Neutrophil recruitment by human IL-17 via C-X-C chemokine release in the airways

IL-17 is a recently discovered cytokine that can be released from activated human CD4+ T lymphocytes. This study assessed the proinflammatory effects of human (h) IL-17 in the airways. In vitro, hIL-17 increased the release of IL-8 in human bronchial epithelial and venous endothelial cells, in a time- and concentration-dependent fashion. This effect of hIL-17 was inhibited by cotreatment with an anti-hIL-17 Ab and was potentiated by hTNF-alpha. In addition, hIL-17 increased the expression of hIL-8 mRNA in bronchial epithelial cells. Conditioned medium from hIL-17-treated bronchial epithelial cells increased human neutrophil migration in vitro. This effect was blocked by an anti-hIL-8 Ab. In vivo, intratracheal instillation of hIL-17 selectively recruited neutrophils into rat airways. This recruitment of neutrophils into the airways was inhibited by an anti-hIL-17 Ab and accompanied by increased levels of rat macrophage inflammatory protein-2 (rMIP-2) in bronchoalveolar lavage (BAL) fluid. The BAL neutrophilia was also blocked by an anti-rMIP-2 Ab. The effect of hIL-17 on the release of hIL-8 and rMIP-2 was also inhibited by glucocorticoids, in vitro and in vivo, respectively. These data demonstrate that hIL-17 can specifically and selectively recruit neutrophils into the airways via the release of C-X-C chemokines from bronchial epithelial cells and suggest a novel mechanism linking the activation of T-lymphocytes to recruitment of neutrophils into the airways.     

New approaches to Pseudomonas aeruginosa lower respiratory tract infections

Since 1973, the occurrence of respiratory tract infections due to P. aeruginosa has increased associated with the development of broad-spectrum penicillins. A clinical entity, diffuse panbronchiolitis (DPB) is a representative disease of chronic P. aeruginosa infections in Japan. In this paper, recent advances of research on pathogenesis and treatments of chronic P. aeruginosa lower respiratory tract infections in our department are reported. We examined sputum from patients with chronic P. aeruginosa infections under the electron microscope. Mucoid type of microcolonies were observed with fibrous matrix of exopolysaccharide. Neutrophils were found to be partially surrounding the microcolony in an attempt to defense. Debris was formed mainly by the destruction of the neutrophils. Most neutrophils were found full of phagocytized debris. These data support that instead of phagocytizing bacteria, neutrophils phagocytized debris and bacteria were not completely eradicated. This might be a factor in the pathogenesis of persistent colonization of P. aeruginosa. In the airways of patients with chronic airway diseases (CAD), neutrophils enhance the recruitment of more neutrophils through the production of neutrophil chemotactic factors such as interleukin-8 (IL-8) and LTB4, perpetuating a cycle of inflammation in the lung. We demonstrated increased levels of IL-8, a chemotactic cytokine, in bronchoalveolar lavage (BAL) fluid from patients with CAD associated with P. aeruginosa infections. We also documented a significant correlation between neutrophil numbers and IL-8 levels or IL-1 beta levels or neutrophil elastase levels in BAL fluids from patients with CAD. By immunohistochemical studies and in vitro data, three major sources of IL-8 in the airways of CAD patients were found to be alveolar macrophages, bronchial epithelial cells, and migrated neutrophils. In Japan, the clinical effectiveness of oral erythromycin (EM) for CAD, including DPB seems to be established, but its pharmacological mechanism remains unclear. In addition, we found a marked decrease of IL-8 levels in BAL fluid from two patients with CAD after treatment with EM. Therefore, we postulated that EM inhibited IL-8 production by stimulated respiratory cells. EM and Roxythromycin, suppressed IL-8 production in Pseudomonas-stimulated neutrophils in a dose-dependent manner. 1 alpha, 25-dihydroxy vitamin D3 also inhibited neutrophil-derived IL-8. Our data encourage the development of new anti-IL-8 agents against persistent P. aeruginosa lower respiratory tract infections.     

