Discovery of localized regions of excess 10-TeV cosmic rays.

The 7 year data set of the Milagro TeV observatory contains 2.2 x 10(11) events of which most are due to hadronic cosmic rays. These data are searched for evidence of intermediate scale structure. Excess emission on angular scales of approximately 10 degrees has been found in two localized regions of unknown origin with greater than 12sigma significance. Both regions are inconsistent with pure gamma-ray emission with high confidence. One of the regions has a different energy spectrum than the isotropic cosmic-ray flux at a level of 4.6sigma, and it is consistent with hard spectrum protons with an exponential cutoff, with the most significant excess at approximately 10 TeV. Potential causes of these excesses are explored, but no compelling explanations are found.

Effect of novel A2A adenosine receptor agonist ATL 313 on Clostridium difficile toxin A-induced murine ileal enteritis.

Clostridium difficile is a spore-forming, anaerobic, gram-positive bacillus that releases two main virulence factors: toxins A and B. Toxin A plays an important pathogenic role in antibiotic-induced diarrhea and pseudomembranous colitis, a condition characterized by intense mucosal inflammation and secretion. Agonist activity at A2A adenosine receptors attenuates inflammation and damage in many tissues. This study evaluated the effects of a new selective A2A adenosine receptor agonist (ATL 313) on toxin A-induced injury in murine ileal loops. ATL 313 (0.5 to 5 nM) and/or the A2A adenosine receptor antagonist (ZM241385; 5 nM) or phosphate-buffered saline (PBS) were injected into ileal loops immediately prior to challenge with toxin A (1 to 10 microg/loop) or PBS. Intestinal fluid volume/length and weight/length ratios were calculated 3 h later. Ileal tissues were collected for the measurement of myeloperoxidase, adenosine deaminase activity, tumor necrosis factor alpha (TNF-alpha) production, histopathology, and detection of cell death by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) method. Toxin A significantly increased volume/length and weight/length ratios in a dose-dependent fashion. ATL 313 treatment significantly (P < 0.05) reduced toxin A-induced secretion and edema, prevented mucosal disruption, and neutrophil infiltration as measured by myeloperoxidase activity. ATL 313 also reduced the toxin A-induced TNF-alpha production and adenosine deaminase activity and prevented toxin A-induced cell death. These protective effects of ATL 313 were reversed by ZM241385. In conclusion, the A2A adenosine receptor agonist, ATL 313, reduces tissue injury and inflammation in mice with toxin A-induced enteritis. The finding of increased ileal adenosine deaminase activity following the administration of toxin A is new and might contribute to the pathogenesis of the toxin A-induced enteritis by deaminating endogenous adenosine.

Evidence for TeV gamma-ray emission from a region of the galactic plane.

Gamma-ray emission from a narrow band at the galactic equator has previously been detected up to 30 GeV. We report evidence for a TeV gamma-ray signal from a region of the galactic plane by Milagro, a large-field-of-view water Cherenkov detector for extensive air showers. An excess with a significance of 4.5 standard deviations has been observed from the region of galactic longitude l E (40 degrees, 100 degrees) and latitude /b/ < 5 degrees. Under the assumption of a simple power law spectrum, with no cutoff in the EGRET-Milagro energy range, the measured integral flux is phi gamma(>3.5 TeV) = (6.4 +/- 1.4 +/- 2.1) x 10(-11) cm(-2) s(-1) sr(-1). This flux is consistent with an extrapolation of the EGRET spectrum between 1 and 30 GeV in this galactic region.

Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists.

