Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 56
Filter
Add more filters










Publication year range
1.
Microbiol Immunol ; 45(3): 241-8, 2001.
Article in English | MEDLINE | ID: mdl-11345534

ABSTRACT

Neutrophils up-regulate beta2 integrins like CD11b/CD18 in response to lipopolysaccharide (LPS). Up-regulation of beta2 integrins causes neutrophils to adhere to surfaces, and to release superoxide anion (O2-). When neutrophils are exposed to LPS plus plasma under conditions not favorable for adherence (absence of Mg2+), the cells do not spontaneously release O2-, but instead they are primed for enhanced release of O2- after subsequent triggering by fMLP. In the presence of Mg2+, neutrophils adhere in response to LPS but fMLP-triggered O2- release by LPS-primed neutrophils is diminished. To understand why adherence interferes with the response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (fMLP), beta2 integrins were cross-linked by mouse monoclonal antibodies that had been immobilized by surface-bound anti-mouse antibody. When unprimed neutrophils were trapped on the surface by these cross-linked monoclonal antibodies, O2- release was triggered, and priming by LPS for fMLP-triggered O2- release was diminished, indicating that this cross-linking of beta2 integrins mimicked adherence. Alkaline phosphatase is up-regulated by LPS or tumor necrosis factor-alpha, and this response was also diminished by the cross-linking antibodies. The diminished alkaline phosphatase up-regulation was reversed by genistein, a general inhibitor of tyrosine kinases, and by piceatannol, an inhibitor for Syk kinase. Piceatannol also inhibited the phosphorylation of Syk caused by cross-linking of beta2 integrins. These results suggested that adherence-induced triggering and Syk kinase activation might be responsible for the diminished response of LPS-primed neutrophils to fMLP when neutrophils were adherent.


Subject(s)
CD18 Antigens/chemistry , Enzyme Precursors/physiology , Neutrophils/drug effects , Protein-Tyrosine Kinases/physiology , Alkaline Phosphatase/metabolism , Antibodies, Monoclonal/pharmacology , Cell Adhesion , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Enzyme Precursors/antagonists & inhibitors , Genistein/pharmacology , Humans , Intracellular Signaling Peptides and Proteins , Lipopolysaccharides , N-Formylmethionine Leucyl-Phenylalanine , Neutrophils/physiology , Phosphorylation/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Stilbenes/pharmacology , Superoxides/analysis , Superoxides/metabolism , Syk Kinase , Time Factors , Tumor Necrosis Factor-alpha
2.
Eur J Pharmacol ; 374(2): 207-11, 1999 Jun 18.
Article in English | MEDLINE | ID: mdl-10422761

ABSTRACT

The effect of serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) was investigated on the prevention of tumor-necrosis-factor-alpha (TNF-alpha)-induced blood-brain barrier opening. TNF-alpha (10,000 IU) was injected intracarotidly to newborn pigs pretreated with 0, 2.4, 4.8, 9.6 and 19.2 mg/kg AEBSF (n = 6 in each group). AEBSF dose-dependently inhibited the TNF-alpha-induced increase in the blood-brain barrier permeability for sodium fluorescein (MW = 376) in all of the five brain regions examined, while only 19.2 mg/kg AEBSF could significantly (P < 0.05) decrease the change in Evan's blue-albumin (MW = 67,000) transport in two regions. In conclusion, AEBSF attenuates vasogenic brain edema formation.


Subject(s)
Blood-Brain Barrier/drug effects , Brain Edema/prevention & control , Serine Proteinase Inhibitors/pharmacology , Sulfones/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Animals, Newborn , Blood-Brain Barrier/physiology , Brain/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Evans Blue/pharmacokinetics , Female , Fluorescein/pharmacokinetics , Male , Swine
3.
Neuroimmunomodulation ; 6(4): 261-83, 1999.
Article in English | MEDLINE | ID: mdl-10393513

ABSTRACT

Muramyl peptides are fragments of peptidoglycan from the cell walls of bacteria. Because of their unique chemistry, the immune system recognizes that muramyl peptides are products of bacteria, and it responds by becoming activated to resist infection. This resistance to infection is nonspecific, and extends to unrelated species of bacteria, fungi, and viruses. A key mechanism of the resistance to infection is activation of macrophages. Macrophage activation results in increased production of microbicidal oxygen radicals like superoxide and peroxide, and in increased secretion of inflammatory cytokines like interleukin-1beta and tumor necrosis factor-alpha. These cytokines, besides activating neutrophils, B lymphocytes, and T lymphocytes, act on the central nervous system to induce physiological responses like fever and sleep. These physiological responses also aid in combating infection. Muramyl peptides also activate macrophages and other cells of the immune system to kill cancer cells. Muramyl peptides and similar agents will become more important as therapeutic agents in the future, due to increasing resistance of microbes to antibiotics, and increasing numbers of patients with immunodeficiencies.


