Increased elastase release by CF neutrophils is mediated by tumor necrosis factor-alpha and interleukin-8

Cystic fibrosis (CF) is a lethal, hereditary disorder characterized by a neutrophil-dominated inflammation of the lung. We sought to determine whether neutrophils from individuals with CF release more neutrophil elastase (NE) than neutrophils from normal subjects. Our results showed that peripheral blood neutrophils (PBNs) from normal subjects and individuals with CF contained similar amounts of NE, but after preincubation with CF bronchoalveolar lavage (BAL) fluid, significantly more NE was released by CF PBNs, a release that was amplified further by incubation with opsonized Escherichia coli. To determine which components of CF BAL fluid stimulated this excessive NE release from CF PBNs, we repeated the experiments after neutralization or immunoprecipitation of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 in CF BAL fluid. We found that subsequent NE release from CF PBNs was reduced significantly when TNF-alpha and IL-8 were removed from CF BAL fluid. When TNF-alpha and IL-8 were used as activating stimuli, CF PBNs released significantly greater amounts of NE compared with PBNs from control subjects and individuals with bronchiectasis. These results indicate that CF PBNs respond abnormally to TNF-alpha and IL-8 in CF BAL fluid and react to opsonized bacteria by releasing more NE. This may help explain the increased NE burden seen in this condition.     

Proteinase 3 mRNA expression is induced in monocytes but not in neutrophils of patients with cystic fibrosis.

Proteinase 3 (PR3), a serine proteinase which can degrade lung tissue, is present in the cystic fibrosis (CF) sputum. In the present study, PR3 protein and mRNA expression was determined in circulating neutrophils and monocytes. CF neutrophils contained similar PR3 concentrations as healthy controls and poorly expressed PR3 mRNA. In contrast, CF monocytes showed significantly higher PR3 concentrations than controls, together with an upregulation of PR3 mRNA expression especially during pulmonary exacerbation. Interestingly, antibiotic treatment fully abrogated PR3 mRNA expression and decreased PR3 protein in monocytes. Our findings highlight a potential role of monocyte-derived PR3 in CF-associated airway inflammation.     

A novel function of serum amyloid A: a potent stimulus for the release of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-8 by human blood neutrophil

High density lipoprotein (HDL) and its main apolipoproteins, AI and serum amyloid A (SAA), present in physiological and acute phase response conditions, respectively, affect the inflammatory process. This study focuses on the effect of AI, SAA, and HDL from healthy (N-HDL) and acute phase individuals (AP-HDL) on the release of TNF-alpha, IL-1beta, and IL-8 by human blood neutrophils. It was observed that SAA (100 microg/mL) causes a dramatic increase (75-400 times) in the basal liberation of the three cytokines assayed. This effect is not triggered by AP-HDL. Although AI (100 microg/ml) increases the release of IL-1beta and IL-8 modestly, N-HDL does not. Both HDLs (0.16-0.32 mg of protein/mL) had an anti-inflammatory action, decreasing the basal and LPS-stimulated cytokine release. Given that the biological role of SAA is still uncertain, the present study adds an important finding potentially pertinent to the biological role of this acute phase protein.     

The acute phase protein serum amyloid A primes neutrophils

We studied here the effect of the acute phase protein serum amyloid A (SAA) on the oxidative burst of neutrophils. Incubation of neutrophils with SAA increased the rate of oxygen uptake and the production of reactive oxygen species of neutrophils activated with opsonized zymosan (OZ). The increment in the neutrophil oxidative burst was dependent on SAA concentration in the range of 3-33 microg protein ml(-1) and was observed only in the presence of a relatively low amount of OZ (1 x 10(6) particles ml(-1)). SAA did not affect oxygen consumption and reactive oxygen production triggered by other stimuli, such as f-Met-Leu-Phe, phorbol myristate acetate or non-opsonized zymosan. Our finding points to a priming effect of SAA probably associated with mobilization of receptors for opsonized particles and strengthens the role of SAA as an effector of neutrophil functions in inflammation.     