Role for macrophage inflammatory protein-2 in lipopolysaccharide-induced lung injury in rats

Macrophage inflammatory protein-2 (MIP-2) is a C-X-C chemokine that possesses chemotactic activity for neutrophils. Rat MIP-2 was cloned and expressed as a 7.9-kDa peptide that exhibited dose-dependent neutrophil chemotactic activity at concentrations from 10 to 250 nM. Rabbit polyclonal Ab to the 7.9-kDa peptide showed reactivity by western blot analysis and suppressed its in vitro chemotactic activity. Cross-desensitization chemotaxis experiments suggested that the chemotactic responses elicited by MIP-2 and the related chemokine, cytokine-induced neutrophil chemoattractant, may be mediated through a common receptor. Also, chemotactic responses to human GRO-alpha were blocked by exposure of human neutrophils to either GRO-alpha or rat MIP-2, suggesting conservation of this receptor-mediated response. After LPS instillation into rat lung, mRNA for MIP-2 was up-regulated in a time-dependent manner, peaking at 6 h. MIP-2 protein was detected in bronchoalveolar lavage fluids of these animals and a significant amount of chemotactic activity present in these fluids was attributed to MIP-2. On the basis of intrapulmonary instillation of Ab to MIP-2, neutrophil accumulation in lungs after airway instillation of LPS was found to be MIP-2-dependent. These data indicate that MIP-2 plays a significant role in LPS-induced inflammatory response in rat lungs and is required for the full recruitment of neutrophils.     

The intravenous administration of tumor necrosis factor alpha, interleukin 8 and macrophage-derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site. 2. Pretreatment of mice with the NO synthase antagonist, NG-monomethyl-L-arginine (L-NMMA, 15-60 mg kg(-1)), but not the inactive enantiomer D-NMMA (30 mg kg(-1)), prevented in a dose-dependent manner the TNF-alpha, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities. 3. Treatment of the neutrophils with TNFalpha (10(-7) M), IL-8 (10(-7) M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50-200 microM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-alpha or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis. 4. Preincubating the neutrophils with L-NMMA (200 microM) significantly increased the TNF-alpha (10(-7) M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10(-7) M)-mediated adhesion. 5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-alpha and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-alpha-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.     

Isolation of bioactive compounds from Helix aspersa nerve tissue and the effect of pQPPLPRYamide on heart, esophagus and central neurons of H. aspersa and rectum of Anodonta woodiana

1. Tumour necrosis factor-alpha (TNF-alpha) is implicated in the pathogenesis of many pulmonary and airway diseases. TNF-alpha stimulation may release interleukin-8 (IL-8) in airways mediated via an increase in intracellular oxidant stress. In the present study, we have assessed leukosequestration and IL-8 release in the airways in response to intratracheal administration of human recombinant TNF-alpha, and examined the modulatory role of endogenous NO by pretreatment with a NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). 2. TNF-alpha (10(2)-10(-4) u) was administered intratracheally in male guinea-pigs which were anaesthetized with urethane and were ventilated artificially. TNF-alpha induced a time- and dose-related increase in neutrophil numbers and a concomitant increase in human IL-8 equivalent level retrieved from bronchoalveolar lavage (BAL) with the peak effect at 10(3) u at 6 h of TNF-alpha injection (late phase). Intratracheal administration of recombinant human (rh)IL-8 (0.025, 0.25, 2.5 ng) producing a similar range of human IL-8 equivalent levels in BAL as measured in our results induced neutrophil recovery in BAL fluid to a similar extent. Administration of anti-IL-8 antibody prevented the late phase of neutrophil recruitment induced by TNF-alpha or rhIL-8. 3. Pretreatment with L-NAME significantly enhanced the TNF-alpha (10(3) u)-induced neutrophil recruitment and human IL-8 equivalents production at 6 h, but not at 1 h of TNF-alpha administration (early phase). L-Arginine reversed the responses to L-NAME. Pretreatment with 0.2% DMSO (i.v.) significantly inhibited TNF-alpha-induced neutrophil recruitment and human IL-8 equivalents release both in the early and late phase of the responses. Pretreatment with DMSO also inhibited the enhancement effect of L-NAME on the late phase of TNF-alpha-induced responses. DMSO failed to modify exogenous rhIL-8-induced neutrophil recruitment. Neither L-NAME nor DMSO alone induced any significant change in neutrophil numbers or human IL-8 equivalent level in BAL fluid. 4. Neutrophil depletion by cyclophosphamide pretreatment failed to modify TNF-alpha-induced human IL-8 equivalent release. 5. The expression of beta 2-integrin, CD11b/CD18 on neutrophils was increased only in the late but not early phase of TNF-alpha stimulation. L-NAME failed to modify these responses. 6. In conclusion, we demonstrated that NO may be an important endogenous inhibitor of TNF-alpha-induced leukocyte chemotaxis via inhibition of IL-8 production. Thus, the production of NO in airway inflammatory diseases may play a negative feedback role in self-limiting the magnitude of inflammatory responses.     