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Macrophages/drug effects , Monokines/drug effects , Muramic Acids/pharmacology , Peptidoglycan/chemistry , Sleep/drug effects , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Animals , Carbohydrate Sequence , Humans , Immune System/cytology , Immune System/drug effects , Molecular Sequence Data
4.
Jpn J Cancer Res ; 89(5): 548-55, 1998 May.
Article in English | MEDLINE | ID: mdl-9685859

ABSTRACT

Sphingosine and its analogs, which inhibit protein kinase C (PKC), are known to be potent inducers of apoptosis in tumor cells. However, we were concerned that sphingosine might also interfere with anti-tumor cells of the immune system. Therefore, we evaluated the effect of sphingosine on activation of human monocytes by interleukin-2 (IL-2) for killing of leukemic cells. Monocytes, purified by elutriation and adherence, were activated with IL-2 or interferon-gamma (IFN-gamma) in the presence or absence of sphingosine or another inhibitor for 18 h. Then the monocytes were washed and the culture medium was replaced with fresh medium to remove the sphingosine. HL- 60 and K562 leukemic cells were added to the monocyte cultures. Over the next 48 h, the cytotoxic activity of the monocytes towards the leukemic cells was assessed by means of an 111-indium-releasing assay. IL-2-activated monocytes lysed 48 +/- 3% of HL-60 cells and 44 +/- 3% of K562 cells. Sphingosine, dihydrosphingosine, N,N-dimethylsphingosine, and the PKC inhibitor H7 inhibited the activation of monocytes by IL-2, blocking cytotoxic activity against the leukemic cells by approximately 75%. These inhibitors were not toxic to monocytes at the concentrations used. In a PKC assay, sphingosine and H7 inhibited PKC activity in IL-2-treated monocytes. Thus, sphingosines, by inhibiting PKC activity, inhibited activation of monocytes by IL-2, which inhibited the killing of leukemic cells.


Subject(s)
Enzyme Inhibitors/pharmacology , Interleukin-2/pharmacology , Leukemia/immunology , Monocytes/immunology , Protein Kinase C/antagonists & inhibitors , Sphingosine/pharmacology , Cytotoxicity Tests, Immunologic , HL-60 Cells , Humans , Interferon-gamma/pharmacology , Leukemia/enzymology , Monocytes/drug effects , Time Factors , Tumor Cells, Cultured
5.
J Leukoc Biol ; 64(2): 177-84, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9715256

ABSTRACT

Lipopolysaccharide (LPS) in solution primes neutrophils for enhanced release of superoxide in response to N-formyl-methionyl-leucyl-phenylalanine. We show that LPS immobilized on polystyrene or polypropylene acted on neutrophils by a mechanism different from that of LPS in solution. Coating the surface with 1% plasma, either before coating with LPS (plasma/LPS) or after coating with LPS (LPS/plasma), was essential to induce the LPS response in neutrophils. However, plasma could be replaced by fibrinogen, type I collagen or type IV collagen, or, to a lesser extent, by fibronectin or vitronectin, which was not true for LPS in solution. About 20% of the LPS added was immobilized on the plastic surfaces, based on its ability to adsorb anti-LPS antibody after extensive washing. The amount of soluble LPS that might have been released from surfaces during the incubation with neutrophils was too low to account for the priming by immobilized LPS. About 13-20 min was needed for neutrophils to become primed after incubation with immobilized LPS. Immobilized LPS induced up-regulation of CD11b/CD18 and latent alkaline phosphatase and also enhanced the adhesive response of neutrophils. Priming by immobilized LPS was inhibited by anti-CD14 antibody or by treatment of neutrophils with the LPS antagonist LA-14-PP. When immobilized LPS was treated with anti-LPS-binding protein (LBP) antibody, the response of neutrophils to LPS/plasma was inhibited but the response to plasma/LPS or fibrinogen/LPS was not. Thus, the LPS in plasma/LPS or fibrinogen/LPS acted on neutrophils in an LBP-independent manner. We conclude that the CD14-dependent LPS receptor system of neutrophils was capable of working in the absence of LBP, but only when LPS was immobilized on a surface coated with protein.


Subject(s)
Acute-Phase Proteins , Carrier Proteins/immunology , Lipopolysaccharides/pharmacology , Membrane Glycoproteins , Neutrophils/cytology , Neutrophils/immunology , Alkaline Phosphatase/metabolism , Antibodies/pharmacology , Cell Adhesion/immunology , Collagen/immunology , Enzyme Activation/immunology , Enzyme-Linked Immunosorbent Assay , Extracellular Matrix/chemistry , Extracellular Matrix/immunology , Fibronectins/pharmacology , Glass , Humans , In Vitro Techniques , Lipopolysaccharide Receptors/immunology , Macrophage-1 Antigen/metabolism , Neutrophil Activation/drug effects , Neutrophil Activation/immunology , Neutrophils/enzymology , Oxygen/metabolism , Plasma/immunology , Polypropylenes , Polystyrenes , Polytetrafluoroethylene
6.
J Mass Spectrom ; 33(12): 1182-91, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9875522