Neutrophil-activating peptide 1/interleukin 8 mRNA expression and protein secretion by human monocytes: effect of cyclosporin A

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8) is a recently described cytokine with potent chemotactic activity for human neutrophil granulocytes (PMN) and T cells. In psoriasis, a chronic hyperproliferative and inflammatory skin disorder, PMN and T cells are found as prominent cells in the inflammatory infiltrate of the lesions; however, monocytes were shown to be the first cells invading a newly formed plaque. NAP-1/IL-8 was found to be present in high amounts in the skin and in scale material of psoriatic patients. Psoriasis responds well to systemic treatment with cyclosporin A (CsA), an immunosuppressive peptide. Therefore, we addressed the question of whether the clinical improvement of psoriatic patients during CsA therapy may be due to an inhibition of NAP-1/IL-8 production and secretion from monocytes. Purified human monocytes were stimulated by lipopolysaccharide in the presence or absence of various concentrations of CsA. Production of NAP-1/IL-8 was determined as expression of specific mRNA by fluorescent in situ hybridization. Secreted peptide was measured by bioassay (PMN chemotaxis) and enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies. The results show that CsA neither inhibited mRNA expression for NAP-1/IL-8 nor secretion of the peptide. These findings support the hypothesis that the pharmacological effect of CsA may be restricted to the inhibition of T-cell activation and proliferation.     

The expression of interleukin-8 and interleukin-8 receptors in endometrial carcinoma

Interleukin-8 (IL-8) is a pro-inflammatory cytokine which exerts its effects via binding to 2 receptors, CXCR1 and CXCR2 and is known to promote angiogenesis, mitogenesis and motogenesis in cancer. IL-8 is over expressed in endometrial carcinoma, but the expression of CXCR1 and CXCR2 in endometrial carcinoma has not been previously investigated. The aim of this study was to determine the expression of IL-8 receptors in endometrial carcinoma. The expression of CXCR1 and CXCR2 was studied in endometrial carcinomas and normal endometrium by immunohistochemistry in 101 tumours. IL-8 and IL-8 receptor expression was also studied by Real-time quantitative PCR (RT-qPCR) in 17 tumours in comparison to normal endometrium. The expression profile was correlated to the clinico-pathological features of the tumours. Immunohistochemistry showed CXCR1 and CXCR2 were expressed in all cases of endometrial carcinoma, with CXCR1 showing stronger expression. There was a statistically significant correlation between CXCR2 staining intensity and tumour grade (P=0.012) and disease free survival (P=0.015) independently. On RT-qPCR, 14/17, 15/17 and 16/17 tumours showed significant increase in IL-8, CXCR1 and CXCR2 expression levels in comparison to normal endometrium, with median fold increase of 42-fold, 51-fold and 27-fold, respectively. This is the first report of the expression of IL-8 receptors in endometrial carcinoma and the results show an association between IL-8 and IL-8 receptors and the pathogenesis of endometrial carcinoma, and represent potential prognostic biomarkers and therapeutic targets.     

Differential synthesis of two interleukin-1 receptor antagonist variants and interleukin-8 by peripheral blood neutrophils

With a short lifespan and containing only few ribosomes and endoplasmic reticulum structures, neutrophils are thought to have a limited capacity for protein synthesis. We here show that peripheral blood polymorphonuclear neutrophils (PMN) are able react to stimulants with differential production of two interleukin (IL)-1 receptor antagonist (IL-1ra) isoforms, secreted IL-1ra (sIL-1ra) and the 16kDa intracellular form of IL-1ra (icIL-1ra3), as well as IL-8. Neutrophils of a high purity and with a low degree of preactivation upregulate mRNA and de novo synthesize protein of both IL-1ra variants and IL-8 in response to granulocyte-macrophage colony-stimulating factor and lipopolysaccharide. The cytokines are differentially regulated and distributed in two intracellular compartments. In comparison with peripheral blood mononuclear cells (PBMC), PMN produce distinctly more sIL-1ra but significantly less IL-8. This may indicate an anti-inflammatory role, enabling PMN to antagonize proinflammatory signals. It is therefore possible that PMN play an important role in immune regulation by counteracting a dysregulation of the inflammatory process.     

