Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst.

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.     

Diverse effects of neutrophil integrin occupation on respiratory burst activation.

Integrin occupation can alter the function of neutrophils (PMN), but the mechanism(s) involved is still unclear. This study demonstrated that the occupation of PMN integrins (especially those of the beta(3) subfamily) strongly enhances TNF stimulation of the respiratory burst but down-regulates that induced by PMA, fMLP, Con A, and serum treated zymosan. Treatment of PMN with genistein, staurosporine, and wortmannin, inhibitors of tyrosine kinases, protein kinase C, and phosphotidylinostol 3-kinase (PI 3-kinase) respectively, completely blocked the TNF-stimulated respiratory burst in PMN. Genistein and wortmannin enhanced the PMA-stimulated respiratory burst but only in cells adherent to RGD peptide. These findings suggest that PMN integrins (beta(3) subfamily) can generate signals that regulate the PMN agonist responses, probably through the activities of tyrosine kinases and PI 3-kinase.     

Macrophage membrane proteins: Possible role in the regulation of priming for enhanced respiratory burst activity

Brief exposure of macrophages to the proteolytic enzymes papain, elastase, or trypsin primed them for enhanced production of superoxide anion (O₂⁻) in response to stimulation by phorbol myristate acetate (PMA). Priming by trypsin was achieved at 0 °C, at which temperature trypsin functions as a protease but is not internalized, supporting the concept that protease priming depends on modification of the plasma membrane. Analysis of external membrane proteins after radioiodination of intact cells and separation by gel electrophoresis indicated that papain treatment of macrophages resulted in the cleavage of a membrane protein with a molecular weight of approximately 305K. Membranes from macrophages primed by elicitation with Corynebacterium parvum also demonstrated a reduced amount of the membrane protein at approximately 305 kDa, as well as a reduction of a protein at about 270 kDa. Lipopolysaccharideelicited macrophages showed a reduced amount of a protein at about 175 kDa. Continuous spectrophotometric assays of O₂⁻ release from adherent macrophages indicated that after exposure to a stimulus, protease-treated cells produced O₂⁻ more quickly than did control cells (reduced lag time). Inhibitors of protein synthesis augmented the priming effect of papain when added with the protease. These results suggest that protease-induced priming results from inactivation of a membrane protein (or proteins) that exerts a down-regulating effect on the respiratory burst.     

The influence of selenium and vitamin E on the enhanced respiratory burst reaction in smokers

The respiratory burst reaction (RBR) of neutrophilic granulocytes of the peripheral blood was estimated by means of the luminol reaction in 10 smokers and in 10 nonsmokers. Compared to the nonsmokers, the RBR of smokers' granulocytes showed a significantly higher rate of RBR. RBR consists of two enzymatic systems, i.e., NADPH-oxidase generating superoxide anions and myeloperoxidase, generating hypochlorous acid. Furthermore the superoxide anion may undergo dismutation to oxygen and peroxide. Thus, since the RBR may cause an oxidative stress, the smokers were supplemented for 10 d with antioxidants, i.e., 200 micrograms L-Se-methionine and 1000 mg vitamin E/d. After 10 d of supplementation with the antioxidants, the RBR of the smokers was significantly decreased by 20-75 percent. Since the oxidative stress associated with RBR may cause autodigestive reactions in the lungs of smokers, it may be beneficial for smokers to use relatively high doses of such antioxidants in order to hamper the pathological processes associated with smoking.     

Turning on the respiratory burst

The respiratory burst is a distinguishing property of phagocytes. It is induced by chemotactic stimulation or phagocytosis and reflects the activation of a membrane-bound enzyme system that transfers electrons from cytosolic NADPH to extracellular oxygen, producing superoxide. The products of the burst are essential for the killing of microorganisms, but are also a cause of tissue damage and inflammation. Studies aimed at a better understanding of the regulation of the respiratory burst should help in the search for new ways to treat infections and inflammation.     

