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G L Mandell

Publications and source records attributed to G L Mandell.

At least 19 recordsLinked to original sources
PubMed1992

A wave of elevated intracellular free calcium spreads through human neutrophils during phagocytosis of zymosan.

The cytosolic concentration of free calcium ([Ca2+]i) plays an important role in the control of many neutrophil functions. In this study, we characterize the early rapid subcellular changes in [Ca2+]i that occur in adherent neutrophils during phagocytosis of zymosan particles, using both dual- and single-excitation wavelength Fura-2 ratio imaging. We observed a wave of elevated cytosolic calcium that began shortly after zymosan contact and propagated from the region of neutrophil contact with the zymosan throughout the cell at a rate of approximately 17 microns/s at 31 degrees C. The wave was initiated by both opsonized and unopsonized zymosan and occurred independently of extracellular calcium. Multiple characteristics of the [Ca2+]i signal (including the absolute and regional [Ca2+]i and wave properties such as amplitude, frequency, duration, and topography) may be responsible for the differential regulation of cellular functions in the neutrophil.

Calcium↗
PubMed1992

Tumor necrosis factor-alpha decreases neutrophil chemotaxis to N-formyl-1-methionyl-1-leucyl-1-phenylalanine: analysis of single cell movement.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine produced by mononuclear cells in response to endotoxin, inhibits neutrophil chemotaxis. We analyzed the effects of TNF-alpha on the orientation and movement of individual neutrophils in a chemoattractant gradient. Neutrophils, treated or untreated with TNF-alpha, were observed migrating in a gradient of the chemotactic peptide N-formyl-1-methionyl-1-leucyl-1-phenylalanine (fMLP) on a specially constructed chamber (Zigmond bridge). The movement of these cells was videotaped, digitized, and then tracked using a newly designed computer algorithm. The data obtained from this algorithm were then utilized to calculate distance traveled, speed and ability to polarize and migrate in a directed manner for each individual cell. TNF-alpha-treated cells behaved like cells not exposed to fMLP in that they failed to orient in a chemotactic gradient and moved in a manner similar to randomly migrating cells. This study provides unique observations of the effect of TNF-alpha on multiple parameters of PMN migration.

Algorithms↗
PubMed1992

Cytokine-induced human multinucleated giant cells have enhanced candidacidal activity and oxidative capacity compared with macrophages.

The granulomatous response to infection is characterized by formation of multinucleated giant cells (MGC). A model has been developed for the study of MGC using fresh human peripheral blood monocytes cultured in medium supplemented with autologous serum and a combination of recombinant human interferon-gamma and interleukin-3 (100 units/mL each). Differential Giemsa staining demonstrated a 53% increase in candidacidal activity of MGC (35.1% +/- 2.0% of ingested organisms were killed by MGC) compared with identically cultured mononuclear macrophages (which killed 22.9% +/- 1.8% of organisms ingested; P less than .05). There was no significant difference in the number of organisms ingested. MGC stimulated with phorbol myristate acetate produced 2.2 times as much superoxide anion per unit of cytoplasmic protein as identically cultured and stimulated macrophages (34.3 vs. 16.2 nmol of superoxide/microgram of cell protein; P less than .01). This was corroborated with single-cell measurements of oxidative activity using digital image analysis. These observations support the hypothesis that MGC have an advantage in microbicidal activity over macrophages that may be due, at least in part, to enhanced oxidative capacity.

Candida albicans↗
PubMed1992

Lipid complexing decreases amphotericin B inflammatory activation of human neutrophils compared with that of a desoxycholate-suspended preparation of amphotericin B (Fungizone).