Crystallization of human interleukin-8. A protein chemotactic for neutrophils and T-lymphocytes

Interleukin-8 is a 72-residue peptide which is chemotactic for neutrophils and T-lymphocytes. It crystallizes in space group P3(1)21 or P3(2)21 with unit cell dimensions of a = b = 40.3 and c = 90.1 A. X-ray diffraction data have been measured to 1.6-A resolution.     

Synthesis of platelet-activating factor by polymorphonuclear neutrophils stimulated with interleukin-8.

Human interleukin-8 (IL-8) was evaluated for its capability to induce the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear neutrophils (PMN). IL-8 promotes in a dose-dependent fashion (1-100 ng/ml) a rapid synthesis of PAF, which is only partially released. The synthesis of PAF is preceded by the activation of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyl-transferase, suggesting that IL-8 activates the remodeling pathway of PAF synthesis. By thin layer chromatography and reverse-phase high pressure liquid chromatography, we demonstrated that PAF synthesized by human PMN stimulated with IL-8 is heterogeneous: the 2-acetylated phospholipids having the biological and physicochemical characteristics of PAF include the 1-O-alkyl form, which is produced in large extent (51%), and the 1-acyl form (20%). The analysis of the individual molecular species of radyl chain indicated nine peaks, 16:0 and 18:0 being the predominant forms. These results identify PAF as a direct product of IL-8 stimulation in PMN.     

Degradation of serum amyloid A and apolipoproteins by serum proteases

We have investigated the protease activity, present in human serum, that digests the serum amyloid A (SAA) protein. SAA radiolabeled with 125I was incubated at 37 degrees C with serum and plasma and analyzed for degradation products by alkaline urea-polyacrylamide gel electrophoresis and gel filtration chromatography. Serum initially digested SAA to intermediates of 3000-5000 in molecular weight, and these were further degraded to smaller peptides with prolonged incubation. SAA was not degraded by plasma anticoagulated with ethylenediaminetetraacetic acid (EDTA) or heparin. Recalcification of plasma anticoagulated with EDTA led to the generation of protease activity against SAA whereas EDTA plasma defibrinated with thrombin was inactive. We employed both nonselective and selective protease inhibitors and synthetic substrates for kallikrein and plasmin to further characterize the serum protease. These studies demonstrated that degradation of SAA is not directly attributable to enzymes involved in coagulation, kinin formation, or fibrinolysis, but the unidentified protease may be activated by one of the clotting factors. The specificity of the SAA degradation was demonstrated in experiments with three of the well-characterized apolipoproteins. Apolipoproteins A-I, C-I, and C-III-1, which also associate with the plasma high-density lipoproteins, were not degraded by serum although they were good substrates for purified thrombin and plasmin.     

Functional expression of IL-9 receptor by human neutrophils from asthmatic donors: role in IL-8 release

Human polymorphonuclear neutrophils (PMNs) express surface receptors for various inflammatory mediators, including IgE and IL-4. Recently, the IL-9R locus has been genetically linked to asthma and bronchial hyperresponsiveness in humans. In this study, we evaluated expression of the IL-9R and the effect of IL-9 on human PMNs. RT-PCR analysis showed the presence of IL-9Ralpha-chain mRNA in PMN RNA preparations from asthmatic patients. Using FACS analysis, surface expression of IL-9Ralpha was detected on PMNs freshly isolated from asthmatics, and to a lesser extent on normal controls. In addition, protein expression of IL-9Ralpha was also detected in peripheral blood and bronchoalveolar lavage PMNs. Furthermore, functional studies showed that IL-9 stimulation of PMNs results in the release of IL-8 in a concentration-dependent manner. The anti-IL-9 neutralizing Ab suppressed this effect, but had no effect on GM-CSF-induced IL-8 release from PMNs. Taken together, these findings suggest a novel role for PMNs in allergic disease through the expression and activation of the IL-9R.     

