Human Neutrophilic Polymorphonuclear Leukocytes Research Articles

E0047 Alphalinonenic acid inhibits high glucosemediated endothelial neutrophil adhesion by decreasing adhesion molecule expression via PI3KAkt pathway

ObjectiveNeutrophil-endothelial adhesion is crucial to vascular injury, the major cause of diabetic vascular complications. We studied the mechanism of cardio-protective effect of Alpha-linolenic acid (ALA).MethodsHuman umbilical vein endothelial cells (HUVECs)...

Leucocyte activation by anti-lactoferrin antibodies bound to vascular endothelium.

Human polymorphonuclear neutrophil leucocytes (PMNL) prestimulated with the formylated tripeptide f-Met-Leu-Phe (fMLP) were activated to an immediate chemiluminescence (CL) response by polyclonal rabbit antibodies against human lactoferrin (Lf). This activation, indicating the formation of reactive oxygen species, was induced by intact IgG antibodies but could not be brought about by F(ab')2 fragments. Human Lf was also shown to adhere to the surface of cultured bovine aorta endothelial cells (BEC). When Lf-coated BEC grown on microcarrier beads were reacted with anti-Lf antibodies, an immediate CL response was achieved also with nonprimed PMNL. Here, too, the reaction required intact IgG antibodies. Also, patient sera containing anti-Lf autoantibodies of IgG class were shown to activate fMLP-treated PMNL. The same effect was obtained (in a dose-dependent manner) with the gammaglobulin fraction from anti-Lf-positive serum. Further, anti-Lf-antibody-positive patient sera incubated with Lf-coated BEC beads were also able to activate non-stimulated PMNL to a chemiluminescence response. The results are discussed in relation to possible mechanisms of cell/tissue damage induced by anti-neutrophil cytoplasmic antibodies (ANCA).

Effects of zafirlukast on the function of humanpolymorphonuclear neutrophil leukocytes in asthmatic patients: A prospective, controlled, in vitro study

Background: Reactive oxygen species (ROSS) play an important role in the pathogenesis of asthma, and oxidative stress contributes to the initiation and worsening of inflammatory respiratory disorders (eg, asthma). Thus, antioxidant drugs may have a role in reducing or preventing damage in asthma. Objective: The aim of the study was to investigate the antioxidant effect of zafirlukast, a leukotriene receptor antagonist, in asthma. Methods: This prospective, controlled, in vitro study was conducted at KingKhalid University Hospital, Riyadh, Saudi Arabia. The generation of ROSS by polymorphonuclear neutrophil leukocytes (PMNs) in patients with mild to moderate asthma (forced expiratory volume in 1 second [FEVI], >70% of the predicted value) and healthy volunteers was assessed using chemiluminescence (CL) with phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OPZ) in the presence of different concentrations of zafirlukast (1.25-60 μg/mL). The xanthine/xanthine oxidase (X-XOD) reaction was used to test the scavenging effect of the drug. Results: Six asthmatic patients (4 women, 2 men; mean age, 30.8 years; meanFEVI, 82.5% of the predicted value) and 8 healthy volunteers (4 women, 4 men; mean age, 28.8 years) were enrolled. A dose-dependent inhibition of the CL response was observed in both groups. However, patients with asthma required higher concentrations of zafirlukast to achieve an inhibitory effect similar to that in healthy controls. This difference was significant at concentrations of 20 to 60 μg/mL (all, P ≤ 0.05). When PMNs were challenged with OPZ, inhibition was also dose dependent in controls at all concentrations (all, P ≤ 0.05), but the inhibitory effect was not significant in the asthmatic patients at any concentration. The difference in the inhibitory effect between the 2 groups was significant at 30, 40, and 60 μg/mL ( P < 0.02, <0.01, and <0.01, respectively). The mean (SEM) viability of the PMNs in the healthy controls was significantly affected only at the highest concentration compared with the control saline dose (86.5% [5.8%] vs 97.0% [8.%]; P < 0.05). No scavenging effect of zafirlukast was found using the X XOD system. Incubating PMA-stimulated cells with zafirlukast (5 and 10 μg/mL) for 10 minutes to 1 hour significantly increased the inhibitory effect of the drug by 15% to 46% (all, P < 0.001). When zafirlukast was tested for reversibility of its inhibitory effect on ROS production, its action was found to be irreversible at a concentration of 30 μg/mL ( P < 0.001) and partially reversible at 60 μg/mL compared with the baseline saline control. Conclusions: Zafirlukast inhibited ROS generation by PMNs in a dose-dependentmanner in asthmatic patients and healthy subjects. However, asthmatic patients required much higher concentrations compared with controls. The incubation of the stimulated cells with zafirlukast increased the inhibitory effect. This finding suggests that the therapeutic effect of zafirlukast in asthma may be in part related to its antioxidant action.

