Topical Application Of Leukotriene B4 Research Articles

Differential effect of 5- and 15-lipoxygenase products on ischemically sensitive abdominal visceral afferents

The effects of 5- and 15-lipoxygenase products, leukotriene B4 (LTB4) and (8R,15S)-dihydroxyeicosa(5E-9,11,13Z)tetraenoic acid (8R,15S-diHETE), on ischemically sensitive abdominal visceral C fiber afferents were evaluated, because this system is important in sensitizing cutaneous afferents. Single-unit activity of abdominal visceral C fiber afferents was recorded from the right thoracic sympathetic chain of anesthetized cats during 5 min of ischemia. Inhibition of 5-lipoxygenase with WY-50295 tromethamine (5 mg/kg iv) augmented the impulse activity from 0.48 +/- 0.15 to 0.79 +/- 0.24 impulses/s (P < 0.05) in seven ischemically sensitive afferents. Conversely, topical application of LTB4 (125 ng) directly onto the receptive field attenuated impulse activity of 10 ischemically sensitive C fiber afferents from 0.82 +/- 0.23 to 0.42 +/- 0.10 impulses/s (P < 0.05). In additional cats, application of 8R,15S-diHETE (125 ng) onto the receptive field augmented the impulse activity of nine ischemically sensitive C fiber afferents (from 0.48 +/- 0.15 to 0.70 +/- 0.15 impulses/s, P < 0.05) and significantly decreased the mechanical threshold of these nine afferents, whereas application of 8S,15S-diHETE (125 ng), a stereoisomer of 8R,15S-diHETE, attenuated the impulse activity from 0.77 +/- 0.48 to 0.45 +/- 0.13 impulses/s (P < 0.05) in six additional ischemically sensitive C fiber afferents. In animals pretreated with aspirin (50 mg/kg iv, n = 6) or 8S,15S-diHETE (125 ng, n = 6), WY-50295 tromethamine (5 mg/kg iv) still potentiated the impulse activity of ischemically sensitive C fiber afferents. These data indicate that 8R,15S-diHETE interacts with stereospecific receptors to sensitize, whereas LTB4 reduces, the response of abdominal visceral afferents to ischemia. Furthermore the data suggest that the augmented response of afferents to abdominal ischemia after inhibition of 5-lipoxygenase is, at least in part, independent of shunting to the cyclooxygenase or 15-lipoxygenase system.

The inter-relation between inflammation and epidermal proliferation in normal skin following epicutaneous application of leukotriene-B4--an immunohistochemical study.

Topical application of leukotriene-B4 (LTB4) on normal skin has been used as an in-vivo model to investigate cutaneous inflammation and epidermal proliferation, which are important phenomena in the pathogenesis of psoriasis. The aim of the present investigation is to further elucidate the interrelation between inflammation and epidermal proliferation, using specific monoclonal antibodies as markers for the different cell types involved. Aliquots of LTB4 were applied on the upperarms of eight healthy volunteers. After LTB4-application, biopsies were taken at consecutive time intervals. On frozen sections, epidermal proliferation was assessed by Ks8.12-(keratin 16) and Ki-67-binding (cycling cells), inflammation was characterized using anti-elastase (PMN), T11 (T-lymphocytes), pan-B (B-lymphocytes), WT 14 (CD14-positive cells) and OKT 6 (Langerhans cells). New observations were that the density of CD14-positive cells was increased even at 8 h and decreased slightly at 72 h. A striking rearrangement of Langerhans cells was seen in close vicinity to intra-epidermal accumulations of PMN. Remarkably an increased density of these cells in the dermis at 72 h was seen and a decrease in the epidermis. In line with previous studies, the accumulation of PMN reached a maximum 24 h after LTB4-challenge. The identity of the mononuclear infiltrate cells which have been reported 48-72 h after LTB4 proved to be T-lymphocytes. No B-lymphocytes were observed. Ki-67-positive nuclei were maximally increased 72 h after LTB4-application, which implies that recruitment of cycling cells is of relevance for the LTB4-induced proliferation in vivo. The hyperproliferation-related keratin 16 was expressed inconsistently in the suprabasal compartment.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition site of dexamethasone on extravasation of polymorphonuclear leukocytes in the hamster cheek pouch microcirculation.