Alternative paths of zinc transepithelial transport in the small intestine

PURPOSE: Neutrophil invasion is a primary event in the development of herpetic keratitis. It has been reported that HSV-1 infection of keratocytes induces the synthesis of IL-8, a potent neutrophil chemoattractant, while corneal epithelium does not. Nevertheless, little is known about the correlation between neutrophil migration and the production of chemotactic factors by HSV-1-infected corneal cells, especially in epithelial cells which form an initial barrier of the ocular surface. We examined whether human corneal epithelial cells as well as keratocytes could induce neutrophil chemotaxis in response to HSV-1 infection. METHODS: Human corneal epithelial cells immortalized with SV40 (HCE) and human keratocytes were infected with HSV-1. The culture fluids collected at 4, 12, 24 h after infection were assayed for human neutrophil chemotaxis using a modified Boyden chamber method. IL-8 levels in these supernatants were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The chemotactic activity induced by HCE and keratocytes after MP strain of HSV-1 infection peaked as early as 4 h postinfection, then declined. Chemotactic activity induced by HSV-1-infected HCE and IL-8 levels on these supernatants paralleled with the infectious virus titer. It was inhibited by monoclonal anti-IL-8 antibody. UV-inactivation of MP strain abrogated neither the induction of chemotactic activity nor IL-8 secretion of infected HCE. CONCLUSIONS: At the early phase of HSV-1 infection, corneal epithelial cells play an important role in inducing neutrophil chemotaxis, which was mediated by IL-8.     

Interaction of fibronectin (FN) cell binding fragments and interleukin-8 (IL-8) in regulating neutrophil chemotaxis

This study investigated the possible interaction of FN fragments in regulating IL-8-mediated neutrophil chemotaxis in vitro using Neuroprobe microchambers. Human neutrophil suspensions were incubated with purified FN fragments or an RGD-containing peptide and allowed to migrate in response to chemotactically active concentrations of human recombinant IL-8. The 120-kD fragment of FN containing the RGD sequence or an RGD peptide (GRGDSP) inhibited IL-8-mediated neutrophil chemotaxis; however, these RGD peptides did not inhibit neutrophil chemotaxis in response to other chemotactic agents. Furthermore, FN fragments not containing the RGD sequence had no effect on IL-8-mediated chemotaxis. These data suggest that directed migration of neutrophils in response to IL-8 is inhibited in the presence of cell-binding fragments of FN and may represent a local mechanism for terminating neutrophil migration at areas of tissue injury.     

