Anti-platelet effects of anti-glaucomatous eye drops: an in vitro study on human platelets.

PURPOSE: Altered platelet aggregability has been implicated in the pathogenesis of glaucoma. This study aims to investigate the anti-platelet potential of intraocular pressure lowering drops, with the possibility of establishing it as an additional mechanism of anti-glaucomatous action. MATERIALS AND METHODS: The anti-aggregating effects of a series of anti-glaucomatous eye drops were determined on human platelets in the platelet aggregation model, using four known aggregating factors (platelet activating factor [PAF], adenosine diphosphate [ADP], thrombin receptor-activating peptide [TRAP], and arachidonic acid [AA]). RESULTS: Almost all of the tested samples inhibited platelet aggregation induced by PAF, ADP, TRAP, and AA, except for Alphagan, which did not demonstrate inhibition of ADP- and TRAP-induced aggregation at a wide range of concentrations. Trusopt, Betoptic, and Azarga eye drops were the most potent inhibitors of all four aggregating factors, while Alphagan was the least potent (P<0.05). CONCLUSION: This study shows that anti-glaucomatous eye drops possess anti-platelet effects, and this was shown for the first time by experimenting on human platelets.

Lipopolysaccharide Cross-Tolerance Delays Platelet-Activating Factor-Induced Sudden Death in Swiss Albino Mice: Involvement of Cyclooxygenase in Cross-Tolerance.

Lipopolysaccharide (LPS) signaling through Toll-like receptor-4 (TLR-4) has been implicated in the pathogenesis of many infectious diseases. Some believe that TLR-mediated pathogenicity is due, in part, to the lipid pro-inflammatory mediator platelet-activating factor (PAF), but this has been questioned. To test the direct contribution of PAF in endotoxemia in murine models, we injected PAF intraperitoneally into Swiss albino mice in the presence and absence of LPS. PAF alone (5 µg/mouse) caused death within 15-20 min, but this could be prevented by pretreating mice with PAF-receptor (PAF-R) antagonists or PAF-acetylhydrolase (PAF-AH). A low dose of LPS (5 mg/kg body wt) did not impair PAF-induced death, whereas higher doses (10 or 20 mg/kg body wt) delayed death, probably via LPS cross-tolerance. Cross-tolerance occurred only when PAF was injected simultaneously with LPS or within 30 min of LPS injection. Tolerance does not appear to be due to an abundant soluble mediator. Histologic examination of lungs and liver and measurement of circulating TNF-α and IL-10 levels suggested that the inflammatory response is not diminished during cross-tolerance. Interestingly, aspirin, a non-specific cyclooxygenase (COX) inhibitor, partially blocked PAF-induced sudden death, whereas NS-398, a specific COX-2 inhibitor, completely protected mice from the lethal effects of PAF. Both COX inhibitors (at 20 mg/kg body wt) independently amplified the cross-tolerance exerted by higher dose of LPS, suggesting that COX-derived eicosanoids may be involved in these events. Thus, PAF does not seem to have a protective role in endotoxemia, but its effects are delayed by LPS in a COX-sensitive way. These findings are likely to shed light on basic aspects of the endotoxin cross-tolerance occurring in many disease conditions and may offer new opportunities for clinical intervention.

The responses of pulmonary and systemic circulation and airway to anaphylactic mediators in anesthetized BALB/c mice.

