A tetrahydrophthalazine derivative 'sodium nucleinate" exerts a potent suppressive effect upon LPS-stimulated mononuclear cells in vitro and in vivo

We described the use of a new chemical substance Sodium nucleinate (SN) as an immunomodulatory substance exhibiting antiinflammatory properties. Sodium nucleinate (SN) registrated in Russian Federation as Tamerit, is 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, derivative of well known chemical substance luminol. To comprehend the mechanisms of SN immunomodulatory activity, we examined the SN modulation of the innate inflammatory cytokine response of human PBMC stimulated with LPS in vitro. Furthermore, we studied the immunomodulatory effects of SN in mice challenged with E. coli LPS in vivo to investigate a possible novel approach to therapy of excessive inflammation that interfere with the response to endotoxin and inflammatory mediators. Our results demonstrated that SN is an efficient inhibitor of sepsis development in mice model of LPS-induced sepsis. The changes induced by SN include decreased mice plasma inflammatory cytokine production. Simmilary we demonstrated a decreased TNF-alpha, IFN-gamma and IL-6 response in human LPS-stimulated PBMNCs. SN was therefore shown to be a promising inhibitor of multiple inflammatory cytokine secretion.     

Heterogeneity of human monocytes: differences in response to IFN-gamma

Human peripheral blood monocytes can be separated into two subpopulations which differ in the efficiency of their adherence to glass after 16 hours of incubation. The adherent subpopulation was found to be about twice as effective in binding mannose-resistant E. coli 0-124, mannose-sensitive E. coli 0-128 and opsonised E. coli than the nonadherent one. In addition, reduction of cytochrome C in response to E. coli binding or 12-myristate 13-acetate (PMA) stimulation was two fold higher in adherent cells. The binding of E. coli O-124 and the superoxide generation stimulated by E. coli were inhibited by the addition of mannose only in the adherent monocytes, indicating the presence of mannose receptors on the cell surface in the adherent subpopulation. The treatment of the nonadherent cells with 0.1-1000 U/ml of Interferon (IFN-gamma) for 24 hours resulted in a dose dependent increase in superoxide generation. After 72 hours of incubation with IFN-gamma (1000 U/ml) the amount of superoxide generated by the nonadherent cells was elevated to 20.5 +/- 1.4 nmoles/10(6) cells/15 min, similar to that of the adherent cells (24.5 +/- 1.2 nmoles/10(6) cells/15 min untreated adherent monocytes). The generation of superoxide in the IFN-gamma treated nonadherent monocytes stimulated by E. coli 0-128 was significantly reduced by addition of mannose.     

Impaired oxidative burst does not affect human monocyte tumoricidal activity

Human peripheral blood monocytes have been shown to lyse neoplastic cells selectively. It has been suggested that the oxidative burst may mediate monocyte tumoricidal activity. Two patients with chronic granulomatous disease were studied. The first patient had no detectable secretion of hydrogen peroxide and superoxide anion after stimulation with 100 ng/ml PMA. Nevertheless, his tumoricidal activity measured against K562 targets in 4-hr 51Cr-release assays by using highly purified monocytes was reproducibly above the 95th percentile of the normal population. His monocytes lysed WEHI-164 targets pretreated with 1 microgram/ml Actinomycin D with similar efficiency. Another patient whose oxidative burst was 15% of normal nevertheless killed Daudi targets efficiently. In addition, oxygen-deprived monocytes of normal donors manifested normal tumoricidal activity, despite failure to produce any detectable oxidative burst after PMA stimulation. We conclude that reactive oxygen metabolites are not the primary mediators of tumor cytolysis by human peripheral blood monocytes.     