The role of neutrophils in the pathogenesis of idiopathic pulmonary fibrosis

STUDY OBJECTIVES: The prognostic value of the neutrophil count in BAL fluid (BALF) has been controversial. The role of neutrophils in this inflammatory lung disease, therefore, was evaluated in this study by additional measures. MATERIALS AND METHODS: We performed BAL in 22 patients with idiopathic pulmonary fibrosis (IPF) diagnosed by open lung biopsy specimen. Percent polymorphonuclear leukocyte (PMN) in BALF and absolute neutrophil counts were compared with those of normal nonsmokers. Elastase complexed to alpha-1-proteinase inhibitor (alpha1-PI) in plasma and BALF was measured as a marker of elastase burden, and neutrophil distribution in 22 lung tissues was observed by immunohistochemistry using antineutrophil elastase antibody. RESULTS: Percent PMN and absolute neutrophil counts in BALF did not increase in patients with IPF as compared with normal nonsmokers (n=15); the plasma elastase-alpha1-PI complex value (mean+/-SE) of patients with IPF (668.5+/-112.4 ng/mL) was significantly high as compared with that of normal nonsmokers (130.3+/-21.3, p<0.001). In addition, the BALF elastase-alpha1-PI complex value (mean+/-SE) of patients with IPF was also significantly high (333.1+/-87.0 ng/mg albumin) as compared with that of normal nonsmokers (83.1+/-29.3 ng/mg albumin, p<0.05). Immunohistochemistry demonstrated considerable numbers of neutrophils infiltrating the lung parenchyma in biopsy specimens obtained by open lung biopsy. CONCLUSIONS: These results suggested that although the neutrophil count in BALF could not represent the distribution of neutrophil in the lung, high levels of neutrophil elastase were demonstrated in lung parenchyma and also in both BALF and sera. Therefore, neutrophils might indeed play an important role in the pathogenesis of IPF.     

Role of cytokines in the modulation of neutrophil chemotaxis in localized juvenile periodontitis

Decreased neutrophil chemotaxis has been implicated in the pathophysiology of the disease, localized juvenile periodontitis (LJP). The biological basis for the altered neutrophil function in LJP has been suggested to be an intrinsic cellular defect, involving a decrease in the number of N-formyl-methionyl-leucyl-phenylalanine (FMLP) receptors on the cell surface. We have investigated the relative contribution of serum-borne factors in the modulation of neutrophil functions in LJP, in a large population of LJP patients and healthy control subjects (HS). Treatment of HS-neutrophils with LJP-sera, resulted in a decreased neutrophil chemotactic response, and down regulation of FMLP receptors on the cell surface. Pretreatment of LJP-sera with anti-TNF and anti-IL-1 antibodies effectively, although incompletely, neutralized the ability of LJP-sera to modulate chemotaxis and FMLP receptor levels in HS-neutrophils. The changes induced by LJP sera were specific and sustained and could not be reversed by placing LJP-serum treated neutrophils in HS-serum. Sera obtained from HS and patients with adult periodontitis (AP), both of which exhibit normal chemotaxis, and patients with clinically diagnosed LJP with normal neutrophil chemotaxis (LJP-nctx) did not modulate HS neutrophil chemotaxis or FMLP receptors. Furthermore, recombinant human TNF-alpha, rhIL-1 alpha and rhIL-1 beta, at very low concentrations (15 pg/ml to 150 pg/ml), modulated the chemotactic response as well as FMLP receptor numbers on HS-neutrophils, in a manner similar to those observed in LJP. The present findings demonstrate that the biologic basis for the altered neutrophil function may not be an intrinsic cellular defect in neutrophils, but at least in part due to quantitatively small but biologically significant elevations in the levels of TNF-alpha and IL-1 in the serum.     

CD11b/CD18 mediates the neutrophil chemotactic activity of fibrin degradation product D domain

Coagulation and fibrinolysis universally accompany tissue injury and repair. The accumulation of regionally generated fibrin degradation products (FDP) may modify the local inflammatory response. We have found FDP to be potent neutrophil chemotaxins. We separated plasmin FDP by chromatofocusing and found chemotactic activity limited to fractions containing the fibrinogen D domain (D-D dimer and D monomer). The bioactivity of the D-D dimer did not require an intact cross link site as removal of this sequence with puff adder venom or hypocalcemic plasmic digestion did not decrease chemotaxis. Peptide inhibition studies confirmed that the chemotactic region did not involve terminal gamma chain sequences or alpha chain RGD motifs. The internal gamma chain peptide KYGWTVFQKRLDGSV (P1), known to bind CD11b/CD18, exhibited concentration dependent chemotactic activity. Similarly, monoclonal antibodies directed against CD11b/CD18 blocked PMN migration to FDP without similar inhibition of chemotaxis to IL-8 or LTB4. Thus, neutrophil chemotaxis to FDP is mediated by interactions between the fibrinogen D domain and CD11b/CD18.     