AIMS: Anaphylactic shock sometimes accompanies pulmonary vaso- and broncho-constriction. We previously reported the hemodynamic features of mouse anaphylaxis (Life Sci. 2014; 116: 98-105). However, the effects of anaphylactic chemical mediators on the hemodynamics of in vivo mice are not well known. Furthermore, it is uncertain whether the mediators exert the same directional actions. Therefore, we determined their effects systematically on total peripheral resistance (TPR), pulmonary vascular resistance (PVR), or airway pressure (AWP) in anesthetized mice. MAIN METHODS: We measured directly pulmonary arterial pressure, left atrial pressure, systemic arterial pressure, central venous pressure and aortic blood flow to determine PVR and TPR, as well as AWP, following injections of platelet-activating factor (PAF), histamine, serotonin, leukotriene (LT) C4, and prostaglandin (PG) D2 in anesthetized open-chest artificially ventilated BALB/c mice. KEY FINDINGS: Consecutive administration of any agents increased PVR dose-dependently with the maximal responsiveness being PAF>LTC4>serotonin>>histamine=PGD2. Histamine caused a biphasic PVR response, an initial decrease, which was abolished by L-NAME, followed by an increase at high doses. PAF, serotonin, and histamine decreased TPR dose-dependently, while LTC4 or PGD2 yielded an increase or no change in TPR, respectively. Serotonin, but not the other agents, increased AWP. SIGNIFICANCE: Anaphylactic mediators exert non-uniform actions on the pulmonary and systemic circulation and airway in anesthetized BALB/c mice: PAF, LTC4 and serotonin cause substantial pulmonary vasoconstriction, while histamine biphasic responses of the initial nitric oxide dependent vasodilation followed by vasoconstriction; PAF, serotonin, and histamine, but not LTC4 or PGD2, evoke systemic vasodilatation; only serotonin induces airway constriction.

Quinidine, but not eicosanoid antagonists or dexamethasone, protect the gut from platelet activating factor-induced vasoconstriction, edema and paralysis.

Intestinal circulatory disturbances, atony, edema and swelling are of great clinical relevance, but the related mechanisms and possible therapeutic options are poorly characterized, in part because of the difficulties to comprehensively analyze these conditions. To overcome these limitations we have developed a model of the isolated perfused rat small intestine where all of these symptoms can be studied simultaneously. Here we used this model to study the role of eicosanoids, steroids and quinidine in platelet-activating factor (PAF)-induced intestinal disorders. A vascular bolus of PAF (0.5 nmol) triggered release of thromboxane and peptidoleukotrienes into the vascular bed (peak concentration 35 nM and 0.8 nM) and reproduced all symptoms of intestinal failure: mesenteric vasoconstriction, translocation of fluid and macromolecules from the vasculature to the lumen and lymphatics, intestinal edema formation, loss of intestinal peristalsis and decreased galactose uptake. All effects of PAF were abolished by the PAF-receptor antagonist ABT491 (2.5 µM). The COX and LOX inhibitors ASA and AA861 (500 µM, 10 µM) did not exhibit barrier-protective effects and the eicosanoid antagonists SQ29548 and MK571 (10 µM, each) only moderately attenuated the loss of vascular fluid, the redistribution to the lumen and the transfer of FITC dextran to the lumen. The steroid dexamethasone (10 µM) showed no barrier-protective properties and failed to prevent edema formation. Quinidine (100 µM) inhibited the increase in arterial pressure, stabilized all the intestinal barriers, and reduced lymph production and the transfer of FITC dextran to the lymph. While quinidine by itself reduced peristalsis, it also obviated paralysis, preserved intestinal functions and prevented edema formation. We conclude that quinidine exerts multiple protective effects against vasoconstriction, edema formation and paralysis in the intestine. The therapeutic use of quinidine for intestinal ailments deserves further study.

C-reactive protein specifically enhances platelet-activating factor-induced inflammatory activity in vivo.

Platelet-activating factor (PAF) is a potent lipid mediator that is implicated in numerous inflammatory diseases. C-reactive protein (CRP) is an acute-phase plasma protein that increases rapidly and dramatically in response to inflammation. In this study, we investigated the effect of the interaction between CRP and PAF on inflammatory responses in vivo. From binding analysis using a time-resolved fluorometric assay, CRP bound to PAF and its precursor/metabolite lyso-PAF in a concentration-dependent manner. In addition, CRP bound to several phospholipids containing lysophosphatidylcholine, which bears structural resemblance to PAF and lyso-PAF, sphingosylphosphorylcholine, and lysophosphatidylethanolamine more readily than to lysophosphatidic acid and lysophosphatidylserine. In in vivo experiments using a rat model of hind paw oedema, CRP increased PAF-induced rat paw oedema in a dose-dependent manner, without causing the oedema itself, but it did not increase histamine and serotonin-induced paw oedema. Furthermore, the receptor for CRP, lectin-like oxidized low-density lipoprotein receptor 1 was not involved in the increase in PAF-induced inflammatory responses caused by CRP. These results indicate that CRP can specifically enhance PAF-induced inflammatory activity through binding to PAF and lyso-PAF. Therefore, CRP may accelerate the pathogenesis of numerous inflammatory diseases caused by PAF.

