Characterization of Immunological Activity of a Low Toxicity Antitumor Lipopolysaccharide from Bordetella pertussis

Immunological properties of a low toxicity lipopolysaccharide (BP-LPS) extracted from Bordetella pertussis (Tohama strain) which was reported to have high antitumor activity against murine tumors were examined and compared with those of LPS extracted from other enterobacteria. The activation or stimulation of murine macrophages and lymphocytes by these LPS, including TNF induction, was found to be similar. However, BP-LPS was clearly less active in its stimulation of murine and human neutrophils as estimated by neutrophil-adherence assay and by their TNF production than E. coli LPS. Furthermore, BP-LPS also suppressed the activation of human neutrophils by Escherichia coli LPS. A comparative study with 7 LPS preparations indicated that their toxicity in terms of animal body weight loss correlated with their ability to induce human neutrophil adherence. The inability of BP-LPS to activate neutrophils may thus have some bearing on its low toxicity.     

Suppression of Anti-Candida Activity of Murine and Human Neutrophils by Glucocorticoids

Effects of glucocorticoid (GC) compounds on inhibitory activity of neutrophils to mycelial growth of Candida albicans were examined by in vitro crystal violet staining method with 14 hr co-culture. Both GC hormones (hydrocortisone ≥6 × 10^–7 m and corticosterone ≥10^–6 m ) and anti-inflammatory GC agents (prednisolone ≥10^–7 m and dexamethasone ≥10^–8 m ) significantly suppressed anti-Candida activity of murine casein-induced neutrophils. Anti-Candida activity of human neutrophils prepared from peripheral blood was also suppressed by hydrocortisone (≥6 × 10^–7 m ). These GC compounds did not affect the Candida growth in the absence of neutrophils. Steroidal compounds without anti-inflammatory activity, cholesterol, cholic acid, aldosterone did not suppress neutrophil activity. These results suggest that GCs at their physiological or clinical concentration may suppress anti-Candida activity of neutrophils in vivo.     

High mannose type N-linked oligosaccharides on endothelial cells may influence β2 integrin mediated neutrophil adherence in vitro

We report herein on the role of N-linked oligosaccharide processing of endothelial cell surface proteins on the adhesion of neutrophils. Monolayers of human umbilical vein endothelial cells were treated for 24 h with deoxymannojirimycin (DMJ), an inhibitor of golgi mannosidase I, which results in changes in glycoprotein processing, and then incubated with neutrophils to examine their ability to adhere to the treated endothelial cells. Treatment with DMJ, which leads to accumulation of high mannose type oligosaccharides, resulted in a twofold increase in adherence of phorbol ester (PMA) activated neutrophils compared to attachment to untreated endothelial cells. This adherence was likely mediated by the β2 integrin, Mac-1, and could be specifically inhibited with monoclonal antibodies to ICAM-1 and to the integrin β2 subunit. Similarly, IL-1 treatment resulted in a β2 integrin mediated increase in neutrophil adherence to the DMJ treated endothelial cells in a dose dependent manner. However, the IL-1 induced adherence was not significantly inhibited by the anti-ICAM-1 antibody, thus, suggesting the presence of other inducible components on the endothelial cell surface. Our results demonstrate that alterations in glycosylation of N-linked oligosaccharides, resulting in the synthesis of high mannose type sugars on molecules that may interact with the β2 integrins, leads to an increased adherence of PMA activated neutrophils to endothelial cells. © 1993 Wiley-Liss, Inc.     

In vitro effect of ascorbic acid on neutrophil–endothelial cell interaction

The effect of different concentrations (0.06, 0.6 and 6.0 mmol/L) of ascorbic acid on neutrophil–endothelial interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. The aim of the study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury, the influence of ascorbic acid on adherence and endothelial cell injury have been investigated. Production of oxygen-derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 6 mmol/L ascorbic acid during coincubation of neutrophils and endothelial cells (p < 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 6 mmol/L (p < 0.0005). The inhibition of neutrophil adherence to endothelial cells was correlated with a diminished neutrophil-mediated endothelial cell injury during incubation with 6 mmol/L ascorbic acid (p < 0.0005). The present results indicate that ascorbic acid might exert a protective effect on neutrophil-mediated endothelial cell injury by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Moreover, the current investigation points to probable protective effect of ascorbic acid on oxidant-mediated cell damage in diseases (e.g., Adult Respiratory Distress Syndrome).     