BAL neutrophilia in asthmatic patients. A by-product of eosinophil recruitment?

Although neutrophil number may be increased in the airways of patients with asthma, its pathogenetic role in this disorder remains unclear. We evaluated BAL of 8 normal control subjects, 30 +/- 2 years of age, and 24 patients with mild asthma: 17 patients with allergic asthma, 24 +/- 1 years of age, and 7 patients with nonallergic asthma, 30 +/- 1 years of age. The BAL of asthmatic patients showed increased numbers of neutrophils (p < 0.01), eosinophils (p < 0.01), and ciliated epithelial cells (p < 0.05) and increased concentrations of myeloperoxidase (MPO) (p < 0.01) compared with control subjects. Positive correlations were observed between the number of BAL neutrophils and eosinophils (Rs = 0.780, p < 0.0001) and between BAL neutrophil numbers and BAL MPO levels (Rs = 0.40, p < 0.05). No correlations were found between the following: (1) BAL eosinophils or neutrophils and BAL epithelial cells (p > 0.05, each comparison); (2) BAL neutrophils or eosinophils and log Pd15 methacholine (MCh) (p > 0.05, each comparison); or (3) BAL epithelial cells or log Pd15 MCh and BAL MPO (p > 0.05, each comparison). Dividing the patient population into two groups, allergic asthmatics and nonallergic asthmatics, similar BAL neutrophil, eosinophil, and epithelial cell numbers and similar MPO levels were found (p > 0.05, each comparison). In addition, the correlations between BAL neutrophils and eosinophils showed similar significance in the two patient subgroups (p > 0.05, each comparison). These results suggest that, both in allergic and nonallergic asthma, airway recruitment and activation of neutrophils occur as does parallel eosinophil migration. However, airway neutrophils do not seem to contribute significantly to epithelial cell injury or to airway hyperresponsiveness in the steady state.     

Stress protein inductions after brain ischemia

To better understand the mechanisms by which neutrophils migrate into the airways, we constructed a novel in vitro model system with human umbilical vein endothelial cell (HUVE) monolayers grown on top of permeable filters and human lung Type II-like alveolar epithelial cell (A549) monolayers grown on the undersurface of the filters. The sequential migration of human neutrophils through the endothelium (apical to basal movement) and subsequently through the epithelium (basal to apical movement) in response to a stimulus located basally to the epithelium was measured. We found that the neutrophil chemoattractants, formylmethionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4), and interleukin-8 (IL-8), induced dose-responsive migration through the double monolayer-filter complex. The pattern of migration was similar to that observed through either a naked filter or single monolayer-filter complex. Maximal chemotaxis through the double monolayer-filter complex was observed by 3 hours. Thus, we have established an in vitro model system to examine the sequential migration of neutrophils through endothelium and the respiratory epithelium in a manner analogous to that occurring with an in vivo airway stimulus causing neutrophil-rich airway inflammatory responses.     

Oleamide potentiates benzodiazepine-sensitive gamma-aminobutyric acid receptor activity but does not alter minimum alveolar anesthetic concentration

