In vitro and in vivo stimulation of neutrophil migration and lymphocyte transformation by thiamine related to inhibition of the peroxidase/H2O2/halide system.

The effects of thiamine on neutrophil functions and mitogen-induced lymphocyte transformation were investigated in vitro and in vivo in adult volunteers following the injection of 50 mg thiamine intramuscularly. Thiamine caused stimulation of neutrophil motility in vitro and in vivo and increased lymphocyte transformation in vivo. Enhancement of these functions was related to inhibition of neutrophil post-phagocytic iodination of Candida albicans by the MPO/H2O2/halide system. The horseradish peroxidase/-H2O2/125 I-mediated iodination of bovine serum albumin was also inhibited by thiamine concentrations which caused increased neutrophil motility. It was found that preincubation of neutrophils and lymphocytes with the horseradish peroxidase/H2O2/halide system caused considerable inhibition of the migratory and proliferative responses respectively. Inclusion of thiamine at concentrations which were found to inhibit the peroxidase/-H2O2/halide system protected the neutrophil migratory and lymphocyte proliferative responses from inactivation by this system. It is suggested that thiamine may cause increased neutrophil migration and lymphocyte transformation by protecting these cells from toxic oxidative products generated by the peroxidase/H2O2/halide system.     

Prevention of peroxidase mediated inhibition of neutrophil motility and lymphocyte transformation by levamisole, OMPI, sodium aurothiomalate, indomethacin and tolmetin in vitro

The effects of sodium aurothiomalate, levamisole, its active metabolite OMPI and the anti-inflammatory agents indomethacin and tolmetin on neutrophil motility and post-phagocytic hexose monophosphate shunt activity, superoxide and H2O2 generation and myeloperoxidase (MPO) mediated iodination of Candida albicans were investigated in vitro. All five agents caused stimulation of neutrophil random motility and migration towards the leucoattractants f-met-met-phe and EAS. Only levamisole caused inhibition of H2O2 and superoxide production, which was associated with inhibition of HMS activity and not related to superoxide scavenging activity. All five agents caused inhibition of MPO mediated iodination of C. albicans. The relationship between inhibition of peroxidase mediated iodination and enhanced motility was further investigated using the horseradish peroxidase (HRP) H2O2/iodide system. Incubation of neutrophils with this system caused inhibition of neutrophil motility. However in the presence of the various drugs neutrophils were protected from inhibition of motility by the HRP/H2O2/iodide system. Further experiments showed that lymphocyte transformation to mitogens was also inhibited by the HRP/H2O2/iodide system. Incubation of lymphocytes with the various drugs prior to exposure to HRP/H2O2/iodide protected the lymphocyte mitogenic responsiveness.     

Levamisole stimulation of neutrophil chemotaxis and chemokinesis by protection of both the leucoattractant and the cellular chemotactic response from inactivation by the peroxidase/hydrogen peroxide/halide system in vitro

The effects of levamisole, at concentrations known to stimulate neutrophil motility, on neutrophil post-phagocytic metabolic activity were investigated. Levamisole at these concentrations caused inhibition of hexose monophosphate shunt (HMS) activity, superoxide production, hydrogen peroxide generation and myeloperoxidase (MPO), mediated iodination of ingested Candida albicans,. The inhibition of MPO-mediated iodination was not solely due to lack of H2O availability as a result of decreased HMS activity but also to a primary inhibition of iodination as shown in a cell-free system with horse-radish peroxidase (HRP) and added H2O2 and sodium iodide. Further experiments were designed to investigate possible relationships between stimulation and motility and levamisole-induced inhibition of superoxide generation and peroxidase-mediated iodination. These showed that the peroxidase/halide/H2O2 system caused inactivation of both the leucoattractant and neutrophil chemotactic responsiveness. However, concentrations of levamisole, which stimulate motility and inhibit superoxide production and peroxidase-mediated iodination, protected both the leucoattractant response and the ability of the cell to respond to the leucoattractant from inactivation by the HRP/H2O2/iodide system.     