Amphotericin B (AmB) has toxic effects and alters neutrophil (polymorphonuclear leukocyte [PMN]) function. A lipid-complexed formulation of AmB (AmB-LC) has been reported (A. S. Janoff, L. T. Boni, M. C. Popescu, S. R. Minchey, P. R. Cullis, T. D. Madden, T. Taraschi, S. M. Gruner, E. Shyamsunder, M. W. Tate, R. Mendelsohn, and D. Bonner, Proc. Natl. Acad. Sci. USA 85:6122-6126, 1988) to be less toxic than a desoxycholate-suspended preparation of AmB (AmB-des; Fungizone). In this study we compared the effects of AmB-des and AmB-LC on in vitro PMN function. Neither form of AmB stimulated PMN chemiluminescence, but AmB-des (2 micrograms/ml) nearly tripled PMN chemiluminescence in response to f-Met-Leu-Phe (fMLP), a phenomenon known as priming. Because AmB stimulates monocytes to release cytokines which can affect PMN function, we studied the effects of AmB on PMNs in mixed leukocyte cultures. AmB-des (1 to 2 micrograms/ml) increased the chemiluminescence of PMNs plus mixed mononuclear leukocytes (MNLs) to fMLP. The activity was about three times that of PMNs plus MNLs and seven times the activity of PMNs stimulated with fMLP in the absence of MNLs. Cell-free AmB-des (2 micrograms/ml)-stimulated, MNL-conditioned medium primed pure PMNs to a level equal to that of whole MNLs treated with AmB-des. AmB-LC was much less potent. AmB-LC (20 micrograms/ml) increased fMLP-stimulated chemiluminescence to two times that of PMNs plus MNLs without AmB-LC. AmB-des (2 micrograms/ml) (but not AmB-LC [2 micrograms/ml]) increased nitroblue tetrazolium reduction by PMNs in whole blood from 31 to 52% of positive cells. Neither form of AmB increased Mac-1 (the CD11b/CD18 integrin) expression of pure PMNs. AmB-des (0.5 to 2 micrograms/ml) (but not AmB-LC [< or = 40 micrograms/ml]) nearly doubled PMN Mac-1 expression in the presence of MNLs, and cell-free AmB-des (2 micrograms/ml)-stimulated, MNL-conditioned medium stimulated PMN Mac-1 to 125% of the control level. AmB-des (0.2 to 2 micrograms/ml) (but not AmB-LC [< or = 40 micrograms/ml]) decreased chemotaxis of pure PMNs to fMLP by as much as 35% and that of PMNs in the presence of MNLs by as much as 50%. Desoxycholate by itself had no effect on PMN function. These differences in activity between AmB-des and AmB-LC may explain the lessened toxicity observed with AmB-LC.

Amphotericin B↗
PubMed1992

In vitro demonstration of transport and delivery of antibiotics by polymorphonuclear leukocytes.

Several antibiotics are concentrated inside polymorphonuclear leukocytes (PMN). To investigate whether PMN could act as vehicles for delivery of antibiotics, we combined an assay measuring PMN chemotaxis under agarose with a bioassay measuring levels of antibiotic in agar. Double-layer plates were made by pouring a layer of chemotaxis agarose into tissue culture plates and then adding a thin layer of Trypticase soy agar. Neutrophils were incubated with antibiotic for 1 h and then were washed and placed in wells made in the plates. After allowing PMN to migrate under the agar toward a chemoattractant well containing formyl-methionine-leucine-phenylalanine for 3 h, Streptococcus pyogenes was streaked on top of the agar and grown overnight. PMN migration and zones of inhibition of bacterial growth were measured. Neutrophils migrated 2.51 +/- 0.16 mm toward the chemoattractant well and 1.48 +/- 0.12 mm toward the medium well; migration was not significantly affected by any of the antibiotics used. Plates with PMN incubated without antibiotic showed insignificant inhibition of bacterial growth toward chemoattractant and medium wells (0.38 +/- 0.18 and 0.14 +/- 0.12 mm, respectively; for both, P > 0.05 for difference from 0). PMN incubated with oxacillin (3 micrograms/ml), a drug not concentrated in PMN, caused a similar lack of inhibition (0.28 +/- 0.09 mm toward chemoattractant; 0.14 +/- 0.03 mm toward medium). Incubation with 30 microns of ciprofloxacin per ml resulted in inhibition that was similar in both directions (1.40 +/- 0.16 versus 1.18 +/- 0.13 mm). However, for PMN incubated with azithromycin (3 micrograms/ml), an agent highly concentrated inside phagocytes, there was a large degree of inhibition which was significantly greater in the direction of chemoattractant than in the direction of medium (3.47 +/- 0.30 versus 1.89 +/- 0.25 mm; P < 0.001), indicating that release of bioactive azithromycin by neutrophils occurred after migration. Likewise, after incubation with rifampin (10 micrograms/ml), which is also concentrated by PMN, inhibition was significantly greater in the direction of chemoattractant than in the direction of medium (1.54 +/- 0.24 versus 0.81 +/- 0.28 mm; P = 0.001). We conclude that for certain antibiotics, PMN may act as vehicles for transport and delivery of active drug to sites of infection.

Anti-Bacterial Agents↗
PubMed1992

Pentoxifylline modulates activation of human neutrophils by amphotericin B in vitro.

