Definition of platelet-derived histamine-releasing factor and histaminergic receptors modulating platelet aggregation.

Endogenous and exogenous free radical scavengers significantly decrease mast cell histamine release induced by coincubation with resting, activated platelets or with platelet-derived supernatant. Histamine and pyridylethylamine dose-dependently enhance platelet aggregation; the effect is potentiated by ranitidine and blocked by mepyramine. Histamine increases also cytosolic calcium concentration in platelets stimulated with thrombin, and binding sites for [3H]-mepyramine are present on platelet membranes.     

Studies on histamine releasing factor (HRF) of human,murine and guinea-pig origin

There has been the lack of small animal models for the studies of histamine releasing factor (HRF). We have found that HRF producedin vitro by lymphocytes of asthmatic patients can act independently from the animal species and induces histamine secretion from mouse peritoneal mast cells. We also found, that spleen cells derived from normal as well as from sensitized mice and guinea-pigs are able to generate HRFin vitro, when cultured in suitable conditions. HRF of mouse and guinea-pig origin released histamine from homologous mast cellsin vitro and its activity was enhanced when spleen cell cultures were stimulated with specific antigens or non-specific mitogen.This paper was supported by Polish Academy of Sciences, (Grant no. 10.5).     

A mononuclear cell derived histamine releasing factor (HRF) in asthmatic patients. Histamine release from basophils in vitro

Mononuclear cells from asthmatic patients produced a histamine releasing factor (HRF). Mononuclear cells produced this factor in culture spontaneously but significantly much more following specific allergen stimulation. This factor released histamine from both sensitized and unsensitized basophils in vitro. Mononuclear cells from healthy subjects produced HRF only after nonspecific mitogen/phytohemagglutinin stimulation. Asthmatic patients on immunotherapy with essential benefits failed to produce HRF.     

Enhanced skin reactivity to platelet-derived permeability factor (PDPF) and exogenous histamine in an acute phase rabbit model

Rabbits made acute phase by sub-cutaneous trauma with 2% croton oil (in mineral oil) were tested by intradermal (ID) injection with platelet-granule extracts containing platelet-derived permeability factor (PDPF). Compared with controls, skin reactivity to PDPF was enhanced in acute phase animals 3–7 days post-trauma, a period of acute inflammation as reflected by the occurrence in the circulation of C-reactive protein; maximal skin responses were observed 3–4 days post-trauma. Individual skin sites reached maximum intensity 15 min–1 hour post-ID injection of PDPF and were sensitive to chlorpheniramine maleate, suggesting a major role for histamine. Intradermal injection of histamine revealed that acute phase animals yielded an initially more intense skin reaction, and were markedly less capable of recovering from the effects of histamine. These data suggest that in the acute phase, there exists a heightened and prolonged sensitivity to the action of histamine which can be exploited by pro-inflammatory agents such as PDPF.This work was supported, in part, by grants from the NIH (HL-23457) and the Institut Pasteur de Lyon. B.A.F. is the recipient of NIH Career Development Award (HL-00614). The majority of these studies were performed on sabbatical at the Institut Pasteur de Lyon (B.A.F.).     

Polymorphonuclear leucocytes release a factor(s) that induces platelet aggregation and ATP release after interaction with insoluble and surface-fixed immune complexes.

We have found that human polymorphonuclear leucocytes (PMN) can be stimulated by large aggregates (heat-aggregated IgG, chemically polymerized IgG, heavily aggregated human immune complexes) and by surface-bound immune complexes (IC) to release enzymes (lysozyme, beta glucuronidase) and a factor(s) able to induce platelet aggregation and ATP release from the platelets. Surface-bound IC were most effective in stimulating the release of this factor(s). We used several substrates for their preparation: plastic-adsorbed antigen. Sepharose-coupled antigen and polymerized antigen. The platelet-aggregating factor(s) released by IC-stimulated PMN and zymosan-stimulated PMN were compared for their susceptibility to inhibition by indomethacin. Both induced a first phase of platelet aggregation that was resistant to indomethacin, but the second phase of aggregation and the release of platelet ATP were inhibited to a variable degree, more pronounced in the case of the factor(s) released after PMN-IC interaction. The lack of inhibition of the early phases of aggregation induced by our factor(s) when platelets were simultaneously exposed to indomethacin suggests that the classical, phospholipid PAF is released under these experimental conditions. Although, further experiments will be necessary to fully characterize the factor(s) involved, our observations suggest a complex interrelationship between human PMN and platelet activation, which may play an important role in the sequence of events that mediate the tissue deposition of IC and appearance of inflammatory changes.     