In vitro stimulation of neutrophil motility by metoprolol and sotalol related to inhibition of both H2O2 production and peroxidase mediated iodination of the cell and leucoattractant

The effects of the beta-receptor blockading agents, metoprolol and sotalol on neutrophil random motility, chemotaxis, post-phagocytic glycolysis, superoxide production, hexose monophosphate shunt activity, myeloperoxidase (MPO) mediated protein iodination and hydrogen peroxide production were assessed in vitro. The concentration range investigated was 10(-8)--10(-2) M for each drug. Both agents caused significant stimulation of neutrophil motility at concentrations of more than 10(-4) M. Increased migration was not associated with increased glycolysis or significant cyclic nucleotide fluctuations, but was inversely related to inhibition of superoxide and hydrogen peroxide generation and MPO mediated iodination with both drugs. In a further series of experiments to determine the relationship between the drug induced inhibition of H2O2 production and MPO mediated protein iodination to stimulation of motility it was found that concentrations of sotalol and metoprolol that caused these effects prevented HRP/H2O2/I- induced inactivation of the leucoattractant and inhibition of neutrophil chemotactic responsiveness. Neither drug inhibited the activity of MPO per se nor the reduction of ferricytochrome c by superoxide generated by the xanthine: xanthine oxidase system in vitro. It is suggested that enhanced neutrophil motility is not related to beta-receptor blockade but rather to restricting the availability of hydrogen peroxide and reactive products of the MPO/H2O2/halide system.     

Assessment of oral ascorbate in three children with chronic granulomatous disease and defective neutrophil motility over a 2-year period

Two brothers and their sister with chronic granulomatous disease, elevated levels of serum IgE and defective neutrophil motility were treated with a single oral daily dose of 1 g sodium ascorbate as a supplement to prophylactic trimethoprim--sulphamethoxazole therapy for 2 years. Laboratory tests of neutrophil functions were performed prior to ascorbate therapy and repeated at 1-monthly intervals for 6 months and at 6-monthly intervals thereafter. Introduction of ascorbate to the therapeutic regimen was accompanied by slight increases in neutrophil hexose monophosphate shunt activity and staphylocidal activity and good improvement of neutrophil motility in all three children. The improved staphylocidal activity was not due to ascorbate-mediated inhibition of neutrophil or serum catalase activities or to detectable increases in superoxide and H2O2 production or activity of the MPO/H2O2/halide system. Both male children have remained free from obvious infection since ascorbate was added to their therapeutic regimen; their sister has experienced one urinary tract infection during a period when treatment with prophylactic co-trimoxazole and ascorbate was inadvertently stopped. All three children have gained weight.     

Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin

Hydroethidine (HE) and 2',7'-dichlorofluorescin (DCFH) were used for the flow cytometric measurement of reactive oxygen metabolites in leukocytes. Hydroethidine and DCFH were both rapidly oxidized in a cell-free cuvette assay to ethidium bromide (EB) and 2',7'-dichlorofluorescein (DCF) by H2O2 and peroxidase, but not by H2O2 alone, while only HE was oxidized by KO2, a source of O2-. Quiescent lymphocytes, monocytes, and neutrophils spontaneously oxidized HE to EB, while DCFH was only oxidized to a low degree. Neutrophils increased 6.9-fold in EB red fluorescence and 12.5-fold in DCF green fluorescence during the respiratory burst induced by phorbol 12-myristate 13-acetate or 6.1-fold and 4.7-fold, respectively, during the respiratory burst induced by Escherichia coli bacteria. The HE or DCFH oxidation during the respiratory burst, unlike the spontaneous HE oxidation, was not inhibitable by 10 mM NaNe indicating a non-mitochondrial source of cellular oxidants during the respiratory burst such as NADPH oxidase, which produces O2-. The oxidation of DCFH, but not of HE, was decreased in stimulated neutrophils, which were simultaneously loaded with HE and DCFH. Intracellular DCFH oxidation induced by incubation of resting neutrophils with extracellular H2O2 was not influenced by the presence of HE. This indicates that HE is oxidized at an earlier step in the reactive oxygen metabolism of neutrophils than DCFH, i.e., by early oxygen metabolites like O2-, while DCFH is oxidized in part by H2O2 and phagosomal peroxidases. The differential oxidation of HE and DCFH during simultaneous cellular staining permits the analysis of up to three functionally different neutrophil populations in septic patients. This is of interest for the determination of disease-related alterations of oxygen metabolism in quiescent and stimulated leukocytes.     

Both recombinant interleukin-1 (beta) and purified human monocyte interleukin-1 prime human neutrophils for increased oxidative activity and promote neutrophil spreading

Both purified human monocyte interleukin-1 and recombinant interleukin-1 (beta) primed neutrophils for increased superoxide production and chemiluminescence in response to f-met-leu-phe. In addition, purified human monocyte interleukin-1 and recombinant interleukin-1 (beta) altered neutrophil shape. Recombinant interleukin-1 (alpha) used at the same concentration of interleukin-1 (beta) did not prime neutrophils for increased superoxide production after stimulation with f-met-leu-phe. Interleukin-1 expressed by monocytes in response to endotoxin stimulation could act as a modulator of neutrophil function.     