ABSTRACT

The structures of major muramyl peptides derived from peptidoglycan of the oral pathogen Streptococcus sanguis were determined and the biological activity of the peptides was tested in vitro on human monocytes. The muramyl peptides, produced by muramidase digestion of the purified peptidoglycan, were separated by reversed-phase high-performance liquid chromatography, either in their native form or after reduction with sodium borohydride. Chemical structures of the peptides were elucidated by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, amino acid analysis, post-source decay analysis and Edman sequencing. The study revealed two distinct monomers: N-acetylglucosaminyl-N-acetylmuramyl-Ala-iGln-Lys(Ala-Ala) (1), where the Ala-Ala is connected to the epsilon-amino group of lysine, and N-acetylglucosaminyl-N-acetylmuramyl-Ala-iGln-Lys(Ala-Ala)-Ala-Ala (2), where an additional dialanyl residue is attached to the lysine alpha-carboxyl group. Two sets of higher oligomers (di-, tri- and tetramers), related structurally to monomers 1 or 2 were also detected. In these oligomers, the monomeric subunits are linked together by Ala-Ala-Ala bridges. The native muramyl peptides primed human monocytes in vitro for the increased production of the microbicidal superoxide radical.


Subject(s)
Muramic Acids/chemistry , Peptidoglycan/chemistry , Streptococcus sanguis/chemistry , Amino Acid Sequence , Biological Assay , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Humans , In Vitro Techniques , Molecular Structure , Monocytes/drug effects , Monocytes/metabolism , Muramic Acids/isolation & purification , Muramic Acids/pharmacology , Peptidoglycan/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Streptococcus sanguis/pathogenicity , Superoxides/metabolism
7.
Brain Inj ; 11(11): 801-14, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9354257

ABSTRACT

This study examined the access and organization of goal-derived categories in semantic memory with a group of chronic traumatic brain injured TBI adults and a group of age and gender-matched neurologically-intact controls. Goal-derived categories are developed by individuals for use in specialized contexts to achieve a goal, such as 'things to take on a camping trip.' Categories were presented to subjects in two task contexts: category verification and exemplar generation. Overall, the TBI subjects were able to accurately identify and organize category exemplars within particular categories. Interestingly, the TBI subjects produced significantly more total responses than the neurologically-intact subjects on exemplar generation; however, a high percentage of their responses (one-third) were inaccurate, consisting of out-of-set responses and repetitions. These findings suggest that difficulties in retrieval may exist in the presence of relatively intact access and organization of goal-derived category structure. The results are discussed relative to deficits in the executive control of verifying goal-directed behaviour and incomplete category representation.


Subject(s)
Brain Injuries/complications , Goals , Language Disorders/etiology , Memory Disorders/etiology , Semantics , Adult , Chronic Disease , Female , Humans , Language Disorders/diagnosis , Language Tests , Male , Memory Disorders/diagnosis , Vocabulary
8.
Immunology ; 90(4): 477-82, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9176098

ABSTRACT

Ceramide acts as an intracellular second messenger in cellular signal transduction. We examined the effects of two cell-permeable ceramides, C2-ceramide and C6-ceramide, on human monocyte functions. After monocytes were primed with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) for 18 hr in suspension culture, they produced a high amount of superoxide (O2-) when triggered by phorbol myristate acetate. C2- or C6-ceramide inhibited O2- release from monocytes primed with LPS (1 ng/ml) or IFN-gamma (100 U/ml), but did not affect unprimed monocytes. An analogue, C2-dihydroceramide, was inactive. C2-ceramide was most effective at 6 microM, and C6-ceramide at 60 microM. C2- or C6-ceramide at these concentrations was not toxic for monocytes, as assessed by trypan blue exclusion and by the 3-[4, 5-dimethylthiazol-2-y1]-2,5 diphenyl tetrazolium bromide (MTT) assay which measures the ability of live cells to produce formazan. C2-ceramide (20 microM) had no effect on the killing of leukaemic cells (HL-60 and K562 cells) by monocytes treated with IFN-gamma, LPS, or both for 18 hr, with killing assessed by an 111 Indium-releasing assay. C2-ceramide (20 microM) induced secretion of low amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) from the monocytes. But C2-ceramide did not alter the higher secretion of TNF-alpha or IL-1 beta from monocytes treated with IFN-gamma or LPS. Thus the cell-permeable ceramides acted like antagonists of LPS, rather than analogues of LPS, as has been proposed. The results here showed that the signal transduction pathway for O2- release by monocytes differed from that for the cytolysis of leukaemic cells, and confirmed that oxygen radicals are not involved in cytolysis.