Novel 2-propynylcyclohexyl-5'-N:-ehtylcarboxamidoadenosines, trans-substituted in the 4-position of the cyclohexyl ring, were evaluated in binding assays to the four subtypes of adenosine receptors (ARs). Two esters, 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester (ATL146e) and acetic acid 4-(3-[6-amino-9-(5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl] -prop-2-ynyl)-cyclohexylmethyl ester (ATL193) were >50 x more potent than 2-[4-(2-carboxyethyl)phenethylamino]-5'-N:-ethylcarboxamidoadenosine (CGS21680) for human A(2A) AR binding. Human A(2A) AR affinity for substituted cyclohexyl-propynyladenosine analogues was inversely correlated with the polarity of the cyclohexyl side chain. There was a comparable order of potency for A(2A) AR agonist stimulation of human neutrophil [cyclic AMP](i), and inhibition of the neutrophil oxidative burst. ATL146e and CGS21680 were approximately equipotent agonists of human A(3) ARs. We measured the effects of selective AR antagonists on agonist stimulated neutrophil [cyclic AMP](i) and the effect of PKA inhibition on A(2A) AR agonist activity. ATL193-stimulated neutrophil [cyclic AMP](i) was blocked by antagonists with the potency order: ZM241385 (A(2A)-selective)>MRS1220 (A(3)-selective)>>N-(4-Cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754; A(2B)-selective) approximately 8-(N-methylisopropyl)amino-N(6)-(5'-endohydroxy-endonorbornyl)-9-methyladenine (WRC0571; A(1)-selective). The type IV phosphodiesterase inhibitor, rolipram (100 nM) potentiated ATL193 inhibition of the oxidative burst, and inhibition by ATL193 was counteracted by the PKA inhibitor H-89. The data indicate that activation of A(2A)ARs inhibits neutrophil oxidative activity by activating [cyclic AMP](i)/PKA.

Design, synthesis, and evaluation of novel A2A adenosine receptor agonists.

We have been interested in the design, synthesis, and evaluation of novel adenosine A2A agonists. Through the use of comparative molecular field analysis (CoMFA) we have generated a training model that includes 78 structurally diverse A2A agonists and correlated their affinity for isolated rat brain receptors with differences in their structural and electrostatic properties. We validated this model by predicting the activity of a test set that included 24 additional A2A agonists. Our CoMFA model, which incorporates the physiochemical property of dipole and selects against A1 receptor activity, generated a correlated final model (r2 = 0.891) that provides for enhanced A2A selectivity and predictability. Synthesis, pharmacological evaluation, and modeling of four novel ligands further validate the utility and predictive power (r2 = 0.626) of the CoMFA model.

The role of inflammation in vascular diseases.

When the body responds to an infectious insult, it initiates an immune response to eliminate the pathogen. The hallmark of the immune response is an inflammatory cascade that can also do extensive damage to host tissues. Inflammation is a major contributing factor to many vascular events, including atherosclerotic plaque development and rupture, aortic aneurysm formation, angiogenesis, and ischemia/reperfusion damage. The immune response is mediated by both circulating and resident leukocytes and the cells with which they interact (e.g., vascular endothelium and smooth muscle cells). The process is orchestrated by the activity of a changing series of released and displayed mediators. These include the expression of adhesion molecules on leukocytes and underlying vascular endothelium and the release of cytokines, chemokines, and tissue-destructive metalloproteases and reactive oxygen species. This review focuses on the causes, the inflammatory processes involved, and possible strategies for decreasing vascular disease through regulation of the inflammatory response.

Neutrophil A2A adenosine receptor inhibits inflammation in a rat model of meningitis: synergy with the type IV phosphodiesterase inhibitor, rolipram.

Bacterial meningitis is a disease worsened by neutrophil-induced damage in the subarachnoid space. In this study, the A2A adenosine receptors on human neutrophils were characterized, and the role of A2A receptors on the trafficking of leukocytes to the cerebrospinal fluid and on blood-brain barrier permeability (BBBP) was assessed in a rat meningitis model. Neutrophils bind the A2A selective antagonist, 125I-ZM241385 (Bmax=843 receptors/neutrophil; KD=0.125 nM). A selective A2A receptor agonist, WRC-0470 (2-cyclohexylmethylidene-hydrazinoadenosine; 0.03-1 microM), alone and synergistically with the type IV phosphodiesterase inhibitor, rolipram, increased neutrophil [cAMP]i and reduced cytokine-enhanced neutrophil adherence, superoxide release, and degranulation. These effects of WRC-0470 were reversed by ZM241385 (100 nM). In a lipopolysaccharide-induced rat meningitis model, WRC-0470 (0-0.9 microgram/kg/h), with or without rolipram (0-0.01 microgram/kg/h), inhibited pleocytosis and reduced the lipopolysaccharide-induced increase in BBBP, indicative of decreased neutrophil-induced damage.