Tumor promoter phorbol-12-myristate-13-acetate induces poly ADP-ribosylation in human monocytes

The tumor promoter phorbol-12-myristate-13-acetate (PMA) induces rapid poly ADP-ribosylation and a drop in cellular NAD concentration in human monocytes. The antioxidants CuZn-superoxide dismutase, catalase, glutathione peroxidase and butylated-hydroxytoluene inhibit the reaction indicating that active oxygen species produced in the PMA-induced oxidative burst represent intermediates. The inhibitor of ADP-ribosyl-transferase, 3-amino-benzamide, inhibited poly ADP-ribosylation but did not prevent the drop in NAD-levels. PMA also causes the slow accumulation of DNA strand breaks in monocytes. The difference in the kinetics of poly ADP-ribosylation and DNA breakage argues against a simple relationship between the two reactions.     

Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry.

The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type- and stimulus-specific differences in signal transduction pathways: 1) n-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B-induced oxidative burst is several-fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; 2) stimulus-induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; 3) the FMLP/cytochalasin B-induced calcium flux in monocytes is less sensitive to the G-protein inhibitor pertussis toxin than the flux in granulocytes; 4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; 5) the PMA-induced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; 6) the G-protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and 7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B-induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10-20 nM is necessary to block PMA-induced oxidative burst.     

Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-alpha stimulation, and effects of a phosphodiesterase inhibitor

Diabetic nephropathy is a major complication of diabetes leading to end-stage renal disease, which requires hemodialysis. Although the mechanism by which it progresses is largely unknown, the role of hyperglycemia-derived oxidative stress has recently been the focus of attention as the cause of diabetic complications. Constituent cells of the renal glomeruli have the capacity to release reactive oxygen species (ROS) upon stimulation of NADPH oxidase activated by protein kinase C (PKC). Hyperglycemia and insulin resistance in the diabetic state are often associated with activation of PKC and tumor necrosis factor (TNF)-alpha, respectively. The aim of this study is to clarify the signaling pathway leading to ROS production by PKC and TNF-alpha in rat glomeruli. Isolated rat glomeruli were stimulated with phorbol 12-myristate 13-acetate (PMA) and TNF-alpha, and the amount of ROS was measured using a chemiluminescence method. Stimulation with PMA (10 ng/ml) generated ROS with a peak value of 136+/-1.2 cpm/mg protein (mean+/-SEM). The PKC inhibitor H-7, the NADPH oxidase inhibitor diphenylene iodonium and the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin inhibited PMA-induced ROS production by 100%, 100% and 80%, respectively. In addition, TNF-alpha stimulated ROS production (283+/-5.8/mg protein/20 min). The phosphodiesterase inhibitor cilostazol activates protein kinase A and is reported to improve albuminuria in diabetic rats. Cilostazol (100 microg/ml) inhibited PMA, and TNF-alpha-induced ROS production by 78+/-1.8, and 19+/-2.7%, respectively. The effects of cilostazol were not additive with wortmannin. Cilostazol arrests oxidative stress induced by PKC activation by inhibiting the PI-3 kinase-dependent pathway, and may thus prevent the development of diabetic nephropathy.     

New data on the tachyphylactic properties of sodium nucleinate]

Sodium nucleinate, when injected into mice in combination with killed E. coli or prodigiozan, caused a decrease in side effects produced by the vaccine or polysaccharide. The combined injection of sodium nucleinate and prodigiozan in doses, ineffective if introduced separately, was accompanied by the potentiation of their tachyphylactic action. The use of sodium nucleinate in combination with polyvinylprrolidone (hemodez) prolonged the tachyphylactic action of sodium nucleinate and increased its effectiveness. The proposed principle is supposed to be suitable for decreasing the reactogenicity of bacterial vaccines and polysaccharides.     