Subject(s)
Ceramides/pharmacology , Cytotoxicity, Immunologic/drug effects , Leukemia/immunology , Monocytes/drug effects , Sphingosine/analogs & derivatives , Superoxides/blood , Humans , Interferon-gamma/immunology , Interleukin-1/biosynthesis , Lipopolysaccharides/immunology , Monocytes/immunology , Monocytes/metabolism , Sphingosine/pharmacology , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/biosynthesis
9.
J Leukoc Biol ; 60(3): 328-36, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8830789

ABSTRACT

Proteases are known to be involved in regulation of macrophage activation and killing. We examined the effect of a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), on lysis of leukemic cells by human macrophages. Monocytes, isolated by Histopaque gradients and centrifugal elutriation, were cultured for 5 days in RPMI-1640 medium with 5% AB serum, and then activated with interferon-gamma (IFN-gamma; 100 U/mL) and lipopolysaccharide (LPS) (5 ng/mL), with or without AEBSF, for 2 days. On day 7, macrophages were washed, fresh medium without AEBSF added, and target cells added for 2 days. Lytic activity against two leukemic cell lines (K562 and HL-60) was assessed by an 111indium-releasing assay. Macrophages treated with IFN-gamma + LPS lysed K562 and HL-60 cells. AEBSF (50-150 microM) blocked the killing of these leukemic cells in a concentration-dependent manner. Other protease inhibitors were not effective. AEBSF was nontoxic at the concentrations used, and did not inhibit tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion from the macrophages. The lytic activity against leukemic cells was inhibited by anti-TNF-alpha antibody, but not by anti-IL-1 beta, nor by superoxide dismutase or catalase. However, the leukemic cells were resistant to being killed by recombinant TNF-alpha alone in the absence of macrophages, indicating that TNF-alpha was required for killing, but that other factors that were inhibited by AEBSF were also required. Serum-free culture supernatant of activated macrophages had significant cytotoxic activity against leukemic cells. This cytotoxic activity was not altered by addition of AEBSF to the culture supernatant, suggesting that AEBSF affected macrophage activation, rather than inhibiting cytotoxic proteases secreted by the macrophages, or affecting the target cells themselves. Thus, a protease, which is susceptible to AEBSF, might be involved in the activation of macrophages, and might regulate the secretion of antitumor effector molecules other than TNF-alpha.


Subject(s)
Macrophages/drug effects , Macrophages/immunology , Sulfones/pharmacology , Trypsin Inhibitors/pharmacology , Cell Death/drug effects , Cell Death/physiology , Cells, Cultured , Cytotoxicity, Immunologic , Endopeptidases/physiology , HL-60 Cells/drug effects , Humans , Interleukin-1/metabolism , Macrophage Activation/drug effects , Macrophages/enzymology , Tumor Necrosis Factor-alpha/metabolism
10.
Neuroimmunomodulation ; 3(2-3): 82-6, 1996.
Article in English | MEDLINE | ID: mdl-8945722

ABSTRACT

Interleukin-1 beta (IL-1 beta) is a key regulatory component of host defense systems, and it is involved in physiological regulation including several central nervous system functions like fever and sleep. Nevertheless, little is known about how IL-1 beta is degraded. Based on their protease secreting capabilities, the ability of microglia, monocytes, and neutrophils to degrade IL-1 beta was studied. Neutrophil cultures degraded Il-1 beta, whereas microglia and monocyte cultures did not. The degradation if IL-1 beta by neutrophils may constitute a downregulation mechanism for IL-1 beta.


Subject(s)
Interleukin-1/metabolism , Neutrophils/metabolism , Animals , Cells, Cultured , Endopeptidases/metabolism , Humans , Lipopolysaccharides/pharmacology , Mice , Microglia/metabolism , Monocytes/metabolism , Recombinant Proteins/metabolism , Tetradecanoylphorbol Acetate/pharmacology
11.
Immunology ; 86(4): 629-35, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8567031

ABSTRACT

Monocytes freshly isolated from human blood produced large amounts of superoxide when triggered by phorbol ester. After monocytes were cultured for 18-24 hr in endotoxin-free, non-adherent conditions, they produced low amounts of superoxide. Addition of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), or platelet-activating factor (PAF) at the beginning of culture 'primed' the monocytes, causing them to maintain a high superoxide response for at least 96 hr. Also, in response to LPS, monocytes secreted TNF-alpha. The ability of LPS, IFN-gamma, TNF-alpha or PAF to maintain the high superoxide response was blocked by addition of inhibitors of serine proteases, either 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) or 3,4-dichloroisocoumarin. AEBSF was most effective at 200 microns, and required 6 hr for maximum effect. AEBSF did not affect phorbol-triggered superoxide release by unprimed monocytes. AEBSF did not affect cell viability, nor did it interfere with the TNF-alpha secretion in response to LPS. An analogue of AEBSF that lacked ability to inhibit proteases did not affect monocyte responses. 3,4-Dichloroisocoumarin blocked priming at a low concentration, 1 microM. We conclude that activity of a monocyte serine protease is required to maintain the high superoxide response in monocytes primed with LPS, IFN-gamma, TNF-alpha, or PAF.