Endogenous adenosine curtails lipopolysaccharide-stimulated tumour necrosis factor synthesis.

Recent studies have demonstrated the inhibitory effect of exogenous adenosine on TNF production. During inflammation endogenous adenosine levels are elevated and may be one of several anti-inflammatory mediators that reduce TNF synthesis. In the present study the authors investigated this role of adenosine in freshly isolated human PBMC. The effect of endogenous adenosine on TNF formation was studied by four different approaches. First, adenosine deaminase was added to LPS-stimulated mononuclear cells. This enzyme specifically deaminates extracellular adenosine to the inactive metabolite inosine. TNF production was augmented from baseline stimulation (LPS alone) of 3.5 +/- 0.4 ng ml-1 -5.2 +/- 0.9 ng ml-1 in the presence of 10 U ml-1 adenosine deaminase. Second, TNF production was determined after stimulation in the presence of dipyridamole, an inhibitor of cellular re-uptake of adenosine which increases extracellular concentrations. TNF synthesis was reduced dose-dependently from 3.1 +/- 0.9 ng ml-1 -1.1 +/- 0.2 ng ml-1 by 10 microM dipyridamole. Third, the adenosine A2 receptor antagonist 8-(3-chlorostyryl)caffeine (100 nM) enhanced TNF synthesis from a baseline of 3.7 +/- 0.5 ng ml-1 -5.5 +/- 0.9 ng ml-1. In contrast, no increase resulted from the addition of 100 nM of the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Finally, the authors were able to show that suppression of TNF formation by the specific type IV phosphodiesterase inhibitor rolipram can be completely reversed by adenosine deaminase or by the application of the A2 receptor antagonist. The authors conclude that endogenous adenosine controls TNF production. This effect of adenosine may not only have a physiological role but also appears to contribute to the pharmacological inhibition of TNF synthesis by exogenous agents such as the specific type IV phosphodiesterase inhibitor rolipram.

Interaction of tumor necrosis factor-alpha and granulocyte colony-stimulating factor on neutrophil apoptosis, receptor expression, and bactericidal function.

Infected patients are likely to have increased levels of tumor necrosis factor-alpha (TNF-alpha) and may be treated with recombinant human granulocyte colony-stimulating factor (G-CSF). Recombinant human TNF-alpha activates polymorphonuclear neutrophil (PMN) inflammatory activity. We examined the effect of exposure to TNF-alpha and G-CSF alone and in combination on PMN apoptosis, receptor expression, phagocytosis, and bactericidal function. The results were compared to those obtained with a promoter of PMN apoptosis, cycloheximide. After 24 hr, 27% of PMNs were nonapoptotic, and TNF-alpha (1 unit/ml) showed no change. Cycloheximide (10 micrograms/ml) decreased the number of nonapoptotic cells to 10% of the initial PMN. In contrast, G-CSF (30 ng/ml) decreased apoptosis (57% nonapoptotic PMN after 24 hr). Both G-CSF and TNF-alpha (but not cycloheximide) induced preservation of PMN Fc gamma RIII (467% and 167% of 24-hr controls, respectively) and beta 2-integrin expression (150% and 168% of 24-hr controls, respectively). G-CSF (but not TNF-alpha or cycloheximide) stimulated expression of Fc gamma RI (191% of 24-hr control) and Fc gamma RII (267% of 24-hr control). G-CSF (but not TNF-alpha) maintained the ability of PMN to ingest and kill opsonized Staphylococcus aureus. TNF-alpha decreased the effect of G-CSF on apoptosis, expression of Fc gamma RIII and Fc gamma RI, and bactericidal function. Thus, TNF-alpha promoted expression of Fc gamma RIII and beta 2-integrin receptors, which are important for phagocytic activity, and G-CSF diminished apoptosis, increased Fc gamma receptor expression, and maintained bactericidal function. TNF-alpha counteracted some effects of G-CSF. Interactions of these cytokines in vivo serve to regulate the PMN inflammatory response and bactericidal capacity.

