl-leucy-phenylalanine (FMLP) and a tetrapeptide: alanine-glycine-sefine-glutamine (AGSG) into rat colon caused erosions and exulcerations. Neutrophils increased secondary to instillation, predominantly with FMLP, and mucus depletion was marked in the cecum. Chloride ion secretion and mucosal permeability were significatively greater in the colonic lumen with the chemotactic peptides. Histamine and serotonin concentration were greater in the colonic fluid in animals treated with the peptides. These observations could suggest that the presence of chemotactic peptides at the colonic lumen produce changes at the mucosal wall, that would participate in generation and perpetuation of the colonic inflammation.

Adhesion-induced generation of oxygen free radical from human alveolar macrophages and its mechanisms / 결핵

BACKGROUND: Neutrophils or monocytes separated in vitro by the adherence to plastic surface are known to be activated by surface adherence itself and subsequent experimental data might be altered by surface adherence. In the process of surface adherence, adhesion molecules have a clear role in intracellular signal pathway of cellular activation. Human alveolar macrophages(HAM) are frequently purified by the adherence procedure after bronchoalveolar lavage. But the experimental data of many reports about alveolar macrophages have ignored the possibility of adhesion-induced cellular activation. METHOD: Bronchoalveolar lavage was performed in the person whose lung of either side was confirmed to be normal by chest CT. With the measurement of hydrogen peroxide release from adherent HAM to plastic surface and non-adherent HAM with or without additional stimulation of phorbol myristate acetate(PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), we observed the effect of the adherence to plastic surface. We also evaluated the effect of various biological surfaces on adhesion-induced activation of HAM. Then, to define the intracellular pathway of signal transduction, pretreatment with cycloheximide, pertussis toxin and anti-CD 11/CD18 monoclonal antibody was done and we measured hydrogen peroxide in the culture supernatant of HAM. RESULTS: 1) The adherence itself to plastic surface directly stimulated hydrogen peroxide release from human alveolar macrophages and chemical stimuli such as phorbol myristate acetate(PMA) or N-formyl-methionyl-leucyl-phenylalanine(fMLP) colud not increase hydrogen peroxide release in these adherent macrophages which is already activated. 2) PMA activated human alveolar macrophages irrespective of the state of adhesion. However, fMLP stimulated the release of hydrogen peroxide from the adherent macrophages, but not from the non-adherent macrophages. 3) HAM adherent to A549 cell(type II alveolar epithelium-like human cell line) monolayer released more hydrogen peroxide in response to both PMA and fMLP. This adherence-dependent effect of fMLP was blocked by pretreatment of macrophages with cycloheximide, pertussis toxin and anti-CD18 monoclonal antibody. CONCLUSION: These results suggest that the stimulatory effect of PMA and fMLP can not be found in adherent macrophage because of the activation of human alveolar macrophage by the adherence to plastic surface and the cells adhered to biologic surface such as alveolar epithelial cells are appropriately responsive to these stimuli. It is also likely that the effect of fMLP on the adherent macrophage requires new protein synthesis via G protein pathway and is dependent on the adhesion between alveolar macrophages and alveolar epithelial cells by virtue of CD11/CD18 adhesion molecules.

Pertussis toxin from Bordetella pertussis blocks neutrophil migration and neutrophil-dependent edema in response to inflammation

Pertussis toxin (Ptx) is a hexameric protein with classical AB architecture produced by Bordetella pertussis. The aim of this study was to investigate the effect og Ptx on migration of polymorphonuclear leukocytes to site of inflamation and on cell- dependent edema. Ptx was purified from the supernatant of the culture medium of B. pertussis using hydroxylapatite chromatography and fetuin affinity chromatography. Ptx induced a maximal clusterin of Chinese hamster ovary cells at concentration as low as 0.1 ng/ ml. Intravenous inection of Ptx (400 ng) significantly blocked the neutrophil migration induced by 200 ng of lipopolysaccharide (LPS from E. coli O111:B4; 2.27 ñ 0.13 vs 0.61 ñ 0.16 per 10**6 neutrophils/ml; P < 0.001; N = 5) and by 200 ng of formylmethionyl-leucyl-phenylalanine(fMLP; 2.53 ñ 0.45 vs 0.75 ñ 0.14 per 10**6 neutrophils/ml; P < 0.01; N=6) into the peritoneal cavities of male Wistar rats (eighing 150-180). In addition, Ptx (400ng) pretreatment also blocked the edema induced by intraplantar injection of 100 µg carrageenin ( increase in volume: 0.667 ñ 0.087 vs 0.313 ñ 0.058 ml; P < 0.01; N = 5) but not the edema induced by 100 µg dextran ( increase in volume: 0.537 ñ 0.06 vs 0.385 ñ 0.076 ml; P > 0.05; N = 5). These data demonstrate that Ptx blocked neutrophil migration induced by a direct f MLP stimulus of a site of inflammation. In addition, this toxin blocks the indirect stimulus of LPS on neutrophil migration. Furthermore, Ptx also inhibits the neutrophil-dependent edema induced by carrageenin, but not the edema induced by dextran that is in part dependent on basophil cells. These results warrant further studies on the mechanisms of Ptx inhibition of neutrophil-dependent edema and cell migration

Efecto del pre-ligamiento del receptor de complemento tipo I en la fagocitosis mediada por el receptor para Fc / Effect of complement type I receptor pre-ligament in the phagocytosis mediated by Fc receptor

El preligamiento del receptor de C3b (CR1) con su ligando potencia la fagocitosis mediada por el receptor para Fc (FcR) en monocitos cultivados, pero no en monocitos frescos. Nuestros estudios se dirigieron a establecer si la cooperación CR1-FcR ocurre en neutrófilos en reposo o activados. Activando neutrófilos con dosis de 1 a 5 ng/ml de PDBu observamos estimulación de la fagocitosis via Fc, mientras que a concentraciones mayores hubo una inhibición de la misma. La adherencia de las células sobre C3 inactivado (iC3) en forma simultánea al tratamiento con dosis estimulatorias o subinhibitorias de PDBu no incrementó la ingestión de eritrocitos de carnero sensibilizados con IgG (E-IgG); aun variando la cantidad de anticuerpo sensibilizante o estimulando a las células con PDBu durante distintos tiempos. La estimulación de los neutrófilos con diferentes concentraciones de fMLP en forma simultánea a la adherencia sobre iC3 tampoco incrementó la fagocitosis de los blancos mediada por el FcR. La comunicación entre el CR1 y el FcR difere en neutrófilos y monocitos, hecho que podría relacionarse con el mecanismo de activación del CR1 y la clase de FcR encontrado en cada tipo de celular