Subject(s)
Monocytes/drug effects , Serine Proteinase Inhibitors/pharmacology , Superoxides/metabolism , Cell Culture Techniques , Coumarins/pharmacology , Dose-Response Relationship, Drug , Humans , Isocoumarins , Kinetics , Lipopolysaccharides/immunology , Monocytes/immunology , Monocytes/metabolism , Sulfonamides/pharmacology , Sulfones/pharmacology , Tumor Necrosis Factor-alpha/metabolism
12.
J Leukoc Biol ; 58(6): 675-82, 1995 Dec.
Article in English | MEDLINE | ID: mdl-7499965

ABSTRACT

When incubated with lipopolysaccharide (LPS) in the presence of plasma, neutrophils become primed for enhanced release of superoxide in response to triggering by formyl-Met-Leu-Phe (fMLP). The effect of LPS on phagocytes is inhibited by a synthetic lipid A precursor, LA-14-PP (lipid IVa) or by LPS from Rhodobacter sphaeroides (Rs). We studied the mechanisms by which LA-14-PP or Rs-LPS inhibited LPS-induced responses. When neutrophils were exposed to LA-14-PP or Rs-LPS for 3 min and then to Escherichia coli-LPS, the antagonists inhibited priming for superoxide release, and also blocked up-regulation of CD11b and adherence. This inhibition was dependent on plasma, was not overcome by higher amounts of E. coli-LPS or plasma, and was not observed at 0 degrees C, suggesting that E. coli-LPS was not able to interact with its receptor or other cellular recognition molecule in neutrophils that had been exposed to the antagonists. The alternative possibility that LA-14-PP or Rs-LPS depleted a plasma cofactor, resulting in inhibition of priming, was investigated by using LPS from Porphyromonas gingivalis (Pg) and Bordetella pertussis (Bp). These LPS primed neutrophils in a plasma-dependent and CD14-dependent manner, but were not blocked by LA-14-PP or Rs-LPS. When sub-optimal concentrations of plasma were exposed to LA-14-PP or Rs-LPS, and then mixed with Pg-LPS or Bp-LPS, followed by incubation with neutrophils, priming and up-regulation of CD11b were inhibited, and this inhibition was overcome by increasing the concentration of plasma. Binding of LPS-binding protein (LBP) in plasma to immobilized E. coli-LPS was inhibited by pre-incubation of plasma with LA-14-PP or Rs-LPS. Together with the result that treatment of plasma with anti-LBP antibody abolished the cofactor activity of plasma, these results indicated that LA-14-PP and Rs-LPS depleted LBP from plasma, resulting in inability of LPS to act on neutrophils. Thus LA-14-PP and Rs-LPS inhibited the action of LPS on neutrophils by at least two mechanisms, blocking of LPS receptor recognition and depletion of the cofactor LBP.


Subject(s)
Acute-Phase Proteins , Carrier Proteins/metabolism , Glycolipids/pharmacology , Lipid A/analogs & derivatives , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Membrane Glycoproteins , Neutrophils/drug effects , Plasma/metabolism , Rhodobacter sphaeroides/chemistry , Humans , Lipid A/pharmacology , Lipopolysaccharides/metabolism , Macrophage-1 Antigen/analysis , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Superoxides/metabolism , Temperature
13.
J Periodontol ; 66(12): 1047-55, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8683417

ABSTRACT

To learn more about the effects of smokeless tobacco on the defensive functions of neutrophils, we studied the influence of nicotine on these cells in vitro, looking at their bactericidal activity against oral pathogens, and at their ability to produce microbicidal reactive oxygen species (oxygen radicals). Exposure of human blood neutrophils to nicotine (0.01% to 0.1%) inhibited their ability to kill Actinomyces naeslundii, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum. Although these concentrations of nicotine are high, such concentrations are relevant to phagocytes in the gingival sulcus, because smokeless tobacco contains 0.5% to 3.5% nicotine by dry weight. Nicotine had no such inhibitory effect when the killing assay was performed in an anaerobic environment, implying that nicotine preferentially affected oxygen-dependent killing mechanisms. To further investigate the effects of nicotine on production of oxygen radicals, neutrophils were primed with lipopolysaccharide and triggered with f-met-leu-phe or phorbol ester in the presence of nicotine. Nicotine inhibited production of superoxide anion (measured by reduction of cytochrome c) and hydrogen peroxide (measured by oxidation of phenol red). Nicotine inhibition of superoxide production was reversible by washing away the nicotine. By observing that nicotine inhibited the reduction of cytochrome c by reagent potassium superoxide, we determined that nicotine directly absorbed superoxide. In addition, by examining nicotine inhibition of the uptake of oxygen by neutrophils, we determined that nicotine also interfered with the production of oxygen radicals by these cells. Nicotine also inhibited production of superoxide and interleukin-1 beta by monocytes. Nicotine did not affect the viability of neutrophils and monocytes, as determined by their ability to exclude trypan blue dye. Inhibition of the aerobic antimicrobial functions of neutrophils and monocytes by nicotine may alter the microbial ecology of the oral cavity, and this might be one mechanism by which nicotine compromises the oral health of users of tobacco products.