Free radicals in myocardial injury: experimental and clinical studies

The exposure of cardiac cells to OFR generated artificially, showed a marked decrease (p < 0.01) in cellular utilization of glucose alongwith a significant decrease in calcium uptake (p < 0.05). We have also provided evidence for a direct relationship of neutrophil OFR production with the extent of myocardial ischemia in patients of myocardial infarction. Our data provides evidence for implication of OFR in myocardial injury and the pivotal role played by modulators like calcium, ECGF and prostaglandins in potentiating damage to the myocardium.     

Reduced ELANE and SLPI expression compromises dental pulp cell activity

BackgroundPatients with ELANE variants and severe congenital neutropenia (SCN) commonly develop oral complications. Whether they are caused only by low neutrophil count or the combination of neutropenia and aberrant dental cells is unknown.MethodsGenetic variant was identified with exome sequencing. Dental pulp cells isolated from the SCN patient with an ELANE mutation were investigated for gene expression, enzyme activity, proliferation, colony formation, wound healing, apoptosis, ROS, attachment, spreading and response to lipopolysaccharide.ResultsELANE cells had diminished expression of ELANE and SLPI and reduced neutrophil elastase activity. Moreover, ELANE cells exhibited impaired proliferation, colony forming, migration, attachment and spreading; and significantly increased ROS formation and apoptosis, corresponding with increased Cyclin D1 and MMP2 levels. The intrinsic levels of TGF‐β1 and TNF‐α were significantly increased; however, IL‐6, IL‐8 and NF‐kB1 were significantly decreased in ELANE cells compared with those in controls. After exposure to lipopolysaccharide, ELANE cells grew larger, progressed to more advanced cell spreading stages and showed significantly increased SLPI, TNF‐α and NF‐kB1 and tremendously increased IL‐6 and IL‐8 expression, compared with controls.ConclusionThis study, for the first time, suggests that in addition to neutropenia, the aberrant levels and functions of ELANE, SLPI and their downstream molecules in pulp cells play an important role in oral complications in SCN patients. In addition, pulp cells with diminished neutrophil elastase and SLPI are highly responsive to inflammation.     

The investigation of the ultrastructural neutrophil changes in alloxan-induced diabetes in rats: response to a chemotactic challenge

Experimental diabetes is one of the most popular conditions in which to study the relation between neutrophil leukocyte activity and periodontal destruction. The aetiology of neutrophil dysfunction in the gingival tissue associated with diabetes has yet to be clarified. Diabetes in rats decreases neutrophil chemotactic activity in proportion to the severity of this systemic disorder. The present study was carried out to evaluate the relationship between the severity of diabetes and the neutrophil response to two chemotactic agents, and to correlate the observed neutrophil defects with the degree of diabetes. In this study two chemotactic agents, casein (0.2 μl, 2 mg ml^?1) or N‐formylmethionylleucylphenylalanine (FMLP; 0.2 μl, 10^?4 M ), were placed into the gingival crevices of alloxan‐induced diabetic rats. Gingival biopsies were taken 15 min later and then at 5‐min intervals up to 45 min and investigated by electron microscopy. Adherence and migration were observed in the rats with moderate diabetes 30 min after the application of casein. There was chemotaxis after 35 min of administration of the peptide FMLP. By 40 min neutrophils with pyknotic nuclei were observed. At 45 min neutrophils with a decreased number of granules were present. As the severity of the diabetes increased, the neutrophils degenerated and were structurally distorted. In the rats which had alloxan‐induced diabetes there was abnormal periodontal damage. This damage is thought to be related to dysfunctional neutrophils. These findings many contribute to an answer to the following question: why is there an apparent variability in the susceptibilty of periodontal breakdown in diabetics? Copyright © 2003 John Wiley & Sons, Ltd.     