A video system was used to investigate the inhibitory effect(s) of the glucocorticoid dexamethasone (DEX) on polymorphonuclear leukocyte (PMN) extravasation in the microcirculation of hamster cheek pouch. DEX was given intraperitoneally at 0.1 or 0.3 mg/kg body weight 2 h before induction of extravasation by topical application of leukotriene B4 (LTB4) or formyl-methionyl-leucyl-phenylalanine (fMLP) on the microvasculature. The number, time course, and behavior of PMNs were examined in the following five steps of extravasation: (1) rolling on the venular endothelium, (2) adhesion on the endothelium, (3) passage between the endothelial cells, (4) staying in the venular wall, and (5) migration from the venular wall into the interstitial space. In either the presence or absence of DEX, topical application of LTB4 (15 pmol/50 microliters) or fMLP (10 nmol/50 microliters) caused an increase in the number of PMNs that adhered to the venules. Thus, DEX did not inhibit the adhesion of PMNs on the endothelial cells. The adhered PMNs induced by these chemoattractants became gradually smaller, finally disappearing in the vascular lumen, as observed on the monitor screen. The whole process took about 10 min. This passage of PMNs between the endothelial cells was also not inhibited by DEX, as over 90% of the adhered PMNs passed through the endothelial cells and advanced to the next step of extravasation in the presence or absence of DEX. When these chemoattractants were applied to DEX-untreated animals, the PMNs that passed through the endothelial cells stayed for about 30 min in the venular wall. Thereafter, PMNs migrated into the interstitial space. The numbers of PMNs in the interstitial space were counted at 30, 60, and 90 min after the application of chemoattractants. In the DEX-untreated animals, PMNs in the interstitial space started to appear by 30 min, and thereafter the number further increased. However, in the DEX-treated animals, the number of PMNs at 60 and 90 min was significantly suppressed. Since DEX may inhibit the synthesis and/or release of a proteinase(s) in the PMN granules, which would normally degrade the basement membrane, this inhibition may be due to the inability of PMNs to penetrate the basement membrane.

微小循環における好中球血管外遊走機構

A series of the process of neutrophil extravasation induced by topical application of leukotriene B4 (LTB4) or formyl-methionyl-leucyl-phenylalanine (fMLP) on microvasculature of the hamster cheek pouch can be divided into five steps; I) rolling on the venular endothelium, II) adhesion on the endothelium, III) passage between the endothelial cells, IV) staying in the venular wall, V) migration from the venular wall to the interstitial space. In step II, topical application of chemoattractants on the microvasculature caused an increase in the number of neutrophils adhered to the venules. In step III, the size of the adhered neutrophils became gradually smaller and finallly disappear from the vascular lumen, as they were observed on the monitor screen. Over 80-90% of the adhered neutrophils passed through the endothelial cells. The whole process from step II to III took about 7 minutes. In step IV, the neutrophils which passed through the endothelial cells stay for about 30 minutes in the venular wall. In step V, neutrophils penetrated the basement membrane and migrated into the interstitial space.Step II and III were suppressed by a cyclic AMP phosphodiesterase inhibitor, fibronectin inhibitor and cytochalasin B. From these results, it was suggested that chemoattractant may cause the contraction of microfilament of neutrophil, and the contraction may induce the expression of adhesive glycoprotein on the neutrophil membrane. The increase of cyclic AMP in the neutrophil may inhibit the contraction of microfilament and/or the expression of adhesive glycoprotein of neutrophil. Dexamethasone did not affect the step I to IV, but suppressed the step V. Dexamethasone may inhibit synthesis and/or release of protease (s) in the neutrophil granules which degrade the basement membrane. In fact, the collagenase inhibitor suppressed the step V, although it did not the step I to IV. Thus, neutophil-derived collagenase may act on the passage of neutrophil through the basement membrane. The process of neutrophil extravasation is the chain of reaction.

Leukotriene B4-induced permeability increase in postcapillary venules and its inhibition by three different antiinflammatory drugs

Leukotriene B4 (LTB4) is a potent chemotactic and chemokinetic substance that causes leukocyte accumulation and increases vascular permeability. We have used the hamster cheek pouch model to study the effect of some different antiinflammatory drugs on the LTB4-induced microvascular permeability. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular leakage, which was measured as the number of venular leaky sites and as efflux of the vascular tracer. Topical application of LTB4 (10(-8) M, 5 min) resulted in a reversible increase of the vascular permeability and could be repeated with 40-min intervals without any significant reduction in permeability response. The LTB4-induced response could be inhibited by budesonide (a glucocorticoid) given locally, by iloprost (a prostacyclin analog) given intravenously and by a combined local and intravenous treatment with terbutaline (a beta 2-receptor agonist) but not by local terbutaline treatment only.