Subject(s)
Blood Bactericidal Activity/drug effects , Immunosuppressive Agents/pharmacology , Neutrophils/drug effects , Nicotine/toxicity , Plants, Toxic , Reactive Oxygen Species/metabolism , Tobacco, Smokeless/toxicity , Actinomyces/immunology , Actinomyces/metabolism , Aggregatibacter actinomycetemcomitans/immunology , Aggregatibacter actinomycetemcomitans/metabolism , Analysis of Variance , Cells, Cultured , Cytochrome c Group/metabolism , Dose-Response Relationship, Drug , Fusobacterium nucleatum/immunology , Fusobacterium nucleatum/metabolism , Humans , Hydrogen Peroxide/metabolism , Interleukin-1/antagonists & inhibitors , Interleukin-1/biosynthesis , Monocytes/drug effects , Monocytes/metabolism , Neutrophils/metabolism , Oxygen Consumption/drug effects , Phagocytosis/drug effects , Superoxides/metabolism
14.
J Leukoc Biol ; 57(6): 865-74, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7790769

ABSTRACT

Neutrophils can inactivate lipopolysaccharide (LPS), thereby blocking the ability of LPS to prime fresh neutrophils for enhanced fMLP-triggered release of superoxide. Here we show that inactivation of LPS by neutrophils was primarily due to lactoferrin. A time course for inactivating LPS showed that neutrophils (5 million/ml) took 30 min to inactivate 10 ng/ml LPS. Mononuclear cells could not inactivate LPS under the same conditions. Experiments with radioactive LPS showed that inactivated LPS remained in the medium and was not taken up or destroyed by the neutrophils during inactivation. Inactivated LPS still gelled Limulus lysate and primed monocytes. Cell-free medium from neutrophil suspensions also inactivated LPS. A single LPS-inactivating factor was purified from medium by heparin-agarose chromatography. SDS-PAGE showed a single band at 80 kDa, which was identified as lactoferrin by immunoblotting. Antilactoferrin immunoglobulin G removed the LPS-inactivating activity from purified lactoferrin and cell-free medium. Surprisingly, even purified neutrophil lactoferrin required 30 min to inactivate LPS, indicating inherently slow binding of lactoferrin to LPS.


Subject(s)
Lactoferrin/physiology , Lipopolysaccharides/metabolism , Neutrophils/metabolism , Culture Media , Humans , Iron/pharmacology , Lipopolysaccharides/pharmacology , Monocytes/drug effects
15.
J Periodontol ; 66(4): 285-90, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7782983

ABSTRACT

To learn more about colonization of the oral epithelium by Fusobacterium nucleatum and the role of fibronectin in mediating adhesion of this microorganism, we studied attachment of this bacterium to cultured gingival epithelial cells that were coated with exogenous, purified plasma fibronectin. The three strains of F. nucleatum studied adhered in large numbers to epithelial cells that had been coated with fibronectin, compared with buffer-coated control cells. Bacterial adherence was also enhanced when epithelial cells were coated with whole human saliva. However, cells coated with saliva depleted of fibronectin did not facilitate adhesion of bacteria. Bacterial adhesion was restored when purified fibronectin was added back. We also tested adherence of bacteria to coverslips coated with fibronectin, saliva, and saliva depleted of fibronectin. The bacteria adhered to coverslips coated with fibronectin or whole human saliva, but did not adhere to coverslips coated with fibronectin-depleted saliva. Bacterial adhesion to coverslips was restored upon addition of purified fibronectin to the fibronectin-depleted saliva. Bacterial attachment to fibronectin-coated coverslips was found to be temperature-dependent, with maximal adhesion observed at 37 degrees C. Pre-treatment of F. nucleatum with soluble fibronectin inhibited attachment of the bacteria by 92%, whereas pre-treatment with bovine serum albumin had no effect. Pre-treatment of bacteria with laminin or type IV collagen caused moderate inhibition of attachment by 60% and 50%, respectively. Treatment of fibronectin-coated coverslips with Fab fragments of anti-fibronectin IgG blocked the attachment of F. nucleatum by 93%. Fab fragments of the other antisera tested had no inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Bacterial Adhesion/physiology , Fibronectins/physiology , Fusobacterium nucleatum/physiology , Gingiva/microbiology , Salivary Proteins and Peptides/physiology , Cells, Cultured , Epithelial Cells , Epithelium/microbiology , Fibronectins/metabolism , Gingiva/cytology , Glass , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/immunology , Protein Binding , Salivary Proteins and Peptides/metabolism
16.
J Periodontal Res ; 30(2): 116-23, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7539837

