The Beta-2-Adrenoreceptor Agonists, Formoterol and Indacaterol, but Not Salbutamol, Effectively Suppress the Reactivity of Human NeutrophilsIn Vitro

The clinical relevance of the anti-inflammatory properties of beta-2 agonists remains contentious possibly due to differences in their molecular structures and agonist activities. The current study has compared the effects of 3 different categories ofβ2-agonists, namely, salbutamol (short-acting), formoterol (long-acting) and indacaterol (ultra-long-acting), at concentrations of 1–1000 nM, with human blood neutrophilsin vitro. Neutrophils were activated with either N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 µM) or platelet-activating factor (PAF, 200 nM) in the absence and presence of theβ2-agonists followed by measurement of the generation of reactive oxygen species and leukotriene B4, release of elastase, and expression of theβ2-integrin, CR3, using a combination of chemiluminescence, ELISA, colorimetric, and flow cytometric procedures respectively. These were correlated with alterations in the concentrations of intracellular cyclic-AMP and cytosolic Ca2+. At the concentrations tested, formoterol and indacaterol caused equivalent, significant (P<0.05at 1–10 nM) dose-related inhibition of all of the pro-inflammatory activities tested, while salbutamol was much less effective (P<0.05at 100 nM and higher). Suppression of neutrophil reactivity was accompanied by elevations in intracellular cAMP and accelerated clearance of Ca2+from the cytosol of activated neutrophils. These findings demonstrate thatβ2-agonists vary with respect to their suppressive effects on activated neutrophils.

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Regulation of human neutrophil aggregation: comparable latent times, activator sensitivities, and exponential decay in aggregability for FMLP, platelet-activating factor, and leukotriene B4

Blood ◽

10.1182/blood.v82.11.3460.3460 ◽

1993 ◽

Vol 82 (11) ◽

pp. 3460-3468 ◽

Cited By ~ 5

Author(s):

YP Rochon ◽

MM Frojmovic

Keyword(s):

Platelet Activating Factor ◽

Leukotriene B4 ◽

Variable Cross ◽

Activator Concentration ◽

Direct Measurements ◽

Formyl Peptide ◽

Activated Neutrophils ◽

Neutrophil Aggregation ◽

Protein Kinase C Activation

Abstract We have recently described a flow cytometry technique, whose sensitivity allows direct measurements of latent times before the onset of aggregation, and of rates, maximal extents, and reversibility of aggregation (J Leuk Biol 50:434, 1991). We report here that activators which stimulate sustained cellular signaling associated with increases in intracellular calcium (ionomycin) or protein kinase C activation (phorbol myristate acetate, PMA) cause complete (> or = 98%) and irreversible neutrophil aggregation, with latent times for the onset of aggregation inversely proportional to the activator concentration. In contrast, the receptor-specific activators leukotriene B4 (LTB4), formyl peptide FMLP, and platelet-activating factor (PAF) gave only partial and reversible aggregatory responses, limited by the following similar properties: latent times of 4.5 seconds +/- 1.5 seconds, independent of activator concentration; similar concentrations for onset of aggregation (approximately 1 nmol/L) that increased over a similar broad range of activator concentration, with one-half maximal rates of aggregation at 10 nmol/L to 30 nmol/L, corresponding to reported dissociation constant values; comparable limited recruitment and spontaneous reversibility of aggregation; absence of interactivator synergism; and similar exponential decays in activated cell stickiness (refractoriness), with t1/2 = 15 to 30 seconds. Variable cross- desensitization was seen between LTB4 and FMLP depending on donor and activator concentrations. In vivo, these properties are expected to provide localization of the aggregatory response, minimizing the otherwise detrimental effects of circulating activated neutrophils.