The antifungal agent amphotericin B (AmB) alters neutrophil (polymorphonuclear leukocyte [PMN]) function, and this may be the mechanism for some of the adverse effects caused by AmB. AmB is a potent inhibitor of PMN migration, increases PMN adherence and aggregation, and primes PMN for increased oxidative activity in response to a second stimulus. AmB also stimulates mononuclear leukocytes (MNLs) to release inflammatory mediators which augment the effects of AmB on PMN function. In the present study, we observed that the methylxanthine derivative pentoxifylline decreased the effects of AmB on PMN function. AmB (2 micrograms/ml) priming doubled PMN chemiluminescence stimulated by fMet-Leu-Phe. In the presence of MNLs, AmB priming increased fMet-Leu-Phe-stimulated PMN chemiluminescence to 622% of unprimed PMN activity. Pentoxifylline (100 microM) blunted the rise in AmB-augmented PMN chemiluminescence in the presence of MNLs to 282% of unprimed PMN activity, and pentoxifylline metabolites were active at 10 microM. Pentoxifylline (100 microM) also blocked AmB-augmented PMN oxidative activity in whole blood, as measured by nitroblue tetrazolium reduction. In the presence of MNL, AmB (2 micrograms/ml) doubled the expression of the important PMN adherence factor Mac-1. Pentoxifylline (1 mM) decreased AmB-stimulated PMN Mac-1 expression back to unstimulated amounts. In the presence of MNLs, AmB (2 micrograms/ml) decreased PMN nondirected and directed migration to fMet-Leu-Phe to 40 and 38% of control PMN migration, respectively. Pentoxifylline (300 microM) counteracted AmB inhibition of nondirected and directed migration to fMet-Leu-Phe, resulting in migration that was 71 and 87% of control PMN migration, respectively. In contrast, the methylxanthine caffeine (100 muM) increased AmB-enhanced chemiluminescence but did not affect AmB-inhibited PMN migration. Pentoxifylline should be evaluated as adjunctive therapy to lessen the inflammatory damage caused by AmB.

Amphotericin B↗
PubMed1992

Regional and generalized changes in cytosolic free calcium in monocytes during phagocytosis.

We measured and visualized cytosolic free calcium ([Ca2+]i) in individual human peripheral blood monocytes during phagocytosis by using the fluorescent indicator fura-2. Monocytes exhibit a rapid rise in [Ca2+]i from a basal level of 75 +/- 11 nM to a peak level of 676 +/- 78 nM (means +/- standard errors of the means; P less than 0.001) by 34 +/- 5 s after contact with opsonized zymosan particles, and a thin rim of high [Ca2+]i was observed surrounding the ingested particle.

Calcium↗
PubMed1992

Induction of multinucleated giant cell formation from in vitro culture of human monocytes with interleukin-3 and interferon-gamma: comparison with other stimulating factors.

One of the hallmarks of the granulomatous response to infection is the formation of multinucleated giant cells (MGC.) In an effort to study MGC, we examined the fusion-promoting effects of a variety of stimulating factors on human peripheral blood monocytes cultured on plastic surfaces in serum-supplemented media. MGC formation was minimally to moderately enhanced by interferon-gamma (IFN-gamma), interleukin (IL)-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), retinoic acid (RA), and IL-6. IL-4 (which has been reported to promote MGC formation from murine macrophages) had an inhibitory effect. IFN-gamma was not required for MGC formation but it significantly increased the fusion-promoting activity of GM-CSF, 1,25-(OH)2D3, RA, and IL-6, IL-3, a hematopoietic growth factor, has been recently shown to induce osteoclast formation from murine bone marrow mononuclear cells. The most striking effect was seen with the combination of IL-3 and IFN-gamma. Fusion index is defined as a percentage of nuclei found within MGC, and an index of 67% at 1 wk was found. The formation of some very large cells with 50 to 100 nuclei was noted. Both Langhans' and foreign-body type cells were seen. Transmission electron micrographs clearly demonstrate the absence of plasma membrane between nuclei. Induction of MGC from peripheral human blood monocytes by IL-3 and IFN-gamma provides an in vitro system for the study of the formation and function of these cells.

Cell Fusion↗
PubMed1992

The Jeremiah Metzger Lecture. Microbial defenses against killing by phagocytes.

Phagocytes are a key feature of defense against microorganisms. Phagocyte function is a complex system with many intricately involved components. Each of these components provides microorganisms with a target for countermeasures against phagocytes. We have discussed examples and purported mechanisms for microbial defenses against the steps involved in killing by phagocytes. Understanding the interplay of these host and pathogen factors leads to a better understanding of both normal host defenses and pathogenesis of disease.

Animals↗
PubMed1991

Adenosine and 2-phenylaminoadenosine (CV-1808) inhibit human neutrophil bactericidal function.