Platelet-activating factor nasal challenge induces nasal congestion and reduces nasal volume in both healthy volunteers and allergic rhinitis patients.

BACKGROUND: Platelet-activating factor (PAF) is a lipid mediator produced by most inflammatory cells. Clinical and experimental findings suggest that PAF participates in allergic rhinitis (AR) pathogenesis. The aim was to assess the PAF ability to induce clinical response in nasal airway after local stimulation. METHOD: Ten nonatopic healthy volunteers (HVs) and 10 AR patients out of pollen season were enrolled. PAF increasing concentrations (100, 200, and 400 nM) were instilled into both nasal cavities (0, 30, and 60 minutes, respectively). Nasal symptoms (congestion, rhinorrhea, sneezing, itching, and total 4 symptom score and nasal volume between the 2nd and 5th cm (Vol(2-5)) using acoustic rhinometry (AcR), were assessed at -30, 0, 30, 60, 90, 120, and 240 minutes. RESULT: PAF increased individual and total nasal symptom score in both HVs and seasonal AR (SAR) patients from 30 to 120 minutes (maximum score at 120', p < 0.05). Nasal obstruction was the most relevant and lasting nasal symptom. PAF also induced a significant reduction of Vol(2-5) at 90' (27%), 120' (38.7%), and 240' (36.4%). No differences in the response to PAF nasal challenge were observed between HVs and SAR subjects in either clinical symptoms or AcR. CONCLUSION: This is the first description of PAF effects on human nasal mucosa using a cumulative dose schedule and evaluated by both nasal symptoms and AcR. Nasal provocation with PAF showed long-lasting effects on nasal symptoms and nasal obstruction in HVs and in patients with SAR. Nasal challenge may be a useful tool to investigate the role of PAF in AR and the potential role of anti-PAF drugs.

The role of PAF/PAFR signaling in zymosan-induced articular inflammatory hyperalgesia.

Platelet-activating factor (PAF) and its receptor (PAFR) have been shown to be involved in several inflammatory events, including neutrophil chemoattraction and nociception. The present study addressed the role of PAF in the genesis of articular hyperalgesia in a model of joint inflammation. Zymosan-induced articular hyperalgesia, oedema and neutrophil migration were dose-dependently reduced following pretreatment with selective PAFR antagonists, UK74505 (5, 10 and 20 mg/kg) and PCA4248 (3, 10, 30 mg/kg). These parameters were also reduced in PAF receptor-deficient mice (PAFR(-/-)). The hyperalgesic action of PAF was further confirmed by the demonstration that joint injection of PAF induces a dose- (0.3, 1 and 3 µg/joint), time- and PAFR-dependent articular hyperalgesia and oedema. The PAF hyperalgesic mechanisms were dependent on prostaglandins, leukotrienes and neutrophils, as PAF-induced articular hyperalgesia was inhibited by indomethacin (COX inhibitor), MK886 (leukotrienes synthesis inhibitor) or fucoidan (leukocyte rolling inhibitor). Furthermore, PAF-induced hyperalgesia was reduced in 5-lypoxigenase-null mice. In corroboration of these findings, intra-articular injection of PAF promotes the production of LTB(4) as well as the recruitment of neutrophils to the joint. These results suggest that PAF may participate in the cascade of events involved in the genesis of articular inflammatory hyperalgesia via stimulation of prostaglandins, leukotrienes and neutrophil migration. Finally, targeting PAF action (e.g., with a PAFR antagonist) might provide a useful therapeutic approach to inhibit articular inflammatory hyperalgesia.

[Study on pharmacokinetics of ginkgolide B injection in Beagle dogs].

OBJECTIVE: To study the pharmacokinetics of ginkgolide B injection in Beagle dogs. METHODS: Determined the serum concentration of ginkgolide B by LC-MS and calculated its parameter of pharmacokinetics via DAS 2.0 software. RESULTS: After intravenous drips of 0.62, 2.07 and 10.35 mg/kg ginkgolide B, parameters of pharmacokinetics of ginkgolide B were as follows: Tmax were 0.444, 1, 1 h; Cmax were 0.764, 3.024, 11.013 mg/L; AUC(0-1) were 1.007, 3.644, 16.646 mg x h/Lo. CONCLUSION: Ginkgolide B has two compartment model in Beagle dogs.