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Leukocyte-induced vascular protein leakage in cat mesentery

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1991.261.6.h1872 ◽

1991 ◽

Vol 261 (6) ◽

pp. H1872-H1879 ◽

Cited By ~ 12

Author(s):

P. Kubes ◽

M. B. Grisham ◽

J. A. Barrowman ◽

T. Gaginella ◽

D. N. Granger

Keyword(s):

Leukocyte Adhesion ◽

Platelet Activating Factor ◽

Leukotriene B4 ◽

Superoxide Production ◽

Vascular Integrity ◽

Fourfold Increase ◽

Protein Leakage ◽

Pmn Functions

The overall objective of this study was to determine whether leukocyte adherence and/or emigration is a prerequisite for the increased vascular protein leakage associated with acute inflammation. An in vivo preparation was used to monitor intestinal vascular protein leakage as well as polymorphonuclear leukocyte (PMN) adhesion and emigration in feline mesenteric microvessels exposed to platelet-activating factor (PAF) and leukotriene B4 (LTB4). Local intra-arterial infusion of PAF (4 ng/min) produced a fourfold increase in vascular protein leakage. A 50-fold higher concentration of LTB4 had no effect on vascular protein efflux. LTB4, however, did potentiate the PAF-induced vascular protein leakage. Both inflammatory mediators caused leukocytes to adhere to endothelial cells in postcapillary venules; however, leukocyte emigration was observed only in the presence of PAF. PAF-induced leukocyte adhesion and emigration and the increased vascular protein leakage were inhibited by a monoclonal antibody (MoAb IB4) directed against the common beta-subunit of the adhesive glycoprotein complex CD11/CD18. MoAb IB4 also prevented LTB4-induced leukocyte adhesion. Both PAF and LTB4 caused degranulation of cat PMNs in vitro, yet superoxide production was stimulated by PAF only. The data derived from these in vivo and in vitro studies indicate that leukocyte adhesion per se does not necessarily lead to increased vascular protein leakage and leukocyte emigration. Adhesion-dependent PMN functions such as emigration and superoxide production may play an important role in producing the alterations in vascular integrity observed in inflamed microvessels.

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Acute lung injury in endotoxemic pigs: role of leukotriene B4

Journal of Applied Physiology ◽

10.1152/jappl.1995.78.3.1121 ◽

1995 ◽

Vol 78 (3) ◽

pp. 1121-1131 ◽

Cited By ~ 23

Author(s):

T. J. VanderMeer ◽

M. J. Menconi ◽

B. P. O'Sullivan ◽

V. A. Larkin ◽

H. Wang ◽

...

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Platelet Activating Factor ◽

Leukotriene B4 ◽

Important Mediator ◽

Preliminary Study ◽

Receptor Upregulation ◽

To Receive

The role of leukotriene B4 (LTB4) in the pathogenesis of acute lung injury was examined in endotoxemic pigs. In a preliminary study, the activity and specificity of an LTB4-receptor antagonist, LY-306669, were evaluated. In vitro, LY-306669 completely blocked the functional upregulation of phagocyte opsonin receptors induced by LTB4 but had a much smaller effect on opsonin receptor upregulation induced by platelet-activating factor. In pigs treatment with LY-306669 prevented leukopenia induced by injection of authentic LTB4 but had no effect on the hematologic or hemodynamic effects of PAF or U-48816, a thromboxane-A2 mimetic. In a second study, pigs received an intravenous priming dose of lipopolysaccharide (LPS) at time (t) = -18 h and were randomized to receive 1) no further treatment (n = 5), 2) LPS (250 micrograms/kg over 1 h beginning at t = 0 h) and LY-306669 (10 mg/kg bolus and 3 mg.kg-1.h-1 infusion beginning at t = -15 min) (n = 7), or 3) LPS and vehicle (n = 6). Treatment with LY-306669 significantly ameliorated LPS-induced hypoxemia, pulmonary edema, and alveolitis. These data suggest that LTB4 is an important mediator of pulmonary dysfunction and transendothelial migration of neutrophils in LPS-induced acute lung injury.

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Pertussis Toxin Shows Distinct Early Signalling Events in Platelet-Activating Factor–, Leukotriene B4–, and C5a-Induced Eosinophil Homotypic Aggregation In Vitro and Recruitment In Vivo

Blood ◽

10.1182/blood.v89.12.4566 ◽

1997 ◽

Vol 89 (12) ◽

pp. 4566-4573 ◽

Cited By ~ 33

Author(s):