Adenosine modulation of tumor necrosis factor-alpha-induced neutrophil activation.

We hypothesized that adenosine, known to be release from inflammatory sites, could lessen the potentially damaging activity of neutrophils (PMN) primed by tumor necrosis factor-alpha (TNF alpha) at sites of infection. We investigated the effect of adenosine on PMN primed with cell-free medium from mononuclear leukocytes (MNL) that had been treated with lipopolysaccharide (LPS) yielding a conditioned medium rich in TNF alpha and on PMN primed with recombinant human TNF alpha (rhTNF alpha). LPS (10 ng/mL) minimally primed PMN, but LPS-MNL-conditioned medium increased PMN chemiluminescence in response to f-Met-Leu-Phe (fMLP) 1242% compared with unprimed PMN. LPS-MNL-conditioned medium contained adenosine (approximately 30 nM). Converting the adenosine in the LPS-MNL-conditioned medium to inosine with adenosine deaminase (ADA) or blocking adenosine binding to PMN with the adenosine receptor antagonist 1,3-dipropyl-8-(phenyl-p-acrylate)-xanthine (BW A1433U) resulted in a near doubling of chemiluminescence. The LPS-MNL-conditioned medium contained TNF alpha (836 pg/mL; approximately 1 U/mL). Recombinant human TNF alpha (1 U/mL) primed PMN for a 1033% increase in chemiluminescence. Added adenosine decreased rhTNF alpha-primed PMN chemiluminescence (IC50 approximately 100 nM), and adenosine (100 nM) decreased both superoxide and myeloperoxidase release from rhTNF alpha-primed fMLP-stimulated PMN. The activity of adenosine was counteracted by ADA and BW A1433U, and the modulating effect of adenosine was on the primed response rather than on priming per se. Thus, physiological concentrations of adenosine reduce the effects of recombinant human TNF alpha and native human TNF alpha (released from LPS-treated MNL) on PMN activity. Endogenous adenosine may preclude or minimize damage to infected tissue by damping the TNF alpha-primed PMN oxidative response.

Methylxanthines with adenosine alter TNF alpha-primed PMN activation.

Methylxanthines are best known as phosphodiesterase inhibitors that cause a rise in intracellular cAMP. One would expect the two methylxanthines, caffeine and pentoxifylline, to have similar actions on neutrophils (PMN). However, caffeine stimulated and pentoxifylline inhibited PMN oxidative activity. Micromolar concentrations of pentoxifylline decreased native and recombinant tumor necrosis factor-alpha (TNF alpha)-primed formyl met-leu-phe (fMLP)-stimulated PMN chemiluminescence, superoxide production and myeloperoxidase (MPO) release. In contrast, equal concentrations of caffeine increased chemiluminescence and MPO release with no effect on superoxide production. These activities of the methylxanthines were only observed in the presence of physiological concentrations of adenosine, and were abolished by the treatment of the PMN with adenosine deaminase. The activities of adenosine, pentoxifylline and caffeine on PMN activity could not be readily explained by changes in PMN [cAMP]. Thus for TNF alpha-primed PMN, pentoxifylline decreases PMN activity by enhancing the effect of adenosine on degranulation and superoxide production; whereas caffeine increases PMN activity by counteracting the effect of adenosine on degranulation.

The specific type IV phosphodiesterase inhibitor rolipram combined with adenosine reduces tumor necrosis factor-alpha-primed neutrophil oxidative activity.