Bacterial lipopolysaccharide, phorbol myristate acetate, and muramyl dipeptide stimulate the expression of a human monocyte surface antigen, Mo3e

Exposure of mononuclear phagocytes to bacterial lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or muramyl dipeptide (MDP) is known to stimulate a variety of cellular activities that include increases in phagocytosis, oxidative metabolism, synthesis and secretion of monokines, and cytotoxicity of microbes and tumor cells. We now report that culture of human peripheral blood monocytes in medium containing LPS, phorbol compounds, or MDP also results in the acquired expression of a plasma membrane antigen. Mo3e, as identified by a murine monoclonal antibody. Mo3e is barely detectable (by immunofluorescence flow cytometry) on freshly isolated monocytes, but is expressed in high antigen density after exposure of cells to E. coli, Salmonella minnesota, or Serratia marcescens LPS (at concentrations exceeding 0.1 ng/ml), PMA (and other biologically active phorbol compounds) (0.5 to 1 X 10(-8) M), or MDP (0.01 to 1 X 10(-6) M). Mo3e expression stimulated by LPS is prevented by pretreatment of LPS with polymyxin B, suggesting that the lipid A portion of LPS is responsible for Mo3e induction (polymyxin B has no effect on Mo3e expression stimulated by PMA or MDP). Culture of monocytes in medium containing protein synthesis inhibitors (or at 4 degrees C) blocks the acquisition of Mo3e. Recombinant IFN-gamma, which is also known to "activate" mononuclear phagocytes, does not stimulate Mo3e expression, although both LPS and IFN induce enhanced expression of monocyte Ia antigen. Analogous to their stimulatory effect on monocytes, LPS and PMA induce Mo3e expression by the human monocytic cell line, U-937. On the basis of these observations, Mo3e may represent an immunologic marker for monocyte activation stimulated in vitro by LPS, PMA (and related compounds), and MDP.     

Sodium nucleinate increase of nonspecific macroorganism resistance to conditionally pathogenic and pathogenic microorganisms

Sodium nucleinate significantly increased the non-specific resistance of mice to E. coli O26, Ps. vulgaris, Ser. marcesens, Ps. aeruginosa, Kl. ozaenae and their associations and total resistance also accompanied by a significant decrease in the number of the bacteria in the spleen and blood, the total number of the cells in the peritoneal exudate and the number of the cells adhering to the glass (macrophages). The preparation is a low molecular RNA consisting mainly of fraction 3S and a small amount of fraction 4S. It contains 1.5 per cent of protein and 2 per cent of DNA and does not contain any polysaccharides. Repeated purification of sodium nucleinate lowering the levels of the admixtures 5 times did not change its efficacy. The low molecular RNA of the rat liver (4S) had a pronounced stimulating activity. On infection of the host stimulated with sodium nucleinate, formation of the post-infection immunity was not decreased.     

Effect of catalase on the proliferation of human lymphocytes to phorbol myristate acetate

Phorbol esters have been documented to stimulate the proliferation of human blood mononuclear cell cultures. In addition, these agents are also known to stimulate the production and release of reactive oxygen species by monocytes. We demonstrated previously that H2O2, one of these oxygen metabolites, impairs the proliferative capacity of human blood lymphocytes. Therefore, in these experiments, we determined whether or not the H2O2 released by monocytes after activation by PMA modifies the proliferation of lymphocytes to this agent. Human blood mononuclear cells (80% lymphocytes and 20% monocytes) were incubated with PMA, and lymphoblastic transformation (LBT) was quantitated at 3 and 5 days by pulsing the cultures with thymidine. Initial experiments established that the concentration of PMA required for optimal LBT was 50 ng/ml. We then demonstrated that this concentration of PMA also induces a burst in hexose monophosphate shunt activity and H2O2 production of mononuclear cells as indicated by the enhanced oxidation of 14C-glucose and 14C-formate, respectively. The amount of H2O2 released into the medium was substantial. Our measurements indicate that the concentration of H2O2 could reach values as high as 0.008 mM during the first 2 hr of the cultures. The addition of catalase to PMA-treated cultures in concentrations sufficient to scavenge the H2O2 released by the monocytes was associated with an enhanced thymidine uptake (mean 79%). These results indicate that the hydrogen peroxide released by the monocytes modifies the response of lymphocytes to the PMA. Paradoxically, mononuclear cell cultures depleted of monocytes also had a lower proliferation to PMA than mononuclear cell cultures. This observation indicates that monocytes also produce factors required for lymphocyte proliferation to PMA such as an interleukin. In contrast, to PMA cultures, catalase did not alter the proliferation of mononuclear cell cultures stimulated by PHA. We previously documented that PHA does not stimulate an immediate burst in the oxidative metabolism of mononuclear cultures. Therefore, the effect of catalase in these two culture systems appears to correlate with the capacity of the mitogen to stimulate the oxidative metabolism of mononuclear cells. These observations suggest that the release of reactive oxygen species by monocytes may modify the response of lymphocytes to antigens both in vitro and in vivo.     