Enhanced neutrophil motility by granulocyte colony-stimulating factor: the role of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase

The effect of granulocyte colony-stimulating factor (G-CSF) on human neutrophil motility was studied using videomicroscopy. Stimulation of neutrophils with G-CSF resulted in enhanced motility with morphological change and increased adherence. Enhanced neutrophil motility was detected within 3-5 min after G-CSF stimulation, reached a maximum at 10 min, and was sustained for approximately 35 min. The maximum migration rate was 84.4 +/- 2.9 microm/5 min. A study using the Boyden chamber method revealed that G-CSF-stimulated neutrophils exhibited random migration but not chemotaxis. Enhanced neutrophil motility and morphological change were inhibited by MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] inhibitors (PD98059 and U0126), and a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), but not by a p38 MAPK inhibitor (SB203580). These findings are consistent with the fact that G-CSF selectively activates MEK/ERK and PI3K, but not p38, in neutrophils. MEK/ERK activation was associated with G-CSF-induced redistribution of F-actin and phosphorylated myosin light chain. Enhanced neutrophil motility was observed even in the presence of neutralizing anti-CD18 antibody, which prevented cell adherence. These findings indicate that G-CSF induces human neutrophil migration via activation of MEK/ERK and PI3K.     

Stimulation of human neutrophil oxidative metabolism by nonopsonized Neisseria gonorrhoeae.

Nonopsonized gonococci possessing opacity-associated (Opa; previously PII) outer membrane proteins stimulate neutrophils to undergo a vigorous oxidative response when measured by luminol-dependent chemiluminescence (LDCL). In these studies, we characterized the mechanism of this stimulation. No gonococci that we tested induced measurable release of neutrophil superoxide anion (O2-) or hydrogen peroxide (H2O2) as measured by reduction of cytochrome c or the oxidation of scopoletin, respectively. Neutrophils pretreated with gonococci and then exposed to phorbol myristate acetate, the chemotactic peptide formylmethionylleucylphenylalanine, or opsonized zymosan released levels of neutrophil O2- and H2O2 comparable to controls, indicating that gonococci were not preventing or inhibiting neutrophil O2- or H2O2 release. To ascertain a possible explanation for these seemingly contradictory observations (i.e., induction of LDCL, but no release of O2- or H2O2), we further characterized the ability of Opa+ gonococci to stimulate LDCL. By using 1 mM azide and 4 U of horseradish peroxidase to monitor extracellular LDCL selectively and 2,000 U of catalase to monitor intracellular LDCL selectively, we determined that greater than 80% of total gonococcus-induced neutrophil LDCL occurred intracellularly. In addition, neutrophils stimulated with Opa+ gonococci showed a marked increase in O2 uptake and hexose monophosphate shunt activity. We conclude that Neisseria gonorrhoeae induces neutrophil oxidative metabolism without causing release of detectable amounts of reactive oxygen intermediates into the surrounding milieu. The gonococcus apparently directs oxidase assembly and activity to the phagolysosomal membrane. This could be a mechanism by which extracellular gonococci persist for extended periods in vivo in the presence of high concentrations of neutrophils.     

The in vitro evaluation of certain neutrophil and lymphocyte functions following the ingestion of 150 mg oral dose of levamisole: assessment of the extent and duration of stimulation of neutrophil chemotaxis, protein iodination and lymphocyte transformation.

Certain functions of human blood neutrophils and lymphocytes were investigated at varying time intervals after the ingestion of a single 150 mg dose of levamisole. The functions tested were neutrophil chemotaxis and post-phagocytic metabolic activity and mitogen-induced DNA and protein synthesis of lymphocytes. It was found that levamisole causes a stimulation of neutrophils motility (cell- and serum-associated) and post-phagocytic hexose monophosphate shunt activity and protein iodination. Increased lymphocyte DNA synthesis, but not protein synthesis, to the mitogen phytohaemagglutinin was observed. The stimulation which was detected almost immediately of these neutrophil and lymphocyte functions was still evident 24 hr later but not at 48 hr, indicating that a single oral dose of levamisole can cause the alteration (stimulation) of leucocyte functions which persists until 24--48 hr after intake of the drug.     