Short-term exposure to ozone at peak ambient levels induces neutrophil influx and impairs lung function in healthy humans. In order to investigate the mechanisms contributing to neutrophil recruitment and to examine the role of T-cells in the acute inflammatory response, we exposed 12 healthy humans to 0.2 parts per million (ppm) of ozone and filtered air on two separate occasions for 2 h with intermittent periods of rest and exercise (minute ventilation = 30 L x min(-1)). Fibreoptic bronchoscopy was performed 6 h after the end of exposures. Total protein, tryptase, histamine, myeloperoxidase, interleukin (IL)-8 and growth-related oncogene-alpha (Gro-alpha) were measured and total and differential cell counts were performed in bronchoalveolar lavage (BAL) fluid. Flow cytometry was performed on BAL cells to study total T-cells, T-cell receptors (alphabeta and gammadelta), T-cell subsets (CD4+ and CD8+ cells) and activated T-cell subsets (CD25+). Using immunohistochemistry, neutrophils, mast cells, total T-cell numbers, T-cell subsets, CD25+ T-cells and leukocyte endothelial adhesion molecules including P-selectin, E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 were quantified in the bronchial biopsies. Paired samples were available from nine subjects. Following ozone exposure there was a threefold increase in the proportion of polymorphonuclear neutrophils (PMNs) (p=0.07) and epithelial cells (p=0.05) in BAL fluid. This was accompanied by increased concentrations of IL-8 (p=0.01), Gro-alpha (p=0.05) and total protein (p=0.058). A significant positive correlation was demonstrated between the two chemokines and proportion of PMNs in BAL fluid. After ozone exposure there was a significant decrease in the CD4/CD8 ratio (p=0.05) and the proportion of activated CD4+ (p=0.01) and CD8+ T-cells (p=0.04). However, no significant changes were demonstrable in any of the inflammatory markers studied in the biopsies. Short-term exposure of healthy humans to 0.2 ppm ozone induced a neutrophil influx in peripheral airways at 6 h post exposure, but no apparent inflammatory response in proximal airways. This response seems to be mediated at least in part by interleukin-8 and growth-related oncogene-alpha.     

The effect of endotoxin on in vivo rat alveolar macrophage phagocytosis

This study was performed to explore whether alveolar macrophage (AM) phagocytosis would be impaired during endotoxemia. Therefore, we characterized in vivo AM phagocytic function in rats following either intravenous (i.v.) or intratracheal (i.t.) administration of lipopolysaccharide (LPS). The i.v. administration of LPS to rats at dosages of 0, 1, 2, and 5 mg/kg showed that increasing LPS doses were significantly associated with increased AM phagocytosis of 198Au colloid (P < .01), decreased recovery of AMs in bronchoalveolar lavage (BAL) (P = .017), no significant differences in neutrophil recovery by lavage (P = .15), or in the concentration of albumin in BAL (P = .14). Across the dosages of LPS administered i.t. (i.e., 0, 1, 5, and 10 mg/kg), there was no difference in AM phagocytosis (P = .29), a significant decrease in AM recovery (P = .002), a significant increase in neutrophil number (P = .01), and little effect on the concentration of albumin (P = .06). Thus, we found that the administration of endotoxin to rats did not impair in vivo AM phagocytic function. In fact, our findings suggest that the i.v. administration of LPS may increase AM phagocytosis of 198Au.     

Effects of thalidomide on neutrophil respiratory burst, chemotaxis, and transmigration of cytokine- and endotoxin-activated endothelium

Vascular endothelium activated by endotoxin and cytokines plays an important role in organ inflammation and blood leukocyte recruitment. Neutrophils, which are a homogeneous population of effector cells, are rapidly attracted in large numbers to sites of inflammation where they form an early response to infection or injury. Excessive production of various interleukins, TNF, arachidonic acid metabolites, and other substances by neutrophils and macrophages results in systemic endothelial cell injury, a fundamental problem. In the present study, we investigated in vitro the effects of thalidomide (THD) on activation of endothelial cells for enhanced transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), and interleukin-1 (IL-1). Modulation of endotoxin- and cytokine-induced neutrophil chemotaxis and respiratory burst by THD were also studied. Treatment of HUVEC with THD in combination with LPS, TNF, and IL-1, respectively, antagonized LPS-activated transmigration of neutrophils but stimulated the effects of TNF and IL-1. All of the agents used-THD, LPS, TNF, and IL-1-inhibited neutrophil chemotaxis. Addition of THD to the neutrophils had no effect on LPS-inhibited chemotaxis whereas the TNF- and IL-1-induced chemotaxis was modulated in a bimodal manner. However, THD failed to influence neutrophil respiratory burst activity. Results demonstrate that THD differentially affects mediator-induced activation of HUVEC and neutrophils.     