Early anti-inflammatory treatment reduces lipid peroxidation and protein nitration after spinal cord injury in rats

We investigated mechanisms by which a monoclonal antibody (mAb) against the CD11d subunit of the leukocyte integrin CD11d/CD18 improves neurological recovery after spinal cord injury (SCI) in the rat. The effects of an anti-CD11d mAb treatment were assessed on ED-1 expression (estimating macrophage infiltration), myeloperoxidase activity (MPO, approximating neutrophil infiltration), lipid peroxidation, inducible nitric oxide synthase (iNOS) and nitrotyrosine (indicating protein nitration) expression in the spinal cord lesion after severe clip-compression injury. Protein expression was evaluated by western blotting and immunocytochemistry. Lipid peroxidation was assessed by thiobarbituric acid reactive substances (TBARS) production. After anti-CD11d mAb treatment, decreased ED-1 expression at 6-72 h after SCI indicated reduced macrophage infiltration. MPO activity (units/g tissue) was reduced significantly from 114 +/- 11 to 75 +/- 8 (- 34%) at 6 h and from 38 +/- 2 to 22 +/- 4 (- 42%) at 72 h. After SCI, anti-CD11d mAb treatment significantly reduced TBARS from 501 +/- 61 to 296 +/- 17 nm (- 41%) at 6 h and to approximately uninjured values (87 nm) at 72 h. The mAb treatment also attenuated the expression of iNOS and formation of nitrotyrosine at 6-72 h after SCI. These data indicate that anti-CD11d mAb treatment blocks intraspinal neutrophil and macrophage infiltration, reducing the intraspinal concentrations of reactive oxygen and nitrogen species. These effects likely underlie improved tissue preservation and neurological function resulting from the mAb treatment.     

An anti-CD11d integrin antibody reduces cyclooxygenase-2 expression and protein and DNA oxidation after spinal cord injury in rats

Our studies have shown that treatment with a monoclonal antibody (mAb) against the CD11d subunit of the leukocyte integrin CD11d/CD18 after spinal cord injury (SCI) decreases intraspinal inflammation, myeloperoxidase activity, lipid peroxidation and protein nitration, improving neurological function in rats. Using severe clip compression SCI in the rat, immunohistochemistry and western blotting were employed to assess the effects of an anti-CD11d mAb treatment on spinal cord cyclooxygenase-2 (COX-2) expression, formation of 8-hydroxy-2-deoxyguanosine (8-OHdG, a marker of RNA and DNA oxidation) and protein carbonylation (a marker of protein oxidation). We also assessed treatment effects on the expression of apurinic/apyrimidinic endonuclease (redox effector factor-1, APE/Ref-1), a multifunctional enzyme involved in the base excision repair of apurinic/apyrimidinic sites in DNA. The expression of COX-2 and formation of 8-OHdG and protein carbonyl groups were increased after SCI while APE/Ref-1 expression was decreased. Anti-CD11d mAb treatment clearly attenuated COX-2 expression and 8-OHdG and protein carbonyl formation and rescued APE/Ref-1 expression after SCI. This study suggests that anti-CD11d mAb treatment significantly reduces intraspinal free radical formation after SCI, thereby reducing protein and DNA oxidative damage. These effects likely underlie tissue preservation and improved neurological function resulting from the mAb treatment.     

Recombinant human interleukin-8, but not human interleukin-1beta, induces bovine neutrophil migration in an in vitro co-culture system

A co-culture system was established by culturing a bovine mammary epithelial cell line (MAC-T) and a bovine aortic endothelial cell line on calf tail collagen pre-coated inserts. This system allowed us to study bovine neutrophil migration across endothelium, extracellular matrix (ECM), and epithelium in the correct sequence and direction in vitro. The effect of recombinant interleukin-1beta (rHIL-1beta) and interleukin-8 (rHIL-8) on bovine neutrophil migration was investigated using this system. rHIL-8 stimulated bovine neutrophil migration in a dose-dependent fashion. The level of migrating bovine neutrophils increased up to approximately 25% when 100 ng/ml of rHIL-8 was used. On the other hand, rHIL-1beta at concentrations up to 100 ng/ml did not directly induce bovine neutrophil migration. Furthermore, pre-incubation with 5 ng/ml of rHIL-1beta in the co-culture system for 4 or 24 h failed to have any effect. These results suggest that IL-8 plays an important role in neutrophil migration into bovine mammary glands during mastitis.