Beta-arrestins regulate a Ral-GDS Ral effector pathway that mediates cytoskeletal reorganization.

beta-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a beta-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with beta-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, beta-arrestin Ral-GDS protein complexes dissociate and Ral-GDS translocates with beta-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement. The subsequent re-association of beta-arrestin Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, beta-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.

A novel anti-inflammatory oligopeptide produced by Entamoeba histolytica

The monocyte locomotion inhibitory factor (MLIF), a heat-stable oligopeptide found in the supernatant fluid of Entamoeba histolytica axenic cultures was isolated by ultra-filtration, gel-sieve chromatography and high powered liquid chromatography (HPLC), and its primary structure (Met-Gln-Cys-Asn-Ser) established by Edman sequencing and mass-spectrometry (MS). A synthetic peptide had the same selective anti-inflammatory features as the native material in comparable concentrations: in vitro inhibition of the locomotion in human peripheral blood monocytes, and of the respiratory burst in the same cells and in human neutrophil polymorphonuclear leucocytes; and in vivo depression of delayed hypersensitivity skin reactions to dinitrochlorobenzene in guinea pigs. This oligopeptide is apparently synthesized by the ameba as suggested by [ 35S]-Cys and Met incorporation, probably as part of a larger molecule, from which it is cleaved by proteolysis. The full sequence was not found in the 431 available E. histolytica protein sequences. The factor may contribute to the unexpected paucity of the late inflammatory reaction found in advanced invasive amebiasis and, perhaps in consequence, to the regeneration without scarring (restitutio ad integrum) of the affected organs that is observed following successful treatment of this disease

Stimulation of Human Neutrophils by Chemotactic Factors Is Associated with the Activation of Phosphatidylinositol 3-Kinase γ

The activation of human polymorphonuclear neutrophil leukocytes (neutrophils) is associated with an increased synthesis of the highly phosphorylated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)). The aims of the present investigation were to determine whether the newly described, G protein-dependent phosphatidylinositol 3-kinase (PI3K), p110gamma, was involved in the responses to chemotactic factors interacting with G protein-coupled receptors. The presence of p110gamma in neutrophils was first established both at the protein and the mRNA level. Stimulation of the cells with fMet-Leu-Phe or interleukin-8 increased the PI3K activity in p110gamma, but not p85, immunoprecipitates. The time course of this effect (threshold within less than 5 s, maximal activation at 10-15 s) was consistent with that of the generation of PtdIns(3,4,5)P(3). Wortmannin, a PI3K inhibitor, abrogated the effects of fMet-Leu-Phe, which were also significantly inhibited by pertussis toxin. Finally, fMet-Leu-Phe also induced a significant translocation of p110gamma to a particulate fraction derived from these cells. These data indicate that p110gamma represent the major PI3K activated by fMet-Leu-Phe and interleukin-8 at very early time points following the stimulation of human neutrophils.

Soluble Fas Ligand Is Chemotactic for Human Neutrophilic Polymorphonuclear Leukocytes

It has been recently shown that Fas ligand (FasL) expression on islet beta grafts results in neutrophilic infiltration and graft rejection. In this study, we show that human recombinant soluble FasL is endowed with potent chemotactic properties toward human neutrophilic polymorphonuclear leukocytes (neutrophils) at concentrations incapable of inducing cell apoptosis. Furthermore, neutrophils exposed to soluble FasL did not display detectable change of intracellular Ca2+ and did not undergo superoxide production or exocytosis of primary and secondary granules. Our results show that FasL is a potent chemoattractant for human neutrophils without evoking their secretory responses. This finding suggests a novel proinflammatory function for this ligand and may help to clarify the mechanism governing FasL-mediated graft rejection, thereby offering rational bases for controlling and modulating FasL-based immunotherapies.