The significance of endothelin in platelet-activating factor-induced fetal growth restriction.

The significance of endothelin-1 (ET-1) in platelet-activating factor (PAF)-induced fetal growth restriction (FGR) was evaluated in timed-pregnant rats receiving intravenous carbamyl-PAF (c-PAF; 0.5, 1.0, or 2.5 µg/kg per h) or vehicle, with or without ET-1 receptor A (ET(A)) antagonist (10 or 20 mg/kg per d) for 7 days beginning on gestation day 14. Tissues were collected on day 21. Carbamyl-PAF reduced fetal weights dose dependently. Placental weights were significantly reduced but not dose dependently. ET(A) antagonism prevented FGR at the 0.5, but not the 1.0 and 2.5 µg/kg per h c-PAF doses. Correspondingly, placental, but not uterine, preproET-1 messenger RNA (mRNA) expression (determined by reverse transcription-polymerase chain reaction) was increased at 0.5 µg/kg per h but not at higher c-PAF doses. In summary, c-PAF infusion results in fetal and placental growth restriction in the rat. At low doses of c-PAF, ET-1 is central to the pathophysiology of PAF-induced FGR. At higher c-PAF doses, FGR is induced by mechanisms other than ET-1 action.

The histopathological effect of thymoquinone on experimentally induced rhinosinusitis in rats.

BACKGROUND: Rhinosinusitis is a common disorder and its treatment includes a variety of topical and systemic drugs. This study was designed to determine the histopathological effect of thymoquinone on experimentally induced rhinosinusitis in rats. METHODS: Sixty rats were randomly allocated into 3 test and 2 control groups, each of which consisted of 12 animals. The rhinosinusitis model was induced using intranasal application of platelet-activating factor. In test groups, the animals were separated into groups: (1) rhinosinusitis-antibiotherapy, (2) rhinosinusitis-thymoquinone, (3) rhinosinusitis-combination therapy. The positive and negative control groups were defined: rhinosinusitis group without any treatment and the group without rhinosinusitis, respectively. The histopathological features (vascular congestion, inflammation, and epithelial injury) in nasal respiratory and olfactory mucosa of animals were examined and graded according to their severity. A quantitative and statistical analysis of histopathological features was performed. RESULTS: All histopathological features showed statistically significant differences between negative and positive control groups, respectively. Conversely, neither the group with rhinosinusitis-antibiotherapy nor the group with rhinosinusitis-thymoquinone had a statistically significant difference with the negative control group. Moreover, none of the histopathological features showed a statistically significant difference, when the group with rhinosinusitis-antibiotherapy and the group with rhinosinusitis-thymoquinone were compared. A statistically significant difference was not determined when the group with rhinosinusitis-combination therapy was compared with the group with rhinosinusitis-thymoquinone. The histopathological features did not show a statistically significant difference between the group with combination therapy and the negative control Conclusion: Thymoquinone is a promising bioactive agent for the treatment of rhinosinusitis, and its histopathological effect is as equivalent as an antibiotic.

Differential effect of platelet activating factor on 1-methyl-4-phenylpyridinium-induced cell death through regulation of apoptosis-related protein activation.

Platelet activating factor (PAF) has been suggested to play a critical role in the pathogenesis of neurological disorders. We assessed the effect of PAF against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), a parkinsonian toxin, in relation to apoptotic process. PAF exhibited differential effect against the MPP(+) toxicity in differentiated PC12 cells depending on concentration. Treatment with 0.75 microM PAF significantly attenuated the MPP(+)-induced increase in Bax levels, decrease in Bid and Bcl-2 levels, and mitochondrial membrane potential loss that lead to the release of cytochrome c and subsequent caspase-3 activation. The inhibitory effect of PAF was not associated with nuclear factor-kappaB activation. In contrast, PAF at the concentrations greater than 2.5 microM exhibited a toxicity and additive effect on the MPP(+) toxicity. The results show that PAF at low concentrations, which does not induce a significant toxicity, may prevent the MPP(+) toxicity by suppressing the apoptosis-related protein activation and mitochondrial membrane permeability change that lead to the cytochrome c release and caspase-3 activation. The preventive effect seems to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH. In contrast, PAF at higher concentrations may exhibit an additive toxic effect against the MPP(+) toxicity by increasing apoptosis-related protein activation.