Adenosine is a natural autocoid and immunomodulator that serves an anti-inflammatory role. Stimulation of polymorphonuclear neutrophils (PMN) with soluble stimuli has been shown to inhibit the PMN oxidative burst. We examined the effects of adenosine and the adenosine analog 2-phenylaminoadenosine (CV-1808) on PMN bactericidal function. Adenosine (10 mM) and CV-1808 (10 to 100 microM) inhibited PMN killing of Staphylococcus aureus. There were more surviving bacteria after 240 min of incubation of PMN with S. aureus and adenosine (10 mM) or CV-1808 (100 microM) (254% +/- 45% and 739% +/- 88% of control, respectively) (P less than 0.05) than there were in the control. In contrast, inosine (10 mM), the major degradation product of adenosine, did not affect killing. Adenosine and CV-1808 did not alter cell association of S. aureus, but S. aureus-activated PMN superoxide release was decreased by adenosine (10 microM) and CV-1808 (10 microM) to 67% +/- 7% and 32% +/- 12% that of the control, respectively (P less than 0.05). Since adenosine inhibited PMN bactericidal function only at approximately 10,000 times peak physiological concentrations, endogenous adenosine levels would not be expected to adversely affect PMN bactericidal function. On the other hand, pharmacological concentrations of adenosine derivatives may decrease the oxidative burst and killing sufficiently to increase host susceptibility to infection.

Adenosine↗
PubMed1990

Adenosine and related compounds counteract tumor necrosis factor-alpha inhibition of neutrophil migration: implication of a novel cyclic AMP-independent action on the cell surface.

Human rTNF-alpha (greater than or equal to U/ml) decreased PMN nondirected and directed migration to FMLP to approximately 50% of control. Adenosine (100 microM) almost completely restored hrTNF-inhibited migration (nondirected from 54 to 92% and directed migration to from 54 to 93% of control). The lowest concentration of adenosine that restored hrTNF-inhibited migration was 3 microM, and the adenosine analogue, 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA) was more potent than adenosine. Although CPCA binds to A2-receptors and stimulates adenylate cyclase, the reversal of hrTNF-inhibited chemotaxis was found to be independent of both PMN cAMP content and binding to A2-receptors, because neither 8-Br-cAMP nor pertussis adenylate cyclase restored hrTNF-inhibited PMN chemotaxis and the A2-receptor antagonist, 1,3-dipropyl-7-methylxanthine decreased CPCA stimulated cAMP but enhanced CPCA-restoration of hrTNF-inhibited chemotaxis. The effect of adenosine could be augmented by inhibition of adenosine uptake and decreased by adenosine deamination. Pentoxifylline, (3,7 dimethyl-1-[5 oxo-hexyl] xanthine), like adenosine also restored PMN chemotaxis inhibited by hrTNF. The adenosine receptor antagonist, 1,3-dipropyl-8(phenyl-p-acrylate)-xanthine (BW A1433U), decreased restoration of hrTNF-inhibited chemotaxis by CPCA or pentoxifylline. Thus, the inhibitory effect of hrTNF on PMN migration can be counteracted by adenosine, CPCA, pentoxifylline, and compounds that increase adenosine availability to the surface of the PMN. Inasmuch as an A1-selective agonist N6-cyclopentyladenosine was less active, and the action of the A2-selective agonist CPCA was enhanced by an A2-receptor antagonist, we hypothesize that neither A1 or A2 receptors are involved in adenosine restoration of hrTNF-inhibited chemotaxis. Further, increased cAMP, an A2-regulated event, does not cause the effect, and adenosine restoration of hrTNF-inhibited migration does not appear to be mediated by changes in PMN [F-actin], FMLP receptor expression, or cytosolic calcium. Hence, the restoration of hrTNF-inhibited chemotaxis is controlled by a novel cyclic AMP-independent action on the PMN surface.

Actins↗
PubMed1989

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

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

Humans↗
PubMed1989

The importance of penetration of antimicrobial agents into cells.

Phagocytes may shelter intracellular pathogens from the otherwise lethal effects of many antibiotics. Research in conditions such as chronic granulomatous disease, tuberculosis, legionellosis, and experimental staphylococcal infection underscores the principle that an antibiotic must enter the cell in order to be active against an intracellular microorganism. Demonstrating entry does not guarantee activity, however. The microenvironment and intracellular distribution of the pathogen and antimicrobial agent, and interactions between agent, pathogen, and host cell all contribute to determining the treatment result.

Animals↗
PubMed1988

Spatial and temporal characteristics of the increase in intracellular Ca2+ induced in cytotoxic T lymphocytes by cellular antigen.