The effects of antiallergic and bronchodilator drugs on platelet-activating factor (PAF-acether) induced bronchospasm and platelet aggregation

Platelet activating factor (PAF-acether) is a potential mediator of asthma and inflammation. Recently, the suggestion was made that inhibition of PAF-acether by disodium cromoglycate (DSCG) might be partly responsible for the effectiveness of DSCG in asthma. We have extended these studies and examined the effects of antiallergic and bronchodilator drugs on PAF-acether induced bronchospasm after i.v. administration in guinea pigs andin vitro platelet aggregation in rabbits. Neither DSCG nor Wy-41,195, a potent orally effective antiallergic, altered either of the PAF-acether responses. Furthermore, aerosolized ipratropium, promethazine, ketotifen and FPL 55712 failed to affect the PAF-acether-induced bronchospasm. The same drugs were also ineffective against platelet aggregation induced by PAF-acether. In contrast, aerosolized thiazinamium chloride inhibited the bronchospasm and also inhibited PAF-acether-induced platelet aggregation. Thiazinamium chloride possessed weak antiaggregatory effects against ADP and was without effect against arachidonic acid-induced platelet aggregation. Both lipoxygenase and cyclooxygenase products of arachidonic acid metabolism appear to be involved in PAF-acether bronchospasm since i.v. administered lipoxygenase inhibitors (phenidone, BW755c and NDGA) and indomethacin independently inhibited thisin vivo response. However, these drugs falled to alter platelet aggregation to PAF-acether. Thiazinamium chloride may be capable of directly antagonizing the PAF-acether-induced platelet aggregatory response and, in addition, inhibiting the synthesis and/or effects of bronchoconstrictor amines and endogenously generated arachidonic acid metabolites.     

Occurrence of platelet-activating factor (PAF) and an endogenous inhibitor of platelet aggregation in diffuse cutaneous mastocytosis.

We have identified PAF in the blister fluid from a patient with bullous mastocytosis, a rare form of mast-cell disease. We have found a novel endogenous inhibitor of platelet aggregation which obscured the presence of the PAF in unprocessed blister fluid and in ethanol or lipid extracts. The PAF was characterized by the demonstration of chromatographic, mass spectral and biological properties identical to those of authentic PAF. Thus this is the first demonstration of PAF in biological fluid from a patient with mastocytosis. High levels of immunoreactive prostaglandin D2 (PGD2) and histamine were also present in the blister fluid. The interaction between PAF and the inhibitor of platelet aggregation in patients with systemic mastocytosis may provide an explanation for some of the manifestations of the disease, in particular the episodic hypotension, cutaneous flushing and pallor.     

Ultrastructural evidence for extravascular platelet recruitment in the lung upon intravenous injection of platelet-activating factor (PAF-acether) to guinea-pigs

Bronchoconstriction and degenerative lesions of the bronchial epithelium were observed microscopically 1 min after the i.v. injection of 100 ng/kg of platelet-activating factor (PAF-acether) to the anaesthesized guinea-pig. Constricted arterioles containing marginated polymorphonuclear neutrophils and platelet aggregates were seen, as well as alveolar capillaries obstructed by thrombi formed by partially or totally degranulated platelets. Three minutes after the injection of PAF-acether, platelet diapedesis to the alveolar septa and lumen was clearly observed. Bronchoconstriction was still present at 3 min, but subsided after 60 min, whereas oedema of the submucosa persisted accompanied by an infiltration of eosinophils and neutrophils. The infusion of prostacyclin prevented the formation of platelet aggregates and platelet diapedesis due to PAF-acether, but the morphological evidence of bronchial constriction was not modified. Aspirin failed completely to modify the effects of PAF-acether. Our results show that PAF-acether causes early formation and deposition of platelet aggregates, accompanied by the margination of polymorphonuclear neutrophil leucocytes in pulmonary vessels of the guinea-pig. Since bronchoconstriction persisted when platelet aggregation was inhibited with prostacyclin, aggregation by itself would not account for this effect. Early platelet diapedesis in the vicinity of bronchial smooth muscle corroborates previous evidence that platelets contain and release bronchoconstrictor substances, which operate by cyclo-oxygenase-independent mechanisms and are possibly involved with the physiopathology of lung inflammation during immediate hypersensitivity.     

Peripheral GABA(A) receptors: evidence for peripheral primary afferent depolarization.

