Graham R. Elliott

Skin irritants and contact sensitizers induce Langerhans cell migration and maturation at irritant concentration

Abstract:  Skin irritants and contact allergens reduce the number of Langerhans cells (LCs). It has been assumed that this reduction is due their migration to the draining lymph node (LN) for initiating immune sensitization in a host. Skin irritation, however, as opposed to contact allergy is not considered to be an immunological disease. Nevertheless, skin irritants are also known for their adjuvant‐like effects on contact allergy, resulting in skin hypersensitivity reactions like toxic dermatitis. The human organotypic skin explant culture (hOSEC) model is used to study the characteristics of chemical‐induced migration of CD1a+ LCs out of the epidermis in relation to irritancy or toxicity. We analysed cells emigrating out of hOSEC for CD1a+ LCs, CD83+ mature dendritic cells (DCs) and CCR7+ LN homing cells. After exposure to a toxic concentration of a non‐immunogenic irritant, an increase of CD1a+CD83+ LCs was found in the culture medium. A non‐toxic concentration of an sensitizer induced an increase of CD1a+ cells. About 50% of skin emigrating CD1a+ LCs were CD83– (immature) but all were CCR7+. Skin irritation by both non‐allergenic and allergenic compounds induces LC migration and maturation. In contrast, only allergenic compounds induced LC migration with partial maturation at subtoxic concentration. This effectively demonstrates that irritation is physiologically needed stimuli for inducing LC maturation.

Prostaglandin E2 inhibits and indomethacin and aspirin enhance, A23187-stimulated leukotriene B4 synthesis by rat peritoneal macrophages

1 The calcium ionophore, A23187, stimulated leukotriene B4 (LTB4), thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) synthesis by 4 day carrageenin‐elicited rat peritoneal macrophages. 2 At concentrations of 2 × 10−7‐2 × 10−5 m indomethacin and aspirin enhanced A23187‐stimulated LTB4 synthesis and inhibited PGE2 and TXB2 formation. 3 PGE2 inhibited A23187‐stimulated LTB4 and TXB2 formation as well as the augmentation of LTB4 release caused by aspirin and indomethacin. However, PGE2 was ineffective when the cells were challenged with arachidonic acid (AA). 4 Dibutyryl adenosine 3′: 5′‐cyclic monophosphate (db‐cyclic AMP) partially inhibited A23187‐stimulated LTB4 production. 5 Our results suggest that PGE2 inhibits macrophage LTB4 synthesis by limiting the availability of AA. Indomethacin and aspirin, possibly by removing the regulatory effect of PGE2, promote the synthesis of the pro‐inflammatory LTB4.

Subtoxic concentrations of allergenic haptens induce LC migration and maturation in a human organotypic skin explant culture model: a novel method for identifying potential contact allergens

Abstract:  The accelerated migration of Langerhans cells (LCs) out of the epidermis and up‐regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten‐specific delayed‐type hypersensitivity reaction. Here we report the findings of a study in which seven chemicals, coded and tested in a blind fashion, were classified as contact allergens or non‐allergens using the human organotypic skin explant culture (hOSEC) model. All chemicals that were identified as a contact sensitizer on decoding induced a definite decrease in the number of CD1a and HLA‐DR‐positive epidermal LCs in the epidermis of the skin explants, as determined by both semiquantitative immunohistochemistry and quantitative flow cytometric analysis. A significant increase in the number of CD83+ cells was accompanied by up‐regulation of activation molecules in the epidermis of hOSEC exposed specifically to contact allergens. In contrast, there were only minor alterations in epidermal LC numbers, expression of CD83 and other activation markers by LCs when the biopsies were treated with non‐toxic concentrations of non‐allergenic irritants and vehicles. The data suggest that an increased epidermal LC migration and maturation accompanied by increased expression of activation markers could be used as end‐point determinants to screen allergens in a non‐animal alternative hOSEC model.

The effect of acute feeding of carnitine, acetyl carnitine and propionyl carnitine on basal and A23187-stimulated eicosanoid release from rat carrageenan-elicited peritoneal macrophages.

Little is known about the ability of carnitine to modulate cell functions. As carnitine plays an important role in lipid metabolism we investigated the acute effect of L-carnitine, L-acetyl carnitine and L-propionyl carnitine (300 mg/kg per d; 4 d) on the basal and calcium-ionophore (A23187)-stimulated release of arachidonic acid metabolites from rat carrageenan-elicited peritoneal macrophages. A decrease in the number of peritoneal carrageenan-elicited macrophages was observed after feeding all three compounds. The basal release of prostaglandin E2, 6 keto-prostaglandin F1 alpha and leukotriene B4 was stimulated by all treatments. In contrast, thromboxane B2 production was diminished by feeding carnitine and acetyl carnitine. A23187-stimulated synthesis of 6 keto-prostaglandin F1 alpha and leukotriene B4 was further enhanced by all three compounds. Acetyl carnitine and propionyl carnitine also enhanced thromboxane B2 synthesis. However, no effects on prostaglandin E2 formation were detected. The 6 keto-prostaglandin F1 alpha:thromboxane B2 ratio, calculated from the basal and A23187-stimulated values, was increased by carnitine treatment. In the presence of A23187 there was also an increase in the 6 keto-prostaglandin F1 alpha:leukotriene B4 ratio. We conclude that carnitine, and possibly some of its derivatives, could modify the macrophage component of an inflammation in vivo.

Indomethacin stimulation of macrophage cytostasis against MOPC-315 tumor cells is inhibited by both prostaglandin E2 and nordihydroguaiaretic acid, a lipoxygenase inhibitor

SummaryIndomethacin enhanced macrophage cytostasis against MOPC-315 tumor cells in vitro. The effect of indomethacin was inhibited by prostaglandin E2 and by the lipoxygenase inhibitor nordihydroguaiaretic acid. Prostaglandin E2 and nordihydroguaiaretic acid also inhibited indomethacin stimulation of macrophage thymidine incorporation. Indomethacin inhibited macrophage prostaglandin E2 formation and stimulated leukotriene B4 synthesis. Nordihydroguaiaretic acid inhibited leukotriene B4 production. Our data indicate that eicosanoids play a role in regulating macrophage cytostasis.

Copper modulation of macrophage cyclooxygenase metabolite synthesis

The effect of copper on the release of cyclooxygenase metabolites from starch elicited, rat, peritoneal macrophages was investigated. Copper sulphate, in the range 10(-6)-10(-5) M, inhibited the formation of prostaglandin (PG) E2 and thromboxane (Tx) B2, the stable metabolite of TxA2, in a dose dependent manner but had no effect on the production of 6-keto-PGF1 alpha, the stable product of prostacyclin. At higher concentrations (5 x 10(-5) and 10(-4) M) the synthesis of all three metabolites of arachidonic acid (AA) was stimulated as was the release of radioactivity from macrophages prelabelled with 14C AA. Copper had no effect on the metabolism of exogenous AA however. At 10(-4) M