Chemotaxin-dependent translocation of immunoreactive ADP-ribosyltransferase-1 to the surface of human neutrophil polymorphs.

mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed with cDNA encoding human skeletal muscle arginine-specific ADP-ribosyltransferase (ART1). A single 2.6-kb transcript was identified, which was similar in size to that observed in human skeletal muscle RNA. An 872-bp cDNA fragment, corresponding to the amino acid sequence of the processed human skeletal muscle enzyme, was generated by reverse transcription-PCR amplification of RNA from human PMNs, and was found to be identical to the ART1 cDNA derived from human skeletal muscle. ART1 was expressed as a fusion protein with glutathione S-transferase (GST) in insect cells, and antibodies were raised against the fusion protein in a rabbit. Following removal of GST immunoreactivity by immunoprecipitation, these antibodies were used to measure the abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs to formyl-Met-Leu-Phe (FMLP) was followed by a rapid increase in the abundance of cell surface ART1 (T1/2 = 1.9 min), and the concentration of FMLP for half-maximum response was 28.6 nM. Similar responses were observed after exposure of the cells to platelet-activating factor or interleukin-8, and we conclude that some of the effects of these chemotaxins are mediated by translocation of an intracellular pool of ART1 to its site of catalytic activity on the outer aspect of the plasma membrane.

Electrochemical detection of nitric oxide production in human polymorphonuclear neutrophil leukocytes

The detection of nitric oxide (NO) release by human polymorphonuclear neutrophil leukocytes (PMNs) presents several difficulties, mainly due to concomitant production of O2- and H2O2, which could interfere with the measurements. A Nafion and nickel porphyrin-coated microelectrode was used to measure NO production in PMNs in vitro. It allowed detection of 6.3 +/- 1.9 nM NO in a PMN-containing system and was unaffected by added chemicals. Addition of the chemotactic oligopeptide f-met-leu-phe (fMLP; 100 nM) induced a NO release which reached a value of 71 +/- 30 pmol NO/10(6) PMN x ml(-1) 5 min after stimulation in the presence of SOD (150 U/ml). If SOD was omitted, the corresponding value was 36 +/- 20 pmol NO/10(6) PMN x ml(-1). Presence or absence of catalase did not alter the amount of NO measured. Addition of the NO-synthase inhibitor N(G)-monomethyl-L-arginine (LNMMA; 1 mM) reduced the current by 82 +/- 20%. These results agree with the rate of NO production in human PMNs when measured spectrophotometrically using the NO-dependent oxidation of oxyhaemoglobin to methaemoglobin. The NO production in human PMN was dependent on fMLP concentrations, but independent of cell-concentrations of 0.5-3.5 x 10(6)/ml. This paper shows that a electrochemical method, e.g. Nafion and porphyrin-coated microelectrode, is suitable for studies of NO release from stimulated human PMNs.

Inhibition of chemotaxis in A7r5 rat smooth muscle cells by a novel panel of inhibitors.

1. Arginine-specific ADP-ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudosubstrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF-BB. 2. The IC50 values for nicotinamide (12 mM) and novobiocin (165 microM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 microM) and K1 (IC50=95 microM) were less potent than previously described in PMNs. The pseudo-substrates for the enzyme (DEA-BAG, agmatine and arginine-methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3. A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4. Evidence for ART activity on the surface of A7r5 cells was investigated using 32P-NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P-AMP, and hydrolysis with NaOH yielded 32P-NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP-ribosylation of agmatine. 5. We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.

Stimulus-dependent transduction mechanisms for nitric oxide release in human polymorphonuclear neutrophil leukocytes