We propose that the primary afferent depolarization that follows GABA(A) receptor activation in the spinal cord also occurs in the periphery. As evidence, the present study localizes beta2/beta3 and alpha1 subunits of the GABA(A) receptor on 10-14% of the unmyelinated primary afferents axons in the glabrous skin of the cat paw. Behavioral studies demonstrate that local peripheral injection of the GABA(A) agonist muscimol at a low concentration (2.0 microM) attenuates, and at a high concentration (1 mM) enhances, formalin-induced nociceptive behaviors. Intraplantar injection of muscimol alone at a high dose evokes thermal hyperalgesia. Bicuculline, a GABA(A) antagonist, prevents these muscimol-induced changes in behavior. The muscimol-induced effects are due to local rather than systemic or central activation of GABA(A) receptors, as such effects are not observed in the contralateral paw. We interpret these findings to indicate that activation of GABA(A) receptors by low concentrations of muscimol depolarizes peripheral primary afferent terminals, a phenomenon we call peripheral primary afferent depolarization, in turn reducing the size of the peripheral action potentials and concomitantly reducing the amount of algogenic substances released from the peripheral terminals of these fibers. This sequence of events presumably results in a reduction in nociceptor activation. Higher concentrations of muscimol further depolarize GABA(A) receptor-containing terminals, which then initiates action potentials in nociceptors analogous to the appearance of dorsal root reflexes that arise following activation of GABA(A) receptors on central primary afferent terminals. These latter events reverse the analgesic effects of GABA(A) ligands and lead to potentiation of nociceptive input. Thus, the present study provides anatomical and behavioral evidence supporting a bimodal role for GABA(A) receptors in the modulation of peripheral nociceptive transmission.     

Corticotropin releasing factor (CRF) and age-related differences in behavior of mice

Aged rodents demonstrate a difference in response to the stress of a novel environment compared to young rodents. Corticotropin releasing factor (CRF) is involved in both the hormonal and motor response to stressful stimuli. The contribution of CRF to age-related differences in behavior was investigated by assessing the response to intracerebroventricular (ICV) injection of CRF in old (27 month) and mature (10 month) C57BL male mice. Open field behavior was also observed. Old mice demonstrated less locomotion and less rearing than mature mice (p less than 0.01). ICV injection of CRF increased corticosterone compared to vehicle control (p less than 0.001) without an age-related difference. CRF decreased food consumption (p less than 0.001) in all animals but had no effect on locomotion (p greater than 0.3); no significant age-related differences were observed. These findings suggest that age-related differences in response to environmental novelty are not due to increased release of CRF.     

Ultrastructural evidence for extravascular platelet recruitment in the lung upon intravenous injection of platelet-activating factor (PAF-acether) to guinea-pigs.

Bronchoconstriction and degenerative lesions of the bronchial epithelium were observed microscopically 1 min after the i.v. injection of 100 ng/kg of platelet-activating factor (PAF-acether) to the anaesthesized guinea-pig. Constricted arterioles containing marginated polymorphonuclear neutrophils and platelet aggregates were seen, as well as alveolar capillaries obstructed by thrombi formed by partially or totally degranulated platelets. Three minutes after the injection of PAF-acether, platelet diapedesis to the alveolar septa and lumen was clearly observed. Bronchoconstriction was still present at 3 min, but subsided after 60 min, whereas oedema of the submucosa persisted accompanied by an infiltration of eosinophils and neutrophils. The infusion of prostacyclin prevented the formation of platelet aggregates and platelet diapedesis due to PAF-acether, but the morphological evidence of bronchial constriction was not modified. Aspirin failed completely to modify the effects of PAF-acether. Our results show that PAF-acether causes early formation and deposition of platelet aggregates, accompanied by the margination of polymorphonuclear neutrophil leucocytes in pulmonary vessels of the guinea-pig. Since bronchoconstriction persisted when platelet aggregation was inhibited with prostacyclin, aggregation by itself would not account for this effect. Early platelet diapedesis in the vicinity of bronchial smooth muscle corroborates previous evidence that platelets contain and release bronchoconstrictor substances, which operate by cyclo-oxygenase-independent mechanisms and are possibly involved with the physiopathology of lung inflammation during immediate hypersensitivity.     

In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4⁺ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.     

Corticotropin-releasing factor type II (CRF-sub-2) receptors in the bed nucleus of the stria terminalis modulate conditioned defeat in Syrian hamsters (Mesocricetus auratus)

In Syrian hamsters (Mesocricetus auratus), social defeat produces a subsequent increase in submissive and defensive behavior and a loss of normal territorial aggression, which the authors have called conditioned defeat. In this study, the authors investigated the effect of blocking corticotropin-releasing factor (CRF) Type I and Type II receptors on conditioned defeat. Intracerebroventricular infusion of the CRF-sub-2 receptor antagonist antisauvagine-30 prior to testing significantly reduced conditioned defeat compared with vehicle controls, whereas the CRF-sub-1 receptor antagonist CP-154,526 had no effect. Also, infusion of antisauvagine-30 into the bed nucleus of the stria terminalis (BNST) 15 min, but not immediately, prior to testing reduced conditioned defeat in a dose-dependent manner. The authors' results provide evidence that CRF-sub-2 receptors in the BNST, but not CRF-sub-1 receptors, are an important component in the neural circuitry regulating conditioned defeat.     