ABSTRACT

When neutrophils are incubated with bacterial lipopolysaccharide (LPS), they become primed for enhanced release of superoxide anion (O2-) in response to stimulation by FMLP. We investigated the human neutrophil-priming activity of LPS from the periodontal pathogens, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Actinobacillus actinomycetemcomitans (Aa) in comparison with that of LPS from Escherichia coli (E. coli). The optimum conditions for LPS to prime neutrophils were assessed for every LPS and found to be as follows: Neutrophils were incubated with LPS in the presence of 10% heat-inactivated plasma and 1 mM EDTA at 37 degrees C for 30 min and then stimulated with 1 microM FMLP at 37 degrees C for 7 min. Under these conditions, half-maximum priming was observed at 6.2 ng/ml Pg-LPS, 45 ng/ml Pi-LPS, 1.5 ng/ml Aa-LPS and 1.5 ng/ml E. coli-LPS. The priming activity of each LPS was neutralized by polymyxin B. Anti-CD14 monoclonal antibody inhibited priming by all LPS. The priming by Aa-LPS and E. coli-LPS was inhibited by LA-14-PP, a synthetic lipid A precursor IVA, but that by Pg-LPS and Pi-LPS was not. Priming by tumor necrosis factor alpha was not affected by polymyxin B, anti-CD14 antibody or LA-14-PP. Gelation of Limulus amebocyte lysate occurred at 10 pg/ml Pg-LPS, 30 pg/ml Pi-LPS, 3 pg/ml Aa-LPS and 3 pg/ml E. coli-LPS. Thus LPS from different periodontal pathogens primed neutrophils with different efficacy.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Aggregatibacter actinomycetemcomitans/immunology , Lipopolysaccharides/immunology , Neutrophil Activation/immunology , Porphyromonas gingivalis/immunology , Prevotella intermedia/immunology , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/immunology , Escherichia coli/immunology , Glycolipids/pharmacology , Humans , Limulus Test , Lipid A/analogs & derivatives , Lipid A/pharmacology , Lipopolysaccharide Receptors , Lipopolysaccharides/antagonists & inhibitors , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Periodontal Diseases/microbiology , Polymyxin B/immunology , Respiratory Burst
17.
J Lab Clin Med ; 123(6): 882-91, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8201267

ABSTRACT

Carbamoylated proteins have been located by using a site-specific polyclonal antihomocitrulline antibody and a fluorescent secondary antibody in leukocytes from patients with end-stage renal disease who were undergoing maintenance continuous ambulatory peritoneal dialysis. A covalent reaction with urea-derived cyanate and the epsilon-amino group of lysine forms homocitrulline residues in carbamoylated proteins. Isocyanic acid, the reactive form of cyanate, is spontaneously formed from urea in aqueous solution at physiologic pH and temperature. In washed, fixed monolayers of cells, an intracellular fluorescent antigen-antibody complex was located throughout the cytoplasm of polymorphonuclear neutrophils (PMNs) and monocytes from 11 patients with blood urea nitrogen (BUN) levels ranging from 32 to 102 mg/dl who were undergoing dialysis for 2 to 135 months. A punctate fluorescence present in the cell surface proteins of living cells demonstrated that lysine residues in the external domain of proteins were carbamoylated, forming homocitrulline. In contrast, we found a perinuclear fluorescence in PMNs in normal subjects with no history of renal insufficiency and BUN levels of 6 to 19 mg/dl. This suggests that homocitrulline is located in carbamoylated proteins within the perinuclear membrane, a structural organelle continuous with the endoplasmic reticulum. It appears that continuous exposure to urea-derived cyanate in low levels results in increasing carbamoylation of stable proteins over the PMN's lifetime. When normal PMNs were exposed to 120 mmol/L cyanate ion in vitro for 10 to 30 minutes, the ability of PMNs to release microbicidal superoxide was strongly inhibited. Thus protein carbamoylation may provide a regulatory mechanism. The altered function of PMNs in renal disease may be due in part to the posttranslational modification of proteins by urea-derived cyanate.


Subject(s)
Blood Proteins/metabolism , Citrulline/analogs & derivatives , Kidney Failure, Chronic/blood , Leukocytes/metabolism , Peritoneal Dialysis , Urea/blood , Adult , Aged , Blood Urea Nitrogen , Citrulline/blood , Cyanates/pharmacology , Female , Fluorescein-5-isothiocyanate , Fluorescent Antibody Technique , Fluorescent Dyes , Humans , Kidney Failure, Chronic/therapy , Male , Middle Aged , Neutrophils/drug effects , Neutrophils/metabolism , Superoxides/blood
18.
Prog Clin Biol Res ; 388: 137-45, 1994.
Article in English | MEDLINE | ID: mdl-7530366

ABSTRACT

The results concerning LPS priming and inactivation are summarized in table 4. This table clearly shows that priming by and inactivation of LPS are mediated via different pathways, because there is no correlation whatsoever between priming and inactivation. LPS priming nicely correlates with CD14 expression. Monocytes express high levels of CD14, and are highly sensitive to LPS. Neutrophils express lower levels of CD14, and also require a higher concentration of LPS to obtain a primed state. Eosinophils express even lower levels of CD14 (if any), and these cells are not primed by LPS up to 150 ng/ml in the presence of serum (data not shown). [table: see text] The inactivation experiments were performed with LPS, but unpublished results from M. Pabst et al. show the same phenomenon with synthetic lipid A. This indicates that the inactivation of LPS is caused by a modification of the lipid A moiety of LPS. As a first step, we tested the hypothesis that the inactivation of LPS is a dephosphorylation of lipid A by alkaline phosphatase (AP). However, AP activity in isolated neutrophils of one of the PNH patients was completely undetectable. Nevertheless, these neutrophils were able to inactivate LPS. This result clearly shows that AP is not the enzyme that inactivates LPS. Secondly, we measured neutral pH 7 phosphatase both in whole cells, and in cell lysate. All cell types tested (neutrophils, eosinophils and monocytes) contained comparable levels of pH 7 phosphatase, but only neutrophils were able to inactivate LPS.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Lipopolysaccharides/pharmacology , Neutrophils/physiology , Phosphatidylinositols/metabolism , Eosinophils/drug effects , Eosinophils/immunology , Eosinophils/physiology , Hemoglobinuria, Paroxysmal/physiopathology , Humans , In Vitro Techniques , Kinetics , Lipopolysaccharide Receptors , Lipopolysaccharides/antagonists & inhibitors , Monocytes/drug effects , Monocytes/immunology , Monocytes/physiology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Neutrophils/immunology , Superoxides/metabolism
19.
Infect Immun ; 59(8): 2542-8, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1649796