Platelet-activating factor-induced intracellular signaling and release of cytokines and prostaglandin E2 in immortalized human corneal epithelial cells.

PURPOSE AND METHODS: Immortalized human corneal epithelial (CEPI-17-CL4) cells were exposed to different concentrations of platelet-activating factor (PAF) and the mobilization of intracellular calcium ([Ca(2+)](i)) was studied using fluorometrics. Additionally, the production of the cytokines [interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha)], and matrix metalloproteinase-1 (MMP-1) and prostaglandin E(2) (PGE(2)) from PAF-stimulated cells was also determined using ELISA assays. RESULTS: While PAF, histamine, and bradykinin stimulated the mobilization of [Ca(2+)](i) in these cells, PAF was the least efficacious. [Ca(2+)](i) mobilization induced by PAF was inhibited by 2 PAF receptor antagonists, PCA-42481 and CV-6209 (both 10 microM), and by a phospholipase C inhibitor, U73122 (60% at 4 microM). PAF increased the production of PGE(2) (with maximum effect at 30 and 100 nM) and GM-CSF (maximum effect at 1 microM) in CEPI-17-CL4 cells. However, PAF did not stimulate the generation of IL-6, IL-8, TNF-alpha, and MMP-1 to any significant level and in a consistent manner. PAF increased the incorporation of [(3)H]-thymidine into CEPI-17-CL4 cells with a maximal effect at 30 nM. CONCLUSIONS: These data indicate that functional PAF receptors are present on CEPI-17-CL4 cells that can activate mobilization of [Ca(2+)](i). Other consequences of PAF receptor activation in CEPI-17-CL4 cells are the generation of PGE(2) and certain proinflammatory cytokines such as GM-CSF, and increasing cell proliferation.

The novel role of platelet-activating factor in protecting mice against lipopolysaccharide-induced endotoxic shock.

BACKGROUND: Platelet-activating factor (PAF) has been long believed to be associated with many pathophysiological processes during septic shock. Here we present novel activities for PAF in protecting mice against LPS-mediated endotoxic shock. PRINCIPAL FINDINGS: In vivo PAF treatment immediately after LPS challenge markedly improved the survival rate against mortality from endotoxic shock. Administration of PAF prominently attenuated LPS-induced organ injury, including profound hypotension, excessive polymorphonuclear neutrophil infiltration, and severe multiple organ failure. In addition, PAF treatment protects against LPS-induced lymphocytes apoptosis. These protective effects of PAF was correlated with significantly decreases in the production of the inflammatory mediators such as TNF-alpha, IL-1beta, IL-12, and IFN-gamma, while increasing production of the anti-inflammatory cytokine IL-10 in vivo and in vitro. CONCLUSIONS: Taken together, these results suggest that PAF may protect mice against endotoxic shock via a complex mechanism involving modulation of inflammatory and anti-inflammatory mediators.

Intravenous infusion of PAF affects ovulation, fertilization and preimplantation embryonic development in NZB x NZW F1 hybrid mice.

Platelet Activating Factor (PAF) is a bioactive phospholipid, which exhibits a variety of biological activities and plays a significant role in all aspects of reproduction. In this work, a single intravenous injection of various concentrations of PAF shortly after Human Chorionic Gonadotropin (HCG) administration as well as 24 and 48 h before HCG administration was studied in NZB x NZW F1 hybrid mice. Optimum results were observed when PAF was injected just after the administration of HCG. In this protocol, the concentrations of PAF exhibited bell-shaped response to every stage of development. Any concentration of PAF between 5.5 x 10(-11) and 5.5 x 10(-15)g/g b.w., caused an improved ovulation rate, an increased fertilization rate, an increased rate of cell cycle and an enhanced hatching blastocyst rate (P<0.05 for all stages). Injection of lyso-PAF had no effect in any stage. Our data show that the effect of PAF on early stages of embryo development in vitro is dependent on its way of administration, on the concentrations used as well as on the time PAF is injected.

Effects of intracerebroventricular injections of free fatty acids, lysophospholipids, or platelet activating factor in a mouse model of orofacial pain.