Mauro M. Teixeira ◽

Mark A. Giembycz ◽

Mark A. Lindsay ◽

Paul G. Hellewell

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Pertussis Toxin ◽

Platelet Activating Factor ◽

Leukotriene B4 ◽

Protein Kinase Inhibitors ◽

Eosinophil Recruitment ◽

Kinase C

Abstract The present study was performed to investigate the early signalling events responsible for eosinophil activation in response to platelet-activating factor (PAF ), C5a, and leukotriene B4 (LTB4 ). We evaluated the effect of pertussis toxin (PTX) on eosinophil aggregation in vitro and cutaneous eosinophil recruitment in vivo. Further studies using the protein kinase inhibitors Ro 31-8220 and staurosporine were performed in vitro to assess in more detail the early signalling events induced by these three mediators. Our results show that C5a and LTB4 signal predominantly or exclusively through a PTX-sensitive G protein that is negatively modulated by protein kinase C, possibly at the level of phospholipase C-β. In contrast, PAF activates eosinophils independent of Gi by a mechanism that is abolished by Ro 31-8220, a selective protein kinase C inhibitor. In addition, these results show for the first time that a receptor-operated event on the eosinophil is essential for chemoattractant-induced eosinophil recruitment in vivo.

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L-selectin function is required for beta 2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1992.263.4.h1034 ◽

1992 ◽

Vol 263 (4) ◽

pp. H1034-H1044 ◽

Cited By ~ 20

Author(s):

U. H. Von Andrian ◽

P. Hansell ◽

J. D. Chambers ◽

E. M. Berger ◽

I. Torres Filho ◽

...

Keyword(s):

Neutrophil Function ◽

Leukotriene B4 ◽

Human Neutrophils ◽

Neutrophil Adhesion ◽

Shear Rates ◽

Sequence Of Events ◽

Multistep Process ◽

Beta 2

In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.

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Identification and characterization of specific receptors for monocyte-derived neutrophil chemotactic factor (MDNCF) on human neutrophils.

Journal of Experimental Medicine ◽

10.1084/jem.169.3.1185 ◽

1989 ◽

Vol 169 (3) ◽

pp. 1185-1189 ◽

Cited By ~ 88

Author(s):

A K Samanta ◽

J J Oppenheim ◽

K Matsushima

Keyword(s):

Human Peripheral Blood ◽

Platelet Activating Factor ◽

Leukotriene B4 ◽

Chemotactic Factor ◽

Single Type ◽

Human Neutrophils ◽

Scatchard Analysis ◽

Specific Receptors ◽

Neutrophil Chemotactic Factor

Specific receptors for a recently purified and cloned monocyte-derived neutrophil chemotactic factor (MDNCF) have been identified on the surface of normal human peripheral blood neutrophils using 125I-labeled recombinant human MDNCF (125I-MDNCF). Competitive binding of 125I-MDNCF to human neutrophils reached a maximal level at 1-3 h at 4 degrees C. The Scatchard analysis showed that there are approximately 20,000 receptors per cell with a single type of high affinity binding (Kd, 8 x 10(-10) M). The receptors for MDNCF are clearly distinct from the receptors for other cytokines and chemotactic agents, e.g., IL-1 alpha, TNF-alpha, and FMLP, C5a, leukotriene B4, and platelet activating factor. Based on the SDS-PAGE analysis of chemically crosslinked 125I-MDNCF receptor complex, there are two polypeptides that bind MDNCF; the molecular weight of these two MDNCF receptors were estimated to be 67,000 and 59,000. Treatment of a promyelocytic cell line, HL60, with 1.25% DMSO for 5 d in vitro increased the number of receptors up to 7,000 receptors/cell with a Kd of 1.2 x 10(-9) M.

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PAF attenuates endothelium-dependent coronary arteriolar vasodilation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.270.6.h2094 ◽

1996 ◽

Vol 270 (6) ◽

pp. H2094-H2099 ◽

Cited By ~ 1

Author(s):

D. V. DeFily ◽

L. Kuo ◽

W. M. Chilian

Keyword(s):