Oxidative inactivation of Actinobacillus actinomycetemcomitans leukotoxin by the neutrophil myeloperoxidase system.

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of inflammatory periodontal disease. We examined a potential mechanism for detoxification of this microbial product by the neutrophil myeloperoxidase system. Exposure to myeloperoxidase, H2O2, and a halide resulted in marked inactivation of leukotoxin, an effect which required each component of the myeloperoxidase system. Toxin inactivation was blocked by agents which inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The latter system, in fact, was more potent than reagent H2O2 in terms of the capacity to inactivate high concentrations of toxin. Toxin inactivation was complete within 1 to 2 min at 37 degrees C. These observations suggest a possible role for oxidative inactivation of leukotoxin by secretory products of neutrophils.     

An in vitro assessment of cellular and humoral immune function in pulmonary tuberculosis: correction of defective neutrophil motility by ascorbate, levamisole, metoprolol and propranolol.

Fifty-six tuberculosis patients and twenty-eight control subjects were evaluated in a comprehensive investigation of cellular and humoral immune function in pulmonary TB. The patient group showed significantly higher levels of secretory IgA and serum IgG, IgA and IgM than did the control group but 7% of patients displayed a selective secretory IgA deficiency. Levels of alpha-1-antitrypsin were also significantly higher in the patient group. There were no significant differences in levels of total haemolytic complement, C'3 and C'4. In moderate to moderately advanced TB patients there were no significant differences in T and B cell numbers nor in mitogen-induced lymphocyte transformation and lymphokine production, when compared with the control group. The range of PPD-induced lymphocyte transformation and lymphokine production levels encountered was similar in both groups although certain patients did not respond to the PPD antigen. Neutrophils from TB patients showed increased random motility in vitro but eight out of ten patients showed impaired directed motility (chemotaxis). Phagocytic and anti-microbial functions were normal in the patient group. The neutrophil chemotactic defect was reversible and could be corrected in vitro when the patients' cells were treated with sodium and calcium ascorbate, levamisole, metoprolol and propranolol.     

Neutrophil priming by hepatocyte growth factor, a novel cytokine.

We demonstrate here that the recently defined cytokine hepatocyte growth factor (HGF) 'primes' human neutrophils. Recombinant human HGF over the concentration range 0.1-20 ng/ml increased the neutrophil response to f-met-leu-phe by up to 200%, and required only a short preincubation, 10 min producing the maximum effect. Priming was independent of changes in cytosolic-free calcium homeostasis. We conclude that HGF may be a physiologically important cytokine with 'priming' activity for neutrophils.     

Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.     

Benoxaprofen: A pro-oxidant anti-inflammatory drug?

Benoxaprofen inhibited the random motility and migration to the leucoattractants endotoxin-activated serum (EAS) and f-met-leu-phe of human polymorphonuclear leucocytes (PMNL)in vitro. Inhibition of random and leucoattractant-induced migration was observed at drug concentrations of >1×110^–6 M and 1×10^–5 M respectively. Benoxaprofenper se was not leucotactic but was pro-oxidative in that it stimulated PMNL hexose-monophosphate shunt activity, chemiluminescence, myeloperoxidase-mediated iodination reactions and degranulation. The drug also mediated auto-oxidation of PMNL as measured by cellular auto-lodination. The relationship between benoxaprofen-mediated inhibition of PMNL migration and activation of oxidative metabolism was investigated using the anti-oxidants ascorbate and levamisole at concentrations of 10^–2 M and 10^–3 M respectively. These agents prevented the decreased motility and auto-oxidation of PMNL induced by 10^–4 M benoxaprofen. Benoxaprofen (10^–4 M) did not inhibit the migration of PMNL from 3 children with chronic granulomatous disease thus showing that intact PMNL oxidative metabolism is required for the induction of drug-mediated inhibition of cell motility. Ingestion of therapeutic doses of benoxaprofen for 7 days by normal adults gave serum drug concentrations greater than those required for detectable effects on PMNL functionsin vitro (mean serum value 126 g/ml). Co-incubation of normal PMNL with serum from individuals who had ingested the drug caused decreased cell migration and increased chemiluminescence. These results show that benoxaprofen inhibits PMNL migration as a consequence of pro-oxidant properties and despite its withdrawal may be the prototype of the pro-oxidative anti-inflammatory drug.     