The production of nitric oxide (NO) may play an important role in functional responses of the human polymorphonuclear neutrophil granulocytes (PMNs). Others have described the presence of both an inducible, Ca 2+-independent and a constitutionally expressed, Ca 2+-dependent nitric oxide synthase (NOS) in human PMNs. However, the conditions for production and release of NO in human PMNs are still largely unknown. We assessed mechanisms for activation of NO release from human PMNs and particularly the dependence on extracellular and intracellular Ca 2+. We addressed this question by applying a variety of agonists with known and differing mechanisms of activation in PMNs and measuring the released NO by two highly sensitive and specific real-time methods for detection of NO, the oxidation of oxyhemoglobin to methemoglobin and an electrochemical method. We found that human PMNs activated with the surface receptor-dependent agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP); the calcium lonophore, A23187; or the direct stimulator of protein kinase C, phorbol myristate acetate (PMA), produced NO which was inhibited by a specific NOS inhibitor, N G-monomethyl- l-arginine. The NO production induced by fMLP or A23187 was dependent on the presence of extracellular Ca 2+, but this was not the case for PMA. The stimulatory effect of fMLP was almost completely inhibited by Bordetella pertussis toxin. These results indicate an NOS activity in purified human PMNs in vitro, and the transduction mechanisms for the agonists used show strong similarity with previously known pathways for other neutrophil functions.

Arginine-specific ADP-ribosyltransferase of human polymorphonuclear neutrophil leucocytes: cDNA cloning and identification of a polymorphic site in the coding region.

Arginine-specific ADP-ribosyltransferase of human polymorphonuclear neutrophil leucocytes: cDNA cloning and identification of a polymorphic site in the coding region.

Methylprednisolone inhibits neutrophil-endothelial cell interactions induced by interleukin-1 β under flow conditions

The effects of methylprednisolone (m-PSL) on IL-1 β-induced neutrophil-endothelial cell interactions, which are normally mediated by increased expression of both intercellular adhesion molecule-1 (ICAM-1) and E-selectin on endothelial cells, were examined using an in vitro flow system. Human neutrophilic polymorphonuclear leukocytes (PMN) were perfused at a shear stress of 1 dyne cm 2 on human umbilical vein endothelial cells (HUVEC) pretreated with IL-1 β (20 U mL ) for 4 hours. Many PMN adhered to IL-1-stimulated HUVEC and then migrated beneath endothelial cell monolayers. Treatment of HUVEC with m-PSL inhibited adherence and migration of PMN in a dose dependent manner. M-PSL also inhibited IL-1 β-induced upregulation of E-selectin and ICAM-1 on HUVEC in a dose dependent manner. These results suggest that m-PSL works as an anti-inflammatory agent through inhibiting PMN-endothelial cell interactions.

Excursions in biomedical mass spectrometry.

1. Mass spectrometry (MS) has played a vital role in the research of the department of clinical pharmacology for over 25 years. 2. MS has been used for trace analysis of endogenous compounds and xenobiotics in plasma and urine, and also for a wide range of structural studies. 3. Examples of current applications are reported, including data from gas chromatography-mass spectrometry (GC-MS) assays for mevalonic acid, the identification of an antibiotic produced by Pseudomonas aeruginosa which is active against Helicobacter pylori, high pressure liquid chromatography (h.p.l.c)-electropray MS studies on steroid sulphates, the aspergillus ciliotoxin, gliotoxin, and ADP ribosyltransferase activity in human polymorphonuclear neutrophil leucocytes (PMNs). The value of electrospray MS in the molecular weight determination of proteins is exemplified by the analysis of human serum amyloid component P.

A possible role for mono(ADP‐ribosyl)transferase in the signalling pathway mediating neutrophil chemotaxis

1. Mono(ADP-ribosyl)transferase activity has been identified on the external surface of human polymorphonuclear neutrophil leucocytes (PMNs). The enzyme is released from the plasma membrane by phosphoinositide-specific phospholipase C, suggesting a glycosylphosphatidylinositol (GPI) linkage of the enzyme to the plasma membrane. Partial sequence of cDNA encoding the enzyme suggests that it is identical to the GPI-linked mono(ADP-ribosyl)-transferase identified previously on human skeletal muscle. 2. A panel of inhibitors of mono(ADP-ribosyl)transferase (including vitamins K1 and K3, novobiocin and nicotinamide) showed a rank order of inhibitory potency similar to that described for other mono(ADP-ribosyl)transferases. Furthermore, the mono(ADP-ribosyl)ation of agmatine was inhibited also by diethylamino (benzylidineamino)guanidine (DEA-BAG), another substrate of the enzyme related structurally to arginine. 3. There was a close linear correlation between the IC50 values for inhibition of mono(ADP-ribosyl)ation of agmatine by DEA-BAG or the enzyme inhibitors and their IC50 values for inhibition of receptor-dependent polymerization of cytoskeletal actin and chemotaxis. 4. These results suggest a role for mono(ADP-ribosyl)transferase in the transduction pathway involved in receptor-dependent re-alignment of the cytoskeleton during neutrophil chemotaxis.

Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes.

An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino-(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1-10 mM) did not yield ADP-ribosyl-DEA-BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD+ by an NAD+ glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The Km of the enzyme for NAD+ was 100.1 30.4 microM and the Vmax 1.4 0.2 pmol of ADP-ribosylagmatine/h per 10(6) cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98% decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [32P]NAD+. Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5'-AMP.

Induction of heat shock protein 72 prevents neutrophil-mediated human endothelial cell necrosis.

To examine the hypothesis that induction of heat shock proteins in human endothelial cells (ECs) by either heat shock or sodium arsenite could prevent subsequent EC necrosis induced by activated human polymorphonuclear neutrophil leukocytes (PMNs). Cultures of ECs were exposed to heat shock (42 degrees C, 30 to 60 minutes) or sodium arsenite (40 to 320 mumol/L) for 6 hours to induce the expression of a heat shock protein of 72-kd molecular weight (HSP-72). Activated PMNs were subsequently added to these ECs for 24 hours to evaluate the ability of HSP-72 to prevent activated PMN-mediated EC necrosis. Neither EC necrosis nor apoptosis was induced by heat shock. Sodium arsenite (40 to 80 mumol/L) did not induce EC necrosis, although 320-mumol/L sodium arsenite caused a significant increase in EC necrosis. Sodium arsenite (80 to 320 mumol/L) also induced dose-dependent EC apoptosis. Endothelial cells exposed to heat shock and sodium arsenite (40 and 80 mumol/L) significantly attenuated subsequent EC necrosis induced by activated PMNs. However, sodium arsenite at 320 mumol/L aggravated activated PMN-mediated EC necrosis. Expression of HSP-72 was detected after ECs were treated both with heat shock and sodium arsenite (40 to 320 mumol/L) for 6 hours. Induction of HSP-72 in ECs by a thermal or nonthermal mechanism could prevent activated PMN-mediated EC necrosis, which may favor increased vascular permeability during systemic inflammatory response syndrome.

Leptospira icterohemorrhagiae and leptospire peptidolgycans induce endothelial cell adhesiveness for polymorphonuclear leukocytes.

We have examined the effect of the virulent Leptospira interrogans strain Teramo, serotype icterohemorrhagiae, on the adherence of human neutrophilic polymorphonuclear leukocytes (PMN) to cultured human umbilical vein endothelial cells (HEC). Selective pretreatment of HEC with intact or sonicated leptospires caused a dose- and time-dependent increase of HEC-PMN adhesion (13.2% +/- 2.5% adherence to untreated HEC versus 46.3% +/- 5.6% adherence to HEC pretreated for 4 h with 10(8) intact leptospires per ml [mean +/- standard error of six experiments; P < 0.001]). In contrast, selective leptospire pretreatment of PMN or the addition of leptospires during the adherence assay did not alter HEC-PMN adherence. Leptospire induction of endothelial-cell adhesiveness occurred without detectable HEC damage and was prevented by RNA and protein synthesis inhibitors and by monoclonal antibodies to the CD11/CD18 adhesion complex of neutrophils and to the endothelial-leukocyte adhesion molecule 1 (ELAM-1) of endothelial cells. Similar results were obtained with pretreatment of HEC with interleukin-1 or with the lipopolysaccharide (LPS) of the gram-negative bacterium Escherichia coli. The possibility that contamination by the LPS of gram-negative bacteria could be involved in the induction of HEC adhesiveness was ruled out by the observation that the LPS inhibitor polymyxin B, which abolished the proadhesive effect of E. coli LPS, was ineffective in inhibiting leptospire- as well as interleukin-1-induced adherence. Similarly, leptospire LPSs seemed to have no role in the increase of endothelial-cell adhesiveness, since pretreatment of HEC with a leptospire LPS extract (phenol-water method) or with a leptospire total lipid extract failed to induce the proadhesive phenotype for neutrophils. Instead, peptidoglycans extracted from our leptospires actively stimulated the endothelial proadhesive activity for neutrophils (16.5% +/- 2.1% adherence to untreated HEC versus 51.2% +/- 2.9% adherence to HEC pretreated for 4 h with 1 microgram of peptidoglycan per ml; [mean +/- standard error of four experiments; P < 0.001]). This peptidoglycan-induced activity was inhibited by monoclonal antibodies to the CD11/CD18 adhesion complex and to ELAM-1 but not by polymyxin B. We conclude that peptidoglycans from pathogenic leptospires are among the molecules that can directly activate vascular endothelial cells to increase their adhesiveness for neutrophilic granulocytes. These observations may contribute to a better understanding of the mechanisms whereby non-gram-negative bacteria modulate the local and systemic inflammatory reaction.