ABSTRACT

In monocytes, sulfatide, a lipid from Mycobacterium tuberculosis, blocked priming for enhanced release of superoxide (O2-) by the macrophage activating factors lipopolysaccharide, gamma interferon, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and muramyl dipeptide. Sulfatide, in the presence of lipopolysaccharide, also caused increased secretion of IL-1 beta and TNF-alpha into monocyte culture medium. Sulfatide altered the pattern of phosphorylation of monocyte proteins. Cell lysates prepared from monocytes treated with sulfatide showed decreased activity of protein kinase C, but sulfatide did not directly inhibit protein kinase C activity when added to lysates. A known inhibitor of protein kinase C, staurosporine, also inhibited O2- release and caused increased secretion of IL-1 beta. Thus, sulfatide appeared to indirectly affect protein kinase C, implicating protein kinase C as part of the mechanism of priming. Because sulfatide blocked priming for enhanced release of O2-, which could interfere with monocyte bactericidal activity, while causing enhanced secretion of IL-1 beta and TNF-alpha, which could promote formation of granulomata, sulfatide might be an important factor in the pathogenesis of M. tuberculosis.


Subject(s)
Interleukin-1/metabolism , Leukocytes, Mononuclear/immunology , Mycobacterium tuberculosis/immunology , Sulfoglycosphingolipids/immunology , Superoxides/metabolism , Tumor Necrosis Factor-alpha/metabolism , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Cells, Cultured , Humans , Kinetics , Leukocytes, Mononuclear/metabolism , Phosphorylation , Protein Biosynthesis , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Proteins/metabolism , Tetradecanoylphorbol Acetate/pharmacology
20.
J Immunol ; 146(4): 1271-6, 1991 Feb 15.
Article in English | MEDLINE | ID: mdl-1846896

ABSTRACT

We studied neutrophil responses to LPS using three methodologic refinements: Teflon bags or serum-coated glass tubes that did not directly trigger neutrophils, LPS-free cytochrome c to measure O2- release, and heat-inactivated serum to inhibit inactivation of LPS by neutrophils. Neutrophils incubated in uncoated glass or plastic tubes adhered to the glass and released O2-, but were not primed for enhanced release of O2- in response to triggering by FMLP. Triggering by the glass or plastic surface did not occur if the neutrophils were stirred to prevent adherence. Adherence to glass or plastic and O2- release were not affected by a mAb (IB4) directed against the beta-chain of the leukocyte adhesion family of surface glycoproteins (CD11/CD18). Neutrophils incubated in glass or plastic did not show enhanced expression of alkaline phosphatase on their surface. When neutrophils were incubated in serum-coated glass tubes or in Teflon bags, there was no O2- release. However, adherence, expression of alkaline phosphatase, and release of O2- were triggered by adding 1 ng/ml LPS plus 1% serum, but not by either LPS or serum alone. In the presence of LPS and serum, O2- release was much higher when the cells were unstirred (adherent) rather than stirred. However, both unstirred and stirred cells expressed a similar elevated level of alkaline phosphatase. LPS-triggered O2- release and adherence were inhibited by antibody IB4. In contrast, priming by LPS for enhanced FMLP-triggered O2- release was greater in stirred cells than in unstirred cells. The antibody enhanced priming of unstirred neutrophils. These results suggested that uncoated glass or plastic triggered O2- release without involvement of leukocyte adhesion glycoproteins. However, neutrophils incubated with LPS and serum expressed alkaline phosphatase and IB4-inhibitable adherence glycoproteins that allowed neutrophils to interact with serum-coated glass or Teflon to trigger O2- release. Priming by LPS for enhanced response to FMLP was suppressed in adherent neutrophils, and this suppression was partly released by IB4. Thus, triggering and priming were reciprocally regulated by neutrophil glycoproteins interacting with surfaces.


Subject(s)
Lipopolysaccharides/pharmacology , Neutrophils/drug effects , Superoxides/blood , Alkaline Phosphatase/drug effects , Antibodies, Monoclonal , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cells, Cultured , Glass , Humans , Macrophage-1 Antigen/physiology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/metabolism , Polytetrafluoroethylene , Up-Regulation
SELECTION OF CITATIONS
SEARCH DETAIL
...