Sensitivity of basophils to histamine releasing factor(s) of various origin: dependency on allergic phenotype of the donor and surface-bound IgE.

Certain species of histamine-releasing factor (HRF) have been demonstrated to distinguish a select group of allergic patients from healthy subjects. An IgE-dependent mechanism of action has been suggested. The donor and IgE dependency of HRF produced by peripheral blood mononuclear cells (PBMCs) has not been clearly demonstrated. In this study, we have compared the response of basophils from normal subjects versus allergic patients with and without asthma. In addition, we have addressed the IgE dependency of HRF recovered from cultures of PBMCs, T cells, B cells, macrophages, and bronchoalveolar lavage fluid. We have demonstrated that basophils from allergic as well as normal subjects respond to PBMC-HRF. The response of basophils from allergic patients with asthma is significantly increased. This heightened response to HRF does not correlate with the severity of disease as assessed by baseline spirometry, medication, and skin test scores. Stripping of the membrane-bound IgE by incubating basophils with lactic acid causes a significant loss of sensitivity to HRF generated by PBMCs, T cells, B cells, and macrophages, as well as to HRF recovered from bronchoalveolar fluid. The loss of response can be restored by sera from patients with asthma but not from normal subjects or by myeloma IgE. In addition, poorly responsive basophils from normal subjects can be rendered sensitive by incubating with sera from patients with asthma. The capacity of a given serum from a patient with asthma to restore the response to HRF is not correlated with the total concentration of IgE in the serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tea polyphenols inhibit acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and platelet-activating factor-induced platelet aggregation

BACKGROUND: Tea extracts have antiallergic and anti-inflammatory actions in rats and mice. However the mechanism through which tea polyphenols act in vivo are still incompletely understood. We found inhibitory effects of black tea extracts on an fMLP-induced aggregating response in a rabbit platelet-polymorphonuclear leukocyte (PMN) system. METHOD: To elucidate whether 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) production in PMNs and/or PAF-stimulated platelet activation were inhibited, the effects of tea polyphenols were investigated on the enzyme activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67), PAF biosynthesis in A23187-activated rabbit PMNs, and rabbit platelet aggregation. By comparing the inhibitory effects of 31 galloyl esters and gallic acid, the structure-inhibitory activity relationship was characterized. RESULTS: Theaflavin and its galloyl esters and pentagalloyl glucose were found to be potent inhibitors of the acetyltransferase (IC(50) = 28-58 microM) and the PAF biosynthesis as well as (-)-epicatechin-3-O-gallate (IC(50) = 72 +/- 13 microM) and (-)-epigallocatechin-3-O-gallate (IC(50) = 46 +/- 6 microM). On the other hand, flavan-3-ols without galloyl group at C-3 and gallic acid did not show significant enzyme inhibition. In addition, theaflavin and its galloyl esters (IC(50) = 32-77 microM) and geranyl gallate, farnesyl gallate and geranylgeranyl gallate (IC(50) = 6.4-7.6 microM) were found to be potent inhibitors of PAF- and TPA-induced rabbit platelet aggregation but not A23187-induced aggregation. CONCLUSIONS: Theaflavin and its galloyl esters in black tea extract, and isoprenyl gallates were potent inhibitors of PAF synthesis and platelet aggregation and these activities may be relevant to the claimed therapeutic effects of tea extracts.     

Evaluation of adenosine 5'-diphosphate (ADP)- and collagen-induced platelet aggregation in canine leishmaniasis

Leishmania-infected dogs, which represent an important reservoir of infection in many parts of the world, frequently suffer from haematological disorders, including thrombocytopenia. In this study, the ability of platelets from healthy (control) dogs (n = 11) and from dogs with naturally acquired clinical leishmaniasis (n = 24) to aggregate in the presence of two different agonists (adenosine 5'-diphosphate [ADP] and collagen) was assayed. Haematological parameters examined consisted of the platelet count, prothrombin time, activated partial thromboplastin time (aPTT), fibrinogen concentration and D-dimer concentration. In dogs with leishmaniasis, a significant decrease in ADP- and collagen-induced platelet aggregation was observed. Compared with platelets from the control dogs, those from leishmania-infected dogs showed a higher sensitivity to collagen, as demonstrated by a reduction in platelet aggregation of up to 20.4%, and a significant (P < 0.0001) difference for all the doses tested. With ADP the reduction was up to 10.4%, the difference reaching a significant level of P < 0.0001 only at the maximum dose used. The nature of this response, which was not accompanied by any clinical signs of bleeding other than an increase in aPTT, emphasizes the role of platelets in the parasite-host cell interaction.