The neuropeptide alpha-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro

The proopiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) has potent anti-inflammatory effects in all animal models of inflammation against which it has been tested. Understanding of the mechanism by which this occurs is incomplete, although there is recent evidence for alpha-MSH receptors in murine and human macrophages and for modulation of production of proinflammatory cytokines and related mediators by alpha-MSH. Because of the prominence of neutrophils in early stages of inflammatory reactions where alpha-MSH is effective, we examined human neutrophils for evidence of mRNA for alpha-MSH receptors and for inhibition of neutrophil chemotaxis. There was accumulation of mRNA for melanocortin receptor 1 (MC1) in RT/PCR product from neutrophils stimulated with interferon and LPS. In subsequent studies alpha-MSH inhibited migration of neutrophils from most normal volunteers when the cells were placed in FMLP or IL-8 gradients. The inhibition by alpha-MSH could be traced to alterations in cAMP in neutrophils. The presence of alpha-MSH receptor message in neutrophils is consistent with the established anti-inflammatory effects of the peptide. Direct inhibition of neutrophil chemotaxis likely contributes to the anti-inflammatory activity of alpha-MSH.     

Induction of tumor necrosis factor-alpha and apoptosis in gastric mucosal injury by indomethacin: effect of omeprazole and ebrotidine

BACKGROUND: Inhalation of swine dust causes airway inflammation with influx of inflammatory cells, predominantly neutrophils, into the lungs. A study was undertaken to determine whether or not exposure to swine dust induces release of interleukin 8 (IL-8) into upper and lower airways and how this possible release is related to cellular influx. A further aim was to study the relationship between the inflammatory response and swine dust exposure. METHODS: Thirty one healthy, non-smoking, previously unexposed subjects were exposed to swine dust during three hours work in a swine house. Bronchoalveolar lavage (BAL) was performed two weeks before and 24 hours after the exposure (n = 16). Nasal lavage and acoustic rhinometry were carried out 1-2 hours before and seven hours after the start of the exposure (n = 31). Exposure measurements were performed with personal sampling equipment. RESULTS: The exposure led to 19-fold and 70-fold increases in the neutrophil concentrations in nasal lavage and BAL fluid, respectively (p < 0.001). In BAL, fluid macrophages, lymphocytes and eosinophils increased significantly. The IL-8 levels in BAL fluid increased from < 31.3 ng/l to 63 (43-109) ng/l (median (25-75th percentile), p < 0.001), and in nasal lavage fluid the concentrations increased from 144 (97-227) ng/l to 1064 (864-1437) ng/l (p < 0.001). IL-8 levels showed a significant correlation with the increase in neutrophils in the nasal lavage fluid but not in the BAL fluid. Acoustic rhinometry demonstrated significant swelling of the nasal mucosa. The air concentration of inhalable dust was 23.3 (20.0-29.3) mg/m3, endotoxin 1.3 (1.1-1.4) micrograms/m3, and muramic acid 0.99 (0.78-2.1) microgram/m3. CONCLUSIONS: The concentration of IL-8 increases in BAL fluid and nasal lavage fluid following exposure to swine dust and may be one of the chemoattractants contributing to the recruitment of neutrophils to the nasal cavity and the alveolar space.     