Bovine interleukin-1 expression by cultured mammary epithelial cells (MAC-T) and its involvement in the release of MAC-T derived interleukin-8

MAC-T cells, an established bovine mammary epithelial cell line, were utilized to investigate both expression of interleukin-1 (IL-1) mRNA and secretion of IL-1 after Escherichia coli lipopolysaccharide (E. coli LPS) stimulation. In addition, recombinant human IL-1beta, recombinant human IL-1 receptor antagonist (IL-1ra) and a neutralizing goat antibody against type I human IL-1 receptor were used to study the involvement of IL-1 in the release of IL-8. The expression of MAC-T derived IL-1alpha mRNA was correlated to production of IL-1alpha protein as measured by an IL-1alpha sandwich ELISA. Secretion of IL-1alpha was dose- and time-dependent, with a maximal level of 600 pg/ml detectable upon 2-h stimulation with 20 microg of LPS per ml. IL-1ra and the neutralizing antibody significantly blocked the ability of IL-1beta to stimulate secretion of IL-8 by MAC-T cells. During this study, we have demonstrated that MAC-T cells secrete IL-1 in response to LPS stimulation and IL-1 is an important mediator for the release of the bovine IL-8 by MAC-T cells. These results further indicate the potential importance of mammary epithelial cells as a source of immunoregulation in the mammary gland via cytokine elaboration.     

Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF-alpha mRNA expression in human monocytes by allicin

Chronic inflammation associated with tumor necrosis factor (TNF)-alpha and reactive oxygen species (ROS) is the hallmark of tuberculosis. Mycobacterium tuberculosis (MTB) directly stimulates human monocytes to secrete TNF-alpha. We show the augmented expression of TNF-alpha mRNA in MTB-infected monocytes by cellular activation and ROS was suppressed by allicin in a dose-dependent manner. Also, allicin enhanced the glutathione peroxidase activity, which correlated inversely with the downregulation of ROS and TNF-alpha in MTB-infected monocytes. Hence, allicin may prove to be a valuable natural antioxidant in combating tuberculosis.     

Macrolide-affected Toll-like receptor 4 expression from Helicobacter pylori-infected monocytes does not modify interleukin-8 production

Macrolide antibiotics have an anti-inflammatory effect by suppressing lipopolysaccharide-induced IL-8 production. IL-8 secretion from monocytes is observed in Helicobacter pylori infection. Although cag gene products are known to induce IL-8 secretion, whether other bacterial substances can initiate the reaction is not determined. In this study, we show that clarithromycin induced down-regulation of Toll-like receptor 4 expression and did not lead to a decrease in IL-8 production and H. pylori lipopolysaccharide. However, Toll-like receptor 4 activation was possibly not the main cause in the induction of inflammation during H. pylori infection.     

Regulation of interleukin-6 receptor expression in human monocytes and hepatocytes

Human blood monocytes normally express the interleukin-6 receptor. Treatment of cultured monocytes with endotoxin, interleukin-1 beta, or interleukin-6 results in a decrease in interleukin-6 receptor mRNA levels. Glucocorticoids aso cause a drop in monocytic interleukin-6 receptor mRNA levels. We also found interleukin-6 receptor expression in cultured human hepatocytes, but in contrast to monocytes, where interleukin-6 receptor mRNA is presented by the ligand and by interleukin-1, treatment of hepatocytes with interleukin-6 or interleukin-1 resulted in increased interleukin-6 receptor mRNA levels. Induction of interleukin-6 receptor mRNA in hepatocytes was less pronounced when glucocorticoids were omitted from the culture medium. We conclude that during noninflammatory homeostasis, blood monocytes are involved in binding of trace amounts of circulating interleukin-6. During inflammatory events, the main target of interleukin-6 may be changed from the monocytic population not only to activated B-cells, but also to the hepatocytes.