Measurement of methemoglobin formation from oxyhemoglobin a real-time, continuous assay of nitric oxide release by human polymorphonuclear leukocytes

We have evaluated the spectrophotometric measurement (at 401 vs. 411 nm) of nitric oxide (NO)-dependent methemoglobin formation from oxygemoglobin in order to assess NO release from human polymorphonuclear neutrophil leukocytes (PMN). S-nitroso- d,l-acetyl-penicillamine (SNAP, 25–200 μM), a donor of NO, induced a dose-dependent methemoglobin formation. Furthermore, when PMN were activated with N-formyl-methionylleucyl-phenylalanine or phorbol myristate acetate in the presence of superoxide dismutase (SOD) and catalase, methemoglobin formation ensued. The amount of methemoglobin formed was dependent on the amounts of oxyhemoglobin and stimulus used, and the number of PMN in the assay. The NO synthase (NOS) inhibitors N G -monomethyl- l-arginine or nitro- l-arginine methyl ester did not affect methemoglobin generation from oxyhemoglobin induced by SNAP but inhibited that mediated by activated PMN with IC 50 values of 250 μM and 340 μM, respectively. The substrate for NO formation from NOS, l-arginine in concentrations up to 1 mM did not significantly influence the methemoglobin formation either induced bu SNAP or activated PMN. Exclusion of SOD did not affect SNAP-dependent oxidation of oxyhemoglobin. Exclusion of SOD from the cell-containing system attenuated methemoglobin formation, and if catalase was also excluded the repsonse was further reduced. Finally, PMN from a patient with X-linked chronic granulomatous disease, unable to produce superoxide anions, showed a similar production of methemoglobin from HbO 2 as did healthy PMN, activated with the respective agonists. We conclude that spectrophotometric measurement of methemoglobin formation from oxyhemoglobin in the presence of SOD and catalase is a suitable method for the measurement of NO release from PMN, with the benefits of a real-time, continuous assay.

Characterization of interstitial collagenases in jaw cyst wall.

Neutral salt extracts of 14 specimens of jaw cysts were prepared. Histopathological analysis showed that the specimens consisted of 6 radicular cysts, 6 dentigerous cysts, 1 residual cyst, and 1 odontogenic keratocyst. One periapical granuloma, 1 dental follicle and a sample of clinically healthy oral mucosa were similarly processed and used as controls. Measurement of collagenase activity by monitoring the formation of specific degradation products of type I and II collagen in solution by SDS-PAGE demonstrated that all the cyst extracts contained collagenase, some of which was endogenously activated. Cyst wall collagenase preferably degraded type I over type II collagen, which suggests that the degradation was due to MMP-1 (matrix metalloproteinase-1) rather than the MMP-8 type. This was further supported by the doxycycline-inhibition profile of cyst collagenase, which was similar to that of MMP-1. Part of the cyst wall collagenase was in latent proenzyme form and probably derived, at least in part, from the newly synthesized intracellular collagenase pool. Latent cyst collagenase was efficiently activated with phenylmercuric chloride and to a lesser extent by gold (I) thioglucose and NaOCl. Western-blotting, using specific antibodies against collagenase from human polymorphonuclear neutrophilic leukocytes (MMP-8) and from fibroblasts (MMP-1), revealed a typical 55/45 kDa doublet; also MMP-8 in the latent 80 kDa form and fragmented to 65 kDa active species were found. These results suggest the presence of MMP-1 and, to a lesser extent, MMP-8 type collagenase in the cyst wall.(ABSTRACT TRUNCATED AT 250 WORDS)