Toll-like receptor agonists stimulate human neutrophil migration via activation of mitogen-activated protein kinases

Human neutrophil migratory responses to Toll-like receptor (TLR) agonists were studied using videomicroscopy. When challenged with lipopolysaccharide (LPS, TLR4 agonist) or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (P3CSK4, TLR2 agonist), neutrophils displayed enhanced motility, which was found to reflect increased random migration but not directed migration (chemotaxis). Enhanced neutrophil motility was detected within 10 min after stimulation with LPS or P3CSK4, and was sustained for more than 80 min. Stimulation of neutrophils with LPS or P3CSK4 resulted in the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), which preceded neutrophil migration. TLR-mediated neutrophil migration was strongly suppressed by pretreatment of cells with U0126 (MAPK/ERK kinase inhibitor) but not with U0124 (an inactive analogue of U0126) or SB203580 (a p38 MAPK inhibitor), and was almost completely abolished by pretreatment of cells with U0126 and SB203580 in combination. Randomly migrating neutrophils in response to LPS or P3CSK4 displayed directed migration when further challenged with gradient concentrations of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF). These findings indicate that TLR agonists stimulate human neutrophil migration via the activation of ERK and p38 MAPK, and FMLP- or PAF-induced neutrophil chemotaxis is not affected by the pre-exposure of cells to TLR agonists.     

The effect of neutrophil migration and prolonged neutrophil contact on epithelial permeability.

The effect of neutrophil migration and prolonged neutrophil contact on epithelial permeability was examined. Although neutrophil migration was not associated with a change in epithelial permeability, prolonged neutrophil-epithelial contact following migration resulted in an increase in epithelial permeability. These results were not altered by catalase, a specific neutrophil elastase inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone or cyclohexamide. This suggests that neutrophil migration does not occur via an H2O2-induced reversible mechanism of junctional opening, which we describe herein.     

Influenza a viruses upregulate neutrophil toll-like receptor 2 expression and function

Neutrophils are involved in the initial host response to influenza A virus (IAV) infection and exhibit both activation and depressed function after exposure to the virus. We demonstrate that IAV causes rapid upregulation of Toll-like receptor 2 (TLR2) expression on neutrophils. The neutrophil agonists, formyl-methylpleucyl-alanine (fMLP), C5a and lipopolysaccharide did not alter neutrophil TLR2 expression, whereas PMA and the microbial TLR2 ligands, peptidoglycan (PGN) and zymosan, reduced it. To determine the functional significance of IAV-induced increase in TLR2 expression, IAV-treated neutrophils were exposed to PGN, Staphylococcus aureus (S. aureus) and zymosan. Pretreatment with IAV resulted in significantly increased uptake of S. aureus and zymosan and accelerated neutrophil apoptosis when combined with S. aureus. IAV-treated cells generated significantly more H(2)O(2) in response to PGN. These results indicate that IAV increases neutrophil surface expression of TLR2 and modulates functional responses to ligands that bind TLR2. These findings may clarify IAV-induced perturbation of neutrophil functions in vivo.     

Novel human neutrophil agonistic properties of arsenic trioxide: involvement of p38 mitogen-activated protein kinase and/or c-jun NH2-terminal MAPK but not extracellular signal-regulated kinases-1/2

Arsenic trioxide (ATO) is known for treating acute promyelocytic leukemia and for inducing apoptosis and mitogen-activated protein kinases (MAPKs) in promyelocytes and cancer cells. We recently reported that ATO induces neutrophil apoptosis. The aim of this study was to establish whether or not ATO recruits MAPKs in neutrophils, as well as to further investigate its agonistic properties. We found that ATO activates p38 and that, unlike H2O2, this response was not inhibited by exogenous catalase. Also, we demonstrated that ATO-induced p38 activation occurs before H2O2 generation and without a calcium burst. We next established that ATO recruits c-jun NH2-terminal (JNK) but not extracellular signal-regulated kinase 1 and 2 (Erk-1/2). Using pharmacological inhibitors, we found that the proapoptotic activity of ATO occurs by a MAPK-independent mechanism. In contrast, the ability of ATO to enhance adhesion, migration, phagocytosis, release, and activity of gelatinase and degranulation of secretory, specific, and gelatinase, but not azurophilic granules, is dependent upon activation of p38 and/or JNK. This is the first study establishing that ATO possesses important agonistic properties in human neutrophils. Given the central role of neutrophils in various inflammatory disorders, we propose that ATO might have broader therapeutic implications in clinics, especially for regulating inflammation.