The present study was carried out to determine the effects of central nervous free fatty acids, lysophospholipids, or platelet activating factor (PAF), in a mouse facial carrageenan injection model of orofacial pain. Mice that received intracerebroventricular (I.C.V.) injection of arachidonic acid or oleic acid showed significantly reduced allodynia and behavioral responses to von Frey hair stimulation of a carrageenan-injected area of the face, at 8 h post-injection, compared to controls that received I.C.V. injection of vehicle. In contrast to free fatty acids, increased responses were observed in mice at 72 h after I.C.V. lysophosphatidic acid or lysophosphatidylcholine injection, and at 8 and 24 h after PAF injection, compared vehicle injected controls. Information regarding pro-nociceptive effect of specific brain lipids may be a useful basis for further studies to explore mechanism.

Lipoxin A4 inhibits acute edema in mice: implications for the anti-edematogenic mechanism induced by aspirin.

Lipoxin A4 (LXA4) is a lipid mediator that plays an important role in the resolution of inflammation. However, the role of LXA4 and aspirin (ASA)-triggered lipoxins (ATLs) in inflammatory edema formation remains unclear. Here, we investigated the inhibitory role played by LXA4 in the carrageenan-induced and other inflammatory mediator-induced edematogenic response in mice, and also assessed the role of ATLs in the anti-edematogenic action of aspirin. Our results showed that LXA4 (1-20 ng/paw or 5 microg/kg i.p.) was effective in inhibiting carrageenan-induced paw edema from 30 min to 2 h. LXA4 (10 ng/paw) was also able to acutely inhibit PAF-, histamine-, PGE2- or bradykinin-induced paw edema, as well as the PAF-induced myeloperoxidase activity increase in the paws. Likewise, LXA4 (10 ng/cavity) also inhibited the pleural edema triggered by histamine (1h), and this response was not followed by leukocyte accumulation. Of note, the lipoxin receptor (ALX-r) antagonist Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe, 200 ng/paw) significantly reverted the anti-edematogenic effect of ASA (300 mg/kg p.o.) against carrageenan, PAF, PGE2 and BK, without affecting the anti-edematogenic action caused by indomethacin (3 mg/kg i.p.) in the carrageenan-induced paw edema. Collectively, our results demonstrate for the first time that LXA4 displays an acute and rapid onset anti-edematogenic activity that does not discriminate among different pro-inflammatory stimuli, an effect that is most likely independent of its action on the leukocyte influx. Finally, the present study demonstrates that ATLs exert a very important role in the acute anti-edematogenic action of ASA.

Mitogen-activated protein kinases regulate platelet-activating factor-induced hyperpermeability.

OBJECTIVE: The authors tested the hypothesis that p42/44- (ERK-1/2) and/or p38-mitogen-activated protein kinases (MAPK) are in vivo regulatory elements in the platelet-activating factor (PAF) activated signaling cascade that stimulates microvascular hyperpermeability. METHODS: FITC-dextran 70 was used as the macromolecular tracer for microvascular permeability in the mouse mesenteric fat tissue. Interstitial integrated optical intensity (IOI) was used as an index of permeability. RESULTS: An application of 10(-7) M PAF increased IOI from 23.1 +/- 3.6 to 70.8 +/- 7.4 (mean +/- SEM). Inhibition of ERK-1/2 with 3 microM and 30 microM AG126 reduced IOI to 32.3 +/- 2.5. Similarly, inhibition of p38-MAPK with 6 nM, 60 nM and 600 nM SB203580 lowered IOI to 29.1 +/- 2.4. CONCLUSIONS: The results demonstrate that ERK-1/2 and p38MAPK participate in the signaling cascade that regulates PAF-induced microvascular hyperpermeability in vivo.

Staphylococcal lipoteichoic acid inhibits delayed-type hypersensitivity reactions via the platelet-activating factor receptor.