Superoxide generation by guinea-pig peritoneal macrophages is inhibited by rolipram, staurosporine and mepacrine in an agonist-dependent manner

Platelet-activated factor (PAF) ( ), formyl-methionyl-leucyl-phenylalanine (fMPL) ( ), phorbol 12-myristate 13 acetate (PMA) ( ), opsonized zymosan (OPZ) (0.01–1 mg/ml) were potent stimuli to superoxide generated by guinea-pig peritoneal macrophages. Superoxide generation by low (≤ −8M) concentrations but not high (≥−7M) concentrations of PAF or fMLP were attenuated by rolipram (100 μM) in the presence of 1 μM prostaglandin E₂ (PGE₂). That stimulated by PMA or OPZ, however, was unaffected. At 1μM, staurosporine was a potent inhibitor of superoxide generation stimulated by both fMLP and PAF but was without effect on that stimulated by OPZ. Superoxide generation stimulated by fMLP, PAF and OPZ was inhibited by 100 μM mepacrine. We conclude that superoxide generation stimulated by the chemoattractants fMLP and PAF involves a cyclic AMP regulated and cyclic AMP independent process. The cyclic AMP independent process is mediated by protein kinase C. Although protein kinase C seems a central element in the respiratory burst stimulated by fMLP, PAF and PMA that stimulated by OPZ bypasses this mechanism. Phospholipase A₂ however, represents a common stage in the signal transduction pathway.     

Nano-sized and micro-sized polystyrene particles affect phagocyte function

Adverse effect of nanoparticles may include impairment of phagocyte function. To identify the effect of nanoparticle size on uptake, cytotoxicity, chemotaxis, cytokine secretion, phagocytosis, oxidative burst, nitric oxide production and myeloperoxidase release, leukocytes isolated from human peripheral blood, monocytes and macrophages were studied. Carboxyl polystyrene (CPS) particles in sizes between 20 and 1,000 nm served as model particles. Twenty nanometers CPS particles were taken up passively, while larger CPS particles entered cells actively and passively. Twenty nanometers CPS were cytotoxic to all phagocytes, ≥500 nm CPS particles only to macrophages. Twenty nanometers CPS particles stimulated IL-8 secretion in human monocytes and induced oxidative burst in monocytes. Five hundred nanometers and 1,000 nm CPS particles stimulated IL-6 and IL-8 secretion in monocytes and macrophages, chemotaxis towards a chemotactic stimulus of monocytes and phagocytosis of bacteria by macrophages and provoked an oxidative burst of granulocytes. At very high concentrations, CPS particles of 20 and 500 nm stimulated myeloperoxidase release of granulocytes and nitric oxide generation in macrophages. Cytotoxic effect could contribute to some of the observed effects. In the absence of cytotoxicity, 500 and 1,000 nm CPS particles appear to influence phagocyte function to a greater extent than particles in other sizes.     