Selective priming of rate and duration of the respiratory burst of neutrophils by 1,2-diacyl and 1-O-alkyl-2-acyl diglycerides. Possible relation to effects on protein kinase C

Both 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-glycerols are released during stimulation of human polymorphonuclear leukocytes (PMNL). 1,2-Diacylglycerols have received intense interest as intracellular "second messengers" due to their ability to activate protein kinase C (Ca2+ phospholipid-dependent enzyme). However, little is known about bioactivities of the alkylacylglycerols. This study compared the ability of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols to modulate the respiratory burst of stimulated PMNL, a response which depends on the activation of an NADPH oxidase to generate bactericidal species of reduced oxygen. Direct stimulation by N-formyl-Met-Leu-Phe caused an abrupt release of H2O2 which ceased within 2.5 min. Preincubation with diacylglycerols (1-oleoyl-2-acetylglycerol,5-30 microM, and 1,2-dioctanoylglycerol,2-5 microM) caused a decrease in lag time, 3-fold increase in initial rate of H2O2 release, and marked prolongation of the response to N-formyl-Met-Leu-Phe (features characteristic of a priming effect). Preincubation with alkylacylglycerols (1-O-delta 9-octadecenyl-2-acetylglycerol, 5-30 microM, and 1-O-octyl-2-octanoylglycerol, 20-50 microM) primed initiation (shortened lag time and increased velocity) but, in contrast to diacylglycerols, did not alter duration of H2O2 release. While low concentrations of diacylglycerols (5-30 microM) primed PMNL, higher concentrations (greater than or equal to 70 microM) stimulated the cells directly. In contrast, higher (70-100 microM) concentrations of alkylacylglycerols did not prime the responses but, in fact, inhibited priming (especially of duration) induced by diacylglycerol. The high concentrations of alkylacylglycerol also inhibited direct stimulation induced by high concentrations of diacylglycerol. Direct stimulation by high concentrations of diacylglycerol probably involves activation of protein kinase C, whereas alkylacylglycerol was found to inhibit activation of protein kinase C by diacylglycerol in vitro. Thus, diacylglycerols are complete priming agonists, altering both rate and duration of the response. In contrast, alkylacylglycerols may have biphasic, concentration-related effects in modulation of functions of PMNL. At low concentrations, they may facilitate initiation of functional events; however, as their concentration increases, they may serve to terminate responses. The distinct priming effects of these diglycerides also reveal that priming can involve at least two distinct events: 1) initiation and 2) prolongation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of the NADPH-binding subunit of the respiratory burst oxidase.

The respiratory burst oxidase is a multicomponent membrane-bound enzyme that uses NADPH to reduce oxygen to O2-. When oxidase-containing membranes from activated neutrophils are treated with 0.3 M KCl, the NADPH-binding component of the oxidase elutes from the membranes in an active form. Treatment of this eluate with [32P]NADPH dialdehyde labels an approximately 32-kDa protein that is absent from eluates obtained from normal resting membranes or from resting or activated membranes from patients with one form of chronic granulomatous disease. We propose that this approximately 32-kDa protein is the NADPH-binding component of the oxidase.     

Temperature influence on stimulated PMN respiratory burst.

Body temperature can modulate the pathogenesis of infectious, metabolic and autoimmune diseases. This effect has been attributed to several hypothesized mechanisms. Body temperature could play an important role in influencing some cellular functions of human white blood cells. In this work we examined the temperature effect on the respiratory burst in human neutrophils. Human polymorphonuclear leucocytes (PMN) were obtained from heparinized venous blood by dextran sedimentation and erythrocyte lysis with NH4Cl (0.87%). Granulocytes were stimulated with opsonized zymosan (OZ), formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate (PMA), and monosodium urate (MSU) crystals at different temperatures (26, 37, 39, 40, 42 degrees C). The technique of luminol dependent chemiluminescence (CL) was used as indicator of oxygen free radicals (OFR) release by stimulated cells. OFR production from PMN stimulated with OZ, PMA, FMLP was higher at 37 degrees C than at 26, 39, 40, 42 degrees C (p < 0.001 OZ stimulated PMN at 40-42 degrees C; p < 0.05 PMA stimulated PMN at 42 degrees C. Significantly different from 37 degrees C value). OFR release from PMN stimulated with MSU crystals was significantly increased at 39 degrees C compared to 37 degrees C value (p < 0.001). This effect could not only be attributed to temperature influence on neutrophil activity. The specific polymorphonuclear leukocyte response to the microcrystals and the temperature influence on chemical and physical characteristics of the crystals may play an important role. We are now studying the temperature effect on activity of PMN exposed to others crystals.     