Neutrophil chemotactic factors produced by malignant fibrous histiocytoma cell lines

The clinicopathological features of malignant cells are sometimes modified by autologous cytokine production. Inflammatory fibrous histiocytoma (IFH) is characterised by leukocyte infiltration and is a variant of malignant fibrous histiocytoma (MFH). We demonstrated that three MFH cell lines (MF-1, MF-3, and MF-4) have the potential to promote neutrophil chemotaxis and to express mRNA for the cytokines, granulocyte-macrophage colony stimulating factor (GM-CSF) and/or interleukin 8/neutrophil attractant/activation protein 1 (IL-8/NAP-1), both with and without interleukin 1 beta (IL-1 beta) stimulation. MF-1 cells showed the spontaneous production of neutrophil chemotactic activity and the expression of both of GM-CSF and IL-8/NAP-1 mRNA, which was enhanced by exogenous IL-1 beta. In contrast, MF-3 cells showed the expression of GM-CSF and IL-8/NAP-1 mRNA with IL-1 beta stimulation but not without it, and MF-4 cells expressed only IL-8/NAP-1 mRNA when stimulated with IL-1 beta (time- and dose-dependent expression). These findings suggest that neutrophil chemotactic cytokines derived from IFH cells might be responsible for the prominent infiltration of neutrophils in this disease.     

Endothelin-1 secretion by alveolar macrophages in systemic sclerosis

Endothelin-1 (ET-1), a potent fibroblast/smooth muscle cells mitogen, has been implicated in the pathogenesis of systemic sclerosis lung disease (SSc). Since monocytes and macrophages are thought to be activated in SSc, we hypothesized that alveolar macrophages (AM) and their precursors blood monocytes from patients with SSc produced more ET-1 than cells from healthy subjects. ET-1 and big ET-1 concentrations were measured in plasma, in bronchoalveolar lavage (BAL) fluids and in cell culture supernatants from monocytes and alveolar macrophages derived from 13 patients with definite SSc with lung involvement and from 10 control subjects. Plasma and BAL fluid ET-1 and big ET-1 levels were similar in both controls and patients with SSc. ET-1 and big ET-1 concentrations in unstimulated alveolar macrophage supernatants were similar in both groups. In contrast, LPS-stimulated alveolar macrophages from patients with SSc secreted higher amounts of ET-1 and big ET-1 than control subjects. ET-1 and big ET-1 concentrations in monocyte supernatants (either LPS-stimulated or not) were not different in patients and controls. These results show that AM from patients with SSc are hyperresponsive to LPS in vitro in terms of ET-1 and big ET-1 production and suggest that AM could participate in vivo in the overproduction of this potentially profibrotic mediator in patients with SSc.     

Treatment of vaginal infections: candidiasis, bacterial vaginosis, and trichomoniasis

Chemokines contribute to the inflammatory response by selective attraction of various leukocytic cell types. Human GCP-2 was originally identified by amino acid sequence analysis as a CXC chemokine co-produced with IL-8 by osteosarcoma cells. Furthermore, the complete coding domain of human GCP-2 was disclosed by means of RT-PCR. Similarly, mouse GCP-2 was isolated from fibroblastoid and epithelial cells and completely identified by sequence analysis. Human and mouse GCP-2 share 61% identical amino acids. Both chemokines occur as multiple NH2-terminally truncated forms. The shorter forms of mouse, but not those of human, GCP-2 showed a higher neutrophil chemotactic potency and gelatinase B releasing capacity. Mouse GCP-2 was a more potent neutrophil activator than human GCP-2, natural mouse KC, and MIP-2. Human GCP-2 was not chemotactic for monocytes, lymphocytes, or eosinophils. Quantitative studies of mRNA expression in diploid fibroblasts revealed GCP-2 induction by IL-1beta. Human GCP-2 induced [Ca2+]i increase in neutrophils, which was reciprocally desensitized by IL-8, GROalpha, and ENA-78. Human GCP-2 induced [Ca2+]i increases and chemotactic responses in both CXCR1- and CXCR2-transfected cells. Finally, GCP-2 provoked neutrophil accumulation and plasma extravasation in rabbit skin. In humans, GCP-2 complements the activity of IL-8 as neutrophil chemoattractant and activator but it constitutes a major neutrophil chemokine in the mouse. GCP-2 induces neutrophil chemotaxis and activation but it might also contribute to detrimental tissue damage in sepsis, acute respiratory distress syndrome, acute hypersensitivity reactions, and autoimmune diseases. It might also influence the invasive capacity of GCP-2-secreting tumor cells.