N-formyl-Met-Leu-Phe-induced oxidative burst in DMSO-differentiated HL-60 cells requires active Hsp90, but not intact microtubules

In this study we examined whether microtubules and heat shock protein 90 (Hsp90) are involved in phorbol myristate acetate (PMA) and N-formyl-Met-Leu-Phe (fMLP)-induced oxidative burst in DMSO-differentiated HL-60 cells. Our results showed that microtubule interfering agents, paclitaxel (1-5 microM), colchicine (1-100 microM), nocodazole (1-20 microM), and vincristine (1-50 microM), did not affect either PMA or fMLP-induced oxidative burst. In contrast, radicicol, an inhibitor of Hsp90, inhibited fMLP-induced oxidative burst in time and concentration-dependent manner where IC50 value for 30 min pre-incubation was 16.5 +/- 3.5 microM radicicol. We conclude that both PMA and fMLP-induced oxidative burst in DMSO-differentiated HL-60 cells is microtubule-independent while the latter requires Hsp90 activity.     

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl-phenylalanine-triggered respiratory burst.

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C-mediated signal transduction mechanisms coupled to the induction of the respiratory burst.     

Surfactant phospholipid DPPC downregulates monocyte respiratory burst via modulation of PKC

Pulmonary surfactant phospholipids have been shown previously to regulate inflammatory functions of human monocytes. This study was undertaken to delineate the mechanisms by which pulmonary surfactant modulates the respiratory burst in a human monocytic cell line, MonoMac-6 (MM6). Preincubation of MM6 cells with the surfactant preparations Survanta, Curosurf, or Exosurf Neonatal inhibited the oxidative response to either lipopolysaccharide (LPS) and zymosan or phorbol 12-myristate 13-acetate (PMA) by up to 50% (P < 0.01). Preincubation of MM6 cells and human peripheral blood monocytes with dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid component of surfactant, inhibited the oxidative response to zymosan. DPPC did not directly affect the activity of the NADPH oxidase in a MM6 reconstituted cell system, suggesting that DPPC does not affect the assembly of the individual components of this enzyme into a functional unit. The effects of DPPC were evaluated on both LPS/zymosan and PMA activation of protein kinase C (PKC), a ubiquitous intracellular kinase, in MM6 cells. We found that DPPC significantly inhibited the activity of PKC in stimulated cells by 70% (P < 0.01). Western blotting experiments demonstrated that DPPC was able to attenuate the activation of the PKCdelta isoform but not PKCalpha. These results suggest that DPPC, the major component of pulmonary surfactant, plays a role in modulating leukocyte inflammatory responses in the lung via downregulation of PKC, a mechanism that may involve the PKCdelta isoform.     

Spin trap determination of free radical burst kinetics in stimulated neutrophils

Electron spin resonance spin-trapping methods were used to investigate the free radical production kinetics of neutrophils stimulated with phorbol myristate acetate (PMA) and opsonized zymosan (OPZ). Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, the principle spin adduct observed is DMPO-OH (trapped hydroxyl radical). The DMPO-OH ESR signal amplitude was observed to decay exponentially. In such cases a simple method may be used to analyze the raw kinetics amplitude data to yield true production rate and net production data. The method, pitfalls, and self-consistency criteria are illustrated with PMA and OPZ-stimulated neutrophils at 25 and 37 degrees C under varying oxygen tensions, and with noise-free simulated data. The simulations demonstrate that rate results are relatively insensitive to the precise choice of decay time constant, tc, while net production results are very sensitive to the choice of tc used to analyze the raw data. OPZ (0.6-2.4 mg/ml) yields a strong, sharp neutrophil burst which peaks in 2 min or less while PMA yields a slower burst which peaks in 3.4-14 min for PMA concentrations of 500-50 ng/ml, respectively. Increased oxygen tension during the PMA experiments increased the spin adduct lifetime. The methods presented are applicable to other cell systems or spin adducts which exhibit first order decay.     

Cyclosporin A inhibits phorbol ester-induced activation of superoxide production in resident mouse peritoneal macrophages.