Characteristics of the Cellular Response of the Rat Conjunctiva to Topically Applied Leukotriene B4

Cellular events in the rat conjunctiva were studied at 1, 6 and 24 h following the topical application of leukotriene B4. A significant increase was noted in the number of eosinophils (p less than 0.01) and neutrophils (p less than 0.05) at 1 h and only in eosinophils at 6 h after leukotriene B4 application at the 2-micrograms dose. At the 200-ng dose, the eosinophil count was significantly elevated at 1 h only, and the number of neutrophils was not significantly changed at any time point. No significant increase in cell counts was observed at the 20-ng dose. In conclusion, certain doses of topically applied leukotriene B4 significantly increased the number of eosinophils and neutrophils in the rat conjunctiva.

Response of polymorphonuclear leukocytes to topical leukotriene B4 in healthy and psoriatic skin.

The in situ infiltration of polymorphonuclear leukocytes following the topical application of leukotriene B4 to the clinically uninvolved skin of psoriatic patients and the skin of normal controls has been quantified using elastase as a marker enzyme. The magnitude of this response in the psoriatic patients proved to be significantly diminished compared with the response in the controls. The time course of the response was similar in both groups, maximum elastase activity being seen about 18 h after application of the LTB4.

The perivascular cell populations in human skin after topical application of leukotriene B4

Fifteen timed biopsies of human skin were performed after application of 100 nanograms of leukotriene B4 in a Finnchamber for six hours. The number of inflammatory cells were counted per high power perivascular field and compared to three control biopsies. At 24 hours a peak of neutrophiles was observed and subsequently lymphocytes predominated. Eosinophils were never prominent. This pattern of successive cell populations has been described in inflammatory and whealing skin disease.

Methotrexate inhibits the leukotriene B4 induced intraepidermal accumulation of polymorphonuclear leukocytes.

The penetration of polymorphonuclear leukocytes (PMNs) into the epidermis following topical application of leukotriene B4 was assessed in the clinically uninvolved skin of psoriatic patients treated with methotrexate and of psoriatic patients without treatment, and in normal controls. Inhibition of PMN infiltration was observed in those patients treated with methotrexate while there was no significant difference between untreated psoriatic patients and normal controls.

Production of Intraepidermal Microabscesses by Topical Application of Leukotriene B4

Leukotriene B4 is a highly potent leukocyte chemotactic compound. It has been identified in chamber fluid and scale from psoriatic skin lesions, in which epidermal neutrophil infiltration is reported to be one of the earliest pathologic events. The ability of leukotriene B4 to reproduce the inflammatory events of psoriasis, by topical application to the skin of normal human volunteers, was thus studied. Persistent visible inflammatory reactions were elicited by application of amounts of leukotriene B4 as low as 5 ng, and the maximum diameters of the reactions were dose-related up to at least 500 ng. The visible reactions appeared 12-24 h after initial application of leukotriene B4, and persisted for several days, leaving brownish pigmentation and scaling at 7 days. Histologic examination showed intraepidermal neutrophil microabscesses at 24 h, but these had resolved by 48 h. A mixed, perivascular neutrophil and mononuclear cell infiltrate was seen in the dermis at 24 h, becoming predominantly mononuclear after 24 h. Nonspecific chemical irritant contact dermatitis was excluded by the absence of reactions to high doses of two chemically similar metabolites of arachidonic acid which lack significant in vitro chemokinetic activity. These experiments provide further evidence for the role of leukotriene B4 in the pathogenesis of psoriasis, and may lead to the development of an experimental model of the inflammatory events in psoriasis, and of a simple in vivo test of neutrophil function.

Increase in vascular permeability induced by leukotriene B4 and the role of polymorphonuclear leukocytes.

Leukotriene B4 (LTB4), a metabolite of arachidonic acid, is known to be a potent chemotactic and chemokinetic substance. We have used the hamster cheek pouch microcirculation model to study the effect of LTB4 on vascular permeability and the involvement of neutrophil granulocytes in this response. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular permeability. Topical application of LTB4 (150 nM-5 microM) to the hamster cheek pouch resulted in an immediate increase in adhering leukocytes in postcapillary venules and later venules. Leukocyte accumulation was reversible, but continued longer the higher the dose of LTB4 used. Subsequently, a dose-dependent increase in vascular permeability was seen at post-capillary and larger venules, with a maximum 10-20 min after application; the maximum occurred later the higher the dose of LTB4. Depletion of neutrophil granulocytes by pretreatment of the animals with antineutrophil serum obtained from immunized rabbits significantly decreased the permeability response to LTB4, whereas the response to histamine was unaffected. These results suggest that neutrophil granulocytes play a role in LTB4-mediated permeability increase. LTB4 may be of importance both for the leukocyte accumulation and for the edema formation seen in inflammatory reactions.