Detection of IgA and IgG but not IgE antibody to respiratory syncytial virus in nasal washes and sera from infants with wheezing.

BACKGROUND AND OBJECTIVE: The capacity of respiratory syncytial virus (RSV) to stimulate an IgE antibody response and enhance the development of atopy and asthma remains controversial. Nasal washes and sera from 40 infants (20 with wheezing, 9 with rhinitis, and 11 without respiratory tract symptoms) were obtained to measure IgE, IgA, and IgG antibody to the immunodominant, F and G, virion proteins from RSV. STUDY DESIGN: Children (aged 6 weeks to 2 years) were enrolled in the emergency department during the mid-winter months and seen at follow-up when they were asymptomatic. All nasal washes were tested for RSV antigen. Determinations of antibody isotypes (IgE, IgA, and IgG) to RSV antigens were done in nasal washes and sera by using an enzyme-linked immunosorbent assay. In a subset of nasal washes, IgE to RSV was also evaluated by using a monoclonal anti-F(c)E antibody-based assay. RESULTS: Fifteen patients with wheezing, two with rhinitis, and one control subject tested positive for RSV antigen at enrollment. Thirteen patients with wheezing were <6 months old, and most (77%) were experiencing their first attack. Among the children with positive test results for RSV antigen, an increase in both nasal wash and serum IgA antibody to RSV-F(a) and G(a) was observed at the follow-up visit. However, there was no evidence for an IgE antibody response to either antigen. CONCLUSION: Both IgA and IgG antibodies to the immunodominant RSV-F(a) and G(a) antigens were readily detected in the nasal washes and sera from patients in this study. We were unable to demonstrate specific IgE antibody to these antigens and conclude that the production of IgE as a manifestation of a T(H)2 lymphocyte response to RSV is unlikely.

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.

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.

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.

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.

In vitro evaluation of a high-efficiency leukocyte adherence filter.

In view of the currently available data, prevention of alloimmunization requires filters with higher efficiency to achieve a reduction in the number of leukocytes below 10 million per transfusion. Two versions (Pall PL-100 and PL-50) of the new generation leukocyte-depletion filters were studied. Single donor (SDPC)- and pooled multiple donor (MDPC) platelets were run in parallel. At a flow rate of 10 ml/min, the PL-100 filter was shown to effectively reduce the number of residual leukocytes to far below the critical immunogenic threshold of 10 million in all SDPC units and in 77% of MDPC units. Apheresis platelets appear not only to be better depleted than pooled multiple donor platelets, but also to have a better post-filtration platelet recovery (96% versus 84%). The efficiency of the smaller version of the filter (Pall-50) was higher than that of the Pall-100 filter for both single and pooled multiple donor platelet concentrates (PC). Leukocytes were absent in more than 92% of units in both types of concentrates. The maximal number of detected leukocytes was 2.2 million in a pool of 6 units. The outcome of filtration of 5-day-old pooled platelets was less favorable than filtration of 1- or 2-day-old pooled platelets, indicating that filtration soon after preparation is preferred to filtration after storage. Post-filtration platelet integrity, activation state, function, and morphology were all well preserved in both single and multiple PCs.

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 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.

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.

Inhibition of the inflammatory action of interleukin-1 and tumor necrosis factor (alpha) on neutrophil function by pentoxifylline.

Inflammatory cytokines, including interleukin-1 and tumor necrosis factor, are produced by monocytes and macrophages in response to microorganisms and microbial products such as endotoxins. The cytokines stimulate neutrophil adherence, degranulation, and superoxide production but inhibit neutrophil migration. We studied the modulation of cytokine-induced neutrophil activation by pentoxifylline and its principle metabolites. Lipopolysaccharide-stimulated mononuclear-leukocyte-conditioned medium containing inflammatory cytokines, purified human interleukin-1, or recombinant human tumor necrosis factor increased neutrophil adherence to nylon fiber, primed neutrophils for increased superoxide production in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), increased neutrophil lysozyme release stimulated by FMLP, and decreased directed migration of neutrophils to FMLP. Pentoxifylline and its principle metabolites at or near therapeutically achievable levels were able to counteract these effects. Pentoxifylline inhibited the increase in free intracellular calcium in polymorphonuclear leukocytes stimulated by FMLP and increased binding of FMLP to neutrophils at 37 degrees C but not at 4 degrees C. By blocking the inflammatory action of interleukin-1 and tumor necrosis factor on neutrophils, pentoxifylline may diminish the tissue damage caused by neutrophils in such conditions as septic shock, adult respiratory distress syndrome, cardiopulmonary bypass lung damage, and myocardial reperfusion injury.

Teicoplanin, vancomycin, rifampicin: in-vivo and in-vitro studies with Staphylococcus aureus.

Groups of mice infected with 3.3 X 10(8) Staphylococcus aureus via the tail vein were treated three days later with rifampicin (13 mg/kg), vancomycin (33 mg/kg), or teicoplanin (33 mg/kg). Rifampicin was the most effective agent (28 out of 29 survivors). Vancomycin and teicoplanin were of equivalent efficacy (21 of 29 and 24 of 29 survivors, respectively). When intraleucocytic staphylococci were incubated with rifampicin (1 or 20 mg/l), vancomycin (100 mg/l), or teicoplanin (100 mg/l), rifampicin was the most active drug. Vancomycin and teicoplanin were similar.