Endothelial Dysfunction ◽

Platelet Activating Factor ◽

Microvascular Dysfunction ◽

Coronary Microvascular Dysfunction ◽

Canine Heart ◽

Activated Neutrophils ◽

Significant Attenuation ◽

Arteriolar Diameter

Platelet-activating factor (PAF) has been reported to play a role in neutrophil activation, microvascular permeability, and endothelial dysfunction in a variety of vascular preparations. Although a majority of the effects of PAF are thought to be mediated by the activation of neutrophils, it is unclear the extent to which the deleterious effects of PAF extend to coronary resistance vessels. Therefore, the purpose of this study was to determine whether PAF causes coronary arteriolar endothelial dysfunction in vivo and whether this dysfunction is independent of activated neutrophils. To test these hypotheses, we measured changes in coronary arteriolar diameter to endothelium-dependent and -independent dilators in vivo by measuring coronary microvascular diameters in a beating canine heart using intravital videomicroscopy following intracoronary infusion of PAF (20 ng.kg-1.min-1). Changes in coronary arteriolar diameter following incubation with PAF were also measured in isolated coronary arterioles. In vivo, incubation with PAF resulted in a significant attenuation of endothelium-dependent dilation to intracoronary acetylcholine (0.1 microgram.kg-1.min-1, 39 +/- 7 vs. 20 +/- 3% dilation) and serotonin (1 microgram.kg-1.min-1, 29 +/- 6 vs. 2 +/- 2% dilation). Papaverine-induced relaxation, however, was unchanged. Likewise, in vitro relaxation to serotonin (10 nM) was significantly reduced (38 +/- 4 vs. 3 +/- 5%) following treatment with PAF, whereas nitroprusside (10 nM)-induced relaxation was unchanged. Because PAF impaired endothelium-dependent arteriolar dilation both in vivo and in vitro, we conclude that the presence of activated neutrophils is not required for PAF-induced coronary microvascular dysfunction.

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Platelet-activating factor—and leukotriene B4-induced release of lactoferrin from blood neutrophils of atopic and nonatopic individuals

Journal of Allergy and Clinical Immunology ◽

10.1016/s0091-6749(05)80178-2 ◽

1990 ◽

Vol 86 (5) ◽

pp. 740-748 ◽

Cited By ~ 4

Author(s):

M TAYLOR ◽

B ZWEIMAN ◽

A MOSKOVITZ ◽

C ALLMEN ◽

P ATKINS

Keyword(s):

Platelet Activating Factor ◽

Leukotriene B4 ◽

Blood Neutrophils

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Regulation of human neutrophil aggregation: comparable latent times, activator sensitivities, and exponential decay in aggregability for FMLP, platelet-activating factor, and leukotriene B4

Blood ◽

10.1182/blood.v82.11.3460.bloodjournal82113460 ◽

1993 ◽

Vol 82 (11) ◽

pp. 3460-3468

Author(s):

YP Rochon ◽

MM Frojmovic

Keyword(s):

Platelet Activating Factor ◽

Leukotriene B4 ◽

Variable Cross ◽

Activator Concentration ◽

Direct Measurements ◽

Formyl Peptide ◽

Activated Neutrophils ◽

Neutrophil Aggregation ◽

Protein Kinase C Activation

We have recently described a flow cytometry technique, whose sensitivity allows direct measurements of latent times before the onset of aggregation, and of rates, maximal extents, and reversibility of aggregation (J Leuk Biol 50:434, 1991). We report here that activators which stimulate sustained cellular signaling associated with increases in intracellular calcium (ionomycin) or protein kinase C activation (phorbol myristate acetate, PMA) cause complete (> or = 98%) and irreversible neutrophil aggregation, with latent times for the onset of aggregation inversely proportional to the activator concentration. In contrast, the receptor-specific activators leukotriene B4 (LTB4), formyl peptide FMLP, and platelet-activating factor (PAF) gave only partial and reversible aggregatory responses, limited by the following similar properties: latent times of 4.5 seconds +/- 1.5 seconds, independent of activator concentration; similar concentrations for onset of aggregation (approximately 1 nmol/L) that increased over a similar broad range of activator concentration, with one-half maximal rates of aggregation at 10 nmol/L to 30 nmol/L, corresponding to reported dissociation constant values; comparable limited recruitment and spontaneous reversibility of aggregation; absence of interactivator synergism; and similar exponential decays in activated cell stickiness (refractoriness), with t1/2 = 15 to 30 seconds. Variable cross- desensitization was seen between LTB4 and FMLP depending on donor and activator concentrations. In vivo, these properties are expected to provide localization of the aggregatory response, minimizing the otherwise detrimental effects of circulating activated neutrophils.

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