Protein phosphorylation and the respiratory burst

The exposure of 32P-loaded neutrophils to any of a variety of activating agents induces changes in the levels of phosphorylation of a large number of phosphoproteins. The uptake of phosphate by one set of phosphoproteins in particular, a family whose members migrate at Mr 48K with near neutral pI values, appears to be closely related to the activation of the respiratory burst oxidase, the O2--producing enzyme of phagocytes that is responsible for the generation of microbicidal oxidants by these cells. Evidence for the relationship between the phosphorylation of these proteins and the activation of the respiratory burst oxidase has been furnished by kinetic studies as well as by studies on protein phosphorylation in neutrophils from patients with chronic granulomatous disease, a group of inherited disorders affecting this oxidase. The details of this relationship are obscure, although the evidence suggests that these phosphoproteins act in substoichiometric amounts with respect to the oxidase.     

Hypericin and photodynamic treatment do not interfere with transport of vitamin C during respiratory burst

Hypericin is a photosensitizing pigment found in St. John's wort (Hypericum perforatum) displaying a high toxicity towards certain tumors. The fact that some non-tumor cells, especially monocytes and granulocytes, are resistant to its photocytotoxic effects, posed the question whether this insensitivity is due to their ability to accumulate vitamin C, an antioxidant which alleviates the deleterious work of free radicals.

HL-60 promyelocytic tumor cells can be differentiated to neutrophilic granulocytes by treatment with dimethylsulfoxide and were used as cell model. In the differentiated cells, treatment with phorbol esters (PMA) stimulates vitamin C (ascorbate) transport. The uptake rates were unaltered by hypericin at concentrations below 1 μM and irradiation with visible light at a light dose of 6 J/cm². Inhibition by higher concentrations of hypericin was most probably due to a combination of photocytotoxic properties of the dye and oxygen radicals generated during respiratory burst. Superoxide production by NADPH oxidase followed by reduction of ferricytochrome c was inhibited by hypericin. The degree of inhibition was dependent on the concentration of hypericin and light intensity: IC₅₀-values were 1.7 and 0.7 μM under light doses of 3.6 and 10.8 J/cm², respectively. Oxidative stress, monitored with 2',7'-dichlorofluorescein (DCF) was only slightly decreased by ascorbate even at higher concentrations of hypericin. In contrast to its effect on the ferricytochrome c-reduction, irradiation had no significant influence on DCF-fluorescence. However, the viability of the cells was strongly decreased after photosensitization and no significant improvement was obtained by ascorbate.

Results from this work indicate that ascorbate transport per se is not altered during photodynamic therapy and vitamin C does not interfere with hypericin-induced photodamage of cellular targets.     