Staphylococcus aureus infections are known triggers for skin inflammation and can modulate immune responses. The present studies used model systems consisting of platelet-activating factor receptor-positive and -negative (PAF-R-positive and -negative) cells and PAF-R-deficient mice to demonstrate that staphylococcal lipoteichoic acid (LTA), a constituent of Gram-positive bacteria cell walls, acts as a PAF-R agonist. We show that LTA stimulates an immediate intracellular Ca2+ flux only in PAF-R-positive cells. Intradermal injections of LTA and the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine (CPAF) induced cutaneous inflammation in wild-type but not PAF-R-deficient mice. Systemic exposure to LTA or CPAF inhibited delayed-type hypersensitivity (DTH) reactions to the chemical dinitrofluorobenzene only in PAF-R-expressing mice. The inhibition of DTH reactions was abrogated by the addition of neutralizing antibodies to IL-10. Finally, we measured levels of LTA that were adequate to stimulate PAF-R in vitro on the skin of subjects with infected atopic dermatitis. Based on these studies, we propose that LTA exerts immunomodulatory effects via the PAF-R through production of the Th2 cytokine IL-10. These findings show a novel mechanism by which staphylococcal infections can inhibit Th1 reactions and thus worsen Th2 skin diseases, such as atopic dermatitis.

Annexin 1-deficient neutrophils exhibit enhanced transmigration in vivo and increased responsiveness in vitro.

The role of the endogenous anti-inflammatory mediator annexin 1 (AnxA1) in controlling polymorphonuclear leukocyte (PMN) trafficking and activation was addressed using the recently generated AnxA1 null mouse. In the zymosan peritonitis model, AnxA1 null mice displayed a higher degree (50-70%) of PMN recruitment compared with wild-type littermate mice, and this was associated with reduced numbers of F4/80+ cells. Intravital microscopy analysis of the cremaster microcirculation inflamed by zymosan (6 h time-point) indicated a greater extent of leukocyte emigration, but not rolling or adhesion, in AnxA1 null mice. Real-time analysis of the cremaster microcirculation did not show spontaneous activation in the absence of AnxA1; however, superfusion with a direct-acting PMN activator (1 nM platelet-activating factor) revealed a subtle yet significant increase in leukocyte emigration, but not rolling or adhesion, in this genotype. Changes in the microcirculation were not secondary to alterations in hemodynamic parameters. The phenotype of the AnxA1 null PMN was investigated in two in vitro assays of cell activation (CD11b membrane expression and chemotaxis): the data obtained indicated a higher degree of cellular responses irrespective of the stimulus used. In conclusion, we have used a combination of inflammatory protocols and in vitro assays to address the specific counter-regulatory role of endogenous AnxA1, demonstrating its inhibitory control on PMN activation and the consequent impact on the inflamed microcirculation.

Rat model of platelet-activating factor-induced rhinosinusitis.

Platelet-activating factor (PAF) is known to be a potent inflammatory mediator, especially in allergic inflammation. However, the exact role of PAF in the pathogenesis of rhinosinusitis has not been clearly established. To understand the role of PAF in the pathogenesis of rhinosinusitis, it is necessary to develop an animal model of PAF-induced rhinosinusitis. The aim of this study was to develop a rat model of rhinosinusitis induced by intranasally applied PAF. Fifty microliters of 16 microg/mL PAF was applied intranasally through each naris in 4-week-old Sprague-Dawley rats, and the same amount of vehicle was applied in control rats. At 1, 3, or 5 days, the animals were painlessly sacrificed, and the nasal cavity and sinuses were prepared for histologic investigation. The histologic sections were examined in a blind manner for the appearance of neutrophil clusters in the sinonasal air space, and the numbers of eosinophils, areas of epithelial loss, goblet cells, and inducible nitric oxide synthase (iNOS)-positive inflammatory cells in the mucosa. Neutrophil clusters were observed in the air space, and the number of eosinophils, areas of epithelial loss, goblet cells, and iNOS-positive inflammatory cells in the mucosa were increased significantly in the PAF-applied rats. The amount of inflammation varied according to the time interval, showing a peak at day 3. We conclude that intranasally applied PAF induces rhinosinusitis in rats. The histologic evidence of rhinosinusitis revealed the appearance of neutrophil clusters in the sinonasal air space, infiltration of eosinophils and iNOS-positive inflammatory cells in the mucosa, areas of epithelial loss, and goblet cell hyperplasia in the epithelium. This rat model of PAF-induced rhinosinusitis may be applied for better understanding of the role of PAF in the pathogenesis of rhinosinusitis.