Peritoneal resident macrophages from mice are sensitive to inhibition by cyclosporin A (CsA) of phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst. Inhibition was assessed in terms of superoxide anion (O2.-) and H2O2 production. Key findings were as follows. (a) CsA inhibited in a dose-dependent manner the production of O2.- when cells were stimulated with PMA. CsA did not alter the respiratory burst induced by other stimuli (zymosan, concanavalin A and fMet-Leu-Phe). It was verified that CsA itself had no scavenger effect. (b) A concomitant decrease in H2O2 liberation following CsA exposure was found. This inhibition was observed both in the initial rate of synthesis and in the accumulation after 15 min of incubation. (c) NADPH oxidase activity in the crude supernatant was unaffected by the previous incubation of macrophages with CsA. CsA does not inhibit glucose transport measured as 14CO2 production. (d) The production of O2.- was strongly dependent on the glucose concentration. Sodium oleate also stimulated O2.- production in resident macrophages. These data might be correlated with the inhibitory effect of CsA upon other functions of macrophages.     

Resistin inhibits essential functions of polymorphonuclear leukocytes

The serum levels of resistin, a 12-kDa protein primarily expressed in inflammatory cells in humans, are increased in patients with chronic kidney disease and in those with diabetes mellitus. Both groups of patients have an increased risk of infections mainly as a result of disturbed polymorphonuclear leukocyte (PMNL) functions. Therefore, we investigated the influence of resistin on human PMNLs. Serum resistin concentrations were determined with a sandwich enzyme immunoassay. Using PMNLs from healthy subjects, chemotaxis was tested by the under-agarose method. Flow cytometric assays to measure oxidative burst and phagocytosis were conducted in whole blood. The uptake of deoxyglucose was determined as measure of the PMNL activation state. The activity of intracellular kinases was assessed by Western blotting and by in vitro kinase assays. Resistin inhibited PMNL chemotaxis and decreased the oxidative burst stimulated by Escherichia coli and by PMA, but did not influence PMNL phagocytosis of opsonized E. coli and PMNL glucose uptake. The inhibition of PMNLs by resistin was observed at concentrations found in serum samples of uremic patients, but not in concentrations measured in healthy subjects. Experiments with specific signal transduction inhibitors and measurements of intracellular kinases suggest that PI3K is a major target of resistin. In conclusion, resistin interferes with the chemotactic movement and the stimulation of the oxidative burst of PMNL, and therefore may contribute to the disturbed immune response in patients with increased resistin serum levels such as uremic and diabetic subjects.     

Relationship between oxidative burst activity and CD11b expression in neutrophils and monocytes from healthy individuals: effects of race and gender

Oxidative burst activity and the expression of adhesion molecules have been used as indicators of leukocyte activation status. The aim of the study was to delineate the relationship of oxidative burst activity and the expression of adhesion molecules in neutrophils and monocytes from a pool of healthy volunteers (n = 96). We also tested the potential role of gender and a racial background in the individual response differences. Basal and phorbol myristate acetate (PMA)-stimulated oxidative burst and CD11b expression were determined using dihydrorhodamine 123 and phycoerythrin (PE)-conjugated anti-CD11b monoclonal antibodies. PMA markedly increased CD11b expression and cellular oxidant content in neutrophils and monocytes in all samples. However, the responses showed considerable variability among individuals. A positive correlation was observed between the responsiveness of neutrophils and monocytes in their basal or PMA-stimulated CD11b expressions and PMA-stimulated oxidative burst activities. In contrast, no correlation was found between the level of adhesion molecule expression and cellular oxidant content in monocytes or neutrophils either under basal or under PMA-stimulated conditions. The reactivity of oxidative burst (i.e., PMA-stimulated over basal) was significantly lower in neutrophils from African American males compared with cells from African American females, white females, or white males. In contrast, reactivity of monocytes was significantly elevated in white males compared with all other groups. These findings indicate that leukocytes with a relatively high degree of adhesion molecule expression may display an average or decreased oxidative burst activity, and vice versa. Our findings also indicate that ethnic background may influence the oxidative burst activity in neutrophils and monocytes. This needs consideration in clinical studies utilizing healthy volunteers with mixed gender and ethnic backgrounds.