The increase in intracellular Ca2+ concentration [( Ca2+]i) in cytolytic T lymphocytes in response to target cell binding was investigated by ratio image fluorescence microscopy. Ca2+ mobilization from intracellular stores occurred at a site distal to target cell contact and was transient. Extracellular Ca2+ influx resulted in an increase in [Ca2+]i that was prolonged and distributed more proximal to the target cell. Oscillations in [Ca2+]i were observed after target cell contact, although the periodicity was dependent on the presence of extracellular Ca2+. The Ag-specific reorientation of cytoplasmic granules occurred well after [Ca2+]i had begun to decline to a resting level, but was dependent on extracellular Ca2+. These studies indicate that the Ag-stimulated increase in [Ca2+]i exhibits considerable spatial and temporal variation, and that these characteristics are altered by the availability of extracellular Ca2+. The results also suggest that these changes in [Ca2+]i may play a role in the cytoplasmic events that accompany T cell-mediated cytolysis.

Animals↗
PubMed1988

Spontaneous oscillations of intracellular calcium and growth hormone secretion.

A novel combination of two single cell assays allowed the simultaneous measurement of intracellular calcium concentration and hormone secretion in normal pituitary cells. [Ca2+]i was recorded using the fluorescent Ca2+ indicator fura-2 and digital imaging microscopy. This technique was combined with a reverse hemolytic plaque assay for growth hormone in order to identify somatotropes and quantitate the amount of hormone released. A dynamic profile of rhythmic calcium oscillations was found in spontaneously secreting somatotropes. Each somatotrope displayed a distinct frequency (one pulse every 5-30 s) and amplitude (range 50-450 nM) generated asynchronously from cell to cell. The amount of growth hormone (GH) released correlated directly with both the frequency and amplitude of calcium oscillations at the level of single GH cells. Furthermore, calcium excursions in somatotropes were rapidly suppressed by either (i) removal of extracellular calcium, (ii) somatostatin (1 mM), or (iii) the calcium channel blockers cobalt (2 mM) and verapamil (100 microM). These observations demonstrate that spontaneous calcium oscillations are characteristic for normal somatotropes. These oscillations are related to spontaneous hormone secretion and due to influx through calcium channels in the membrane. Somatostatin, the physiologic inhibitor of GH secretion, suppresses calcium transients. These findings suggest that the intracellular signaling information may be encoded both in the frequency and amplitude of calcium oscillations.

Animals↗
PubMed1988

Dual stimulation of phospholipase activity in human monocytes. Role of calcium-dependent and calcium-independent pathways in arachidonic acid release and eicosanoid formation.

Human peripheral blood monocytes, prelabeled with [3H]arachidonic acid (AA), release labeled eicosanoids in response to soluble or particulate stimuli. Treatment with 12-O-tetradecanoate phorbol-13 acetate (20 nM), calcium ionophores, A23187 (2 microM) or ionomycin (1 microM), or serum-treated zymosan (300 micrograms) resulted in production of cyclooxygenase (CO) metabolites, 6-keto-PG-F1 alpha, thromboxane-B2, PGE2, PGF2 alpha, PGD2, PGB2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; 15-lipoxygenase products, including 15-hydroxyeicosatetraenoic acid (HETE); and unmetabolized AA. Labeled 5-lipoxygenase (LO) products, 5-HETE, and leukotriene-B4 were detected only after exposure to ionophore or serum-treated zymosan. The calcium dependence of 5-LO activation was confirmed in experiments where calcium was omitted from the incubation medium, and EGTA (0.5 mM) was added, as well as by direct measurement of increased intracellular calcium in phagocytosing monocytes. Combined or sequential treatment with two stimuli increased the release of unmetabolized AA without a commensurate augmentation of labeled metabolites, indicating that release of CO and LO metabolites does not necessarily reflect the extent of phospholipase activation. Quantitation of individual eicosanoids by RIA confirmed results by using radionuclides. These studies show the following. Activation of human monocyte phospholipase may be regulated by at least two pathways, one "12-O-tetradecanoate phorbol-13 acetate-like," which is largely independent of calcium, and another which is mediated by increased intracellular Ca2+ ("ionophore-like"). "Physiologic" stimulation of monocyte arachidonate release, such as that seen accompanying phagocytosis of opsonized particles, may occur via either a calcium-sensitive or calcium-insensitive pathway or both. Calcium may regulate eicosanoid formation at the level of phospholipase or 5-LO. Free AA, CO products, and 12- or 15-LO products are ordinarily released after phagocytosis, but leukotriene-B4, 5-HETE, or other 5-LO metabolites are produced only under conditions where calcium concentrations are optimal.

Arachidonic Acid↗