Free zinc inhibits transport of vitamin C in differentiated HL-60 cells during respiratory burst

Zinc is an essential trace element for the immune system. It is known to be essential for highly proliferating cells, especially for cells of the immune system. However, zinc and other divalent cations are known to inhibit the human neutrophilic NADPH oxidase. Differentiated HL-60 cells were found to accumulate large quantities of vitamin C (ascorbate) after activation of the NADPH oxidase by phorbol esters (PMA). This increase in vitamin C transport is due to the generation of superoxide and subsequent oxidation of ascorbate to dehydroascorbate (DHA) which is preferentially taken up by the cells. We found that zinc reversibly inhibits both PMA-stimulated ascorbate uptake and superoxide generation with a half-maximal effect at 20 microM of free zinc ions. Higher residual extracellular ascorbate concentrations were measured with increasing zinc concentrations, indicating that less ascorbate was oxidized and taken up by the cells. When the fluorescent dye diSC3(5) was used to monitor shifts in membrane potential, we found that depolarization with PMA was prolonged after preincubation of the cells with zinc. Suppression of the respiratory burst as well as inhibition of the uptake of the antioxidant vitamin C may disturb the balance between oxidative damage of invading particles and antioxidant protection in activated neutrophils.     

Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients

In neutrophils of patients with essential hypertension the NADPH-dependent O2- production elicited by stimulation with f-Met-Leu-Phe is three to four fold higher in comparison with neutrophils of normotensive control subjects. Neutrophils from hypertensive patients are less responsive to priming, by non-stimulating doses of the agonist, as compared to control cells, which following this pretreatment augment superoxide anion production up to levels close to those expressed by neutrophils from hypertensive patients. No difference in NADPH oxidase activity, between neutrophils from the two groups of subjects, was observed when the rate of O2- production was evaluated in a reconstructed cell-free system containing the membrane fraction and the cytosolic cofactors. These results are consistent with the hypothesis that differences in the functional organization of the oxidase at the membrane level in neutrophils of hypertensive are responsible for the enhanced O2- production following agonist stimulation.     

The bactericidal effects of the respiratory burst and the myeloperoxidase system isolated in neutrophil cytoplasts

Neutrophil polymorphonuclear leucocytes kill bacteria by oxygen-dependent and oxygen-independent mechanisms. Many potentially toxic mechanisms have been described, but the complexity of the phagosomal environment and the synergy between oxidative and non-oxidative systems hamper the investigation of individual bactericidal mechanism in whole cells. Neutrophil cytoplasts are greatly depleted of granule proteins and permit the investigation of the bactericidal effects of the respiratory burst in isolation. In this study they have been used to examine the role of the respiratory burst and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus. Cytoplasts generated oxygen radicals at comparable rates to human neutrophils and phagocytosed but did not kill S. aureus. The selective reconstitution of the myeloperoxidase-hydrogen peroxide-halide system by coating bacteria with myeloperoxidase conferred on cytoplasts the ability to kill intracellular bacteria. However, extracellular killing by diffusible bactericidal factors was not detected in this system.     

Dual mechanisms in priming of the chemoattractant-induced respiratory burst in human granulocytes. A Ca2+-dependent and a Ca2+-independent route

After interaction with so-called priming agents, the respiratory burst in human granulocytes does not become activated, but is enhanced upon subsequent stimulation with the chemoattractant FMLP. Investigating the mechanism of the priming reaction, we found that a transient rise in the cytosolic free calcium concentration [( Ca2+]i) suffices to irreversibly prime human granulocytes. Thus, platelet-activating factor (PAF) induced a transient increase in [Ca2+]i and primed the cells to an enhanced respiratory burst upon subsequent interaction with FMLP. Artificially, the transient rise in [Ca2+]i was mimicked by addition and subsequent removal of the Ca2+ ionophore ionomycin; this treatment too, primed the respiratory burst of the granulocytes. The priming induced by ionomycin was completely abolished when [Ca2+]i changes were buffered during exposure of the cells to the ionophore. The priming induced by PAF was only partially inhibited under [Ca2+]i-buffering conditions during priming, indicating that multiple pathways exist in the priming of granulocytes by PAF.     

Chromatographic separation of vitamin D3 sulfate and vitamin D3.

This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin D3 sulfate from free vitamin D3 and its metabolites. This technique has been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin D3 sulfate (35S or 35S and 3H) only the peak of vitamin D sulfoconjugate was found in chromatographic elution of serum extracts.