Monocytes and macrophages produce tumor necrosis factor-alpha (TNF alpha) in response to microbial products including endotoxin. TNF alpha is a potent primer of neutrophil (PMN) oxidative activity. Certain xanthine phosphodiesterase (PDE) inhibitors such as pentoxifylline have been shown to inhibit stimulated oxidative activity in PMN. In the present study, the non-xanthine PDE type IV inhibitor rolipram (4-[3'-cyclopentyloxy-4'-methoxyphenyl]-2-pyrrolidone) alone and in combination with adenosine is examined as a potential modulator of TNF alpha-primed PMN oxidative activity. Attainable in vivo concentrations of rolipram and physiological concentrations of adenosine alone and together synergistically decreased rhTNF alpha-primed suspended PMN oxidative activity stimulated by the chemoattractant f-met-leu-phe. The rolipram effect was reversible by washing, and rolipram had a comparable effect if added before or after priming, indicating that its effect was on the primed response rather than on priming per se. In addition, rolipram especially when combined with adenosine, decreased rhTNF alpha-stimulated PMN adherence to a fibrinogen-coated surface, and the oxidative burst of rhTNF alpha-stimulated adherent PMN. The specific adenosine A2a receptor agonists CGS 21680 and WRC-0474 had comparable activity to adenosine in these experiments. Adenosine (or CGS 21680) combined with rolipram synergistically increased f-met-leu-phe-stimulated PMN cAMP content. The effects of both adenosine and rolipram with adenosine could be only partly counteracted by treatment of the PMN with the protein kinase A inhibitor KT 5720, indicating that protein phosphorylation is only partially involved. Rolipram activity was about 1000 x (by molar concentration) greater than pentoxifylline in comparable assays. Thus, rolipram, especially when combined with adenosine, has potent modulating effects on PMN activation and may be useful in decreasing inflammatory tissue damage in patients with sepsis.

Inhibition of phospholipase D blocks activation of fibrinogen-adherent neutrophils by tumor necrosis factor.

Tumor necrosis factor stimulates fibrinogen-adherent neutrophils to produce a dramatic oxidative burst; the resulting superoxide and other products may contribute to tissue damage in severe infection. Inhibitors of the phospholipase D pathway blocked this activation without affecting cell adherence or nonspecific activation by phorbol myristate acetate. The phospholipase D pathway appears to be involved in the activation of adherent neutrophils by tumor necrosis factor, and this pathway may be a target for modulation of tumor necrosis factor's effects.

The effect of three human recombinant hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and interleukin-3) on phagocyte oxidative activity.

Hematopoietic growth factors not only modulate blood progenitor cell activity but also alter the function of mature phagocytes. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 1 ng/mL for 60 min) did not stimulate luminol-enhanced chemiluminescence of polymorphonuclear leukocytes (PMNs) in suspension but primed PMN for as much as a 15-fold increase in chemiluminescence in response to f-met-leu-phe (fMLP). Mixed mononuclear leukocytes (monocytes [approximately 20%] and lymphocytes [approximately 80%]; MNL) chemiluminescence was very low even after rhGM-CSF priming, but MNLs added to the PMNs (PMN-MNL) resulted in near doubling of rhGM-CSF-primed PMN fMLP-stimulated chemiluminescence. The enhancing factor(s) from MNLs were inherent rather than induced by the GM-CSF, and purified lymphocytes increased GM-CSF-primed PMN chemiluminescence equal to mixed MNLs. We could not detect cell-free "enhancing factor(s)," but cell-to-cell contact further enhanced rhGM-CSF-primed fMLP-stimulated PMN-MNL oxidative activity by 40%. Polyclonal rabbit anti-tumor necrosis factor (TNF) (but not preimmune serum) decreased both fMLP-stimulated rhGM-CSF-primed PMNs and PMN-MNL chemiluminescence, suggesting that TNF on the PMN surface is enhancing GM-CSF-primed chemiluminescence. GM-CSF priming markedly increased PMN superoxide release (sevenfold), but PMN superoxide release was not further enhanced by the presence of MNLs. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) and interleukin-3 (rhIL-3) displayed much smaller effects on pure PMNs and mixed PMN-MNL chemiluminescence and superoxide release than rhGM-CSF. rhGM-CSF primes PMNs for increased oxidative activity more than rhG-CSF and rhIL-3. Maximal oxidative activity was observed when mixed PMN-MNL were primed with GM-CSF in a cell pellet-promoting cell-to-cell contact. This enhanced activity can be attributed, in part, to both inherent enhancing factor(s) on lymphocytes and PMN-associated TNF induced by GM-CSF.

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.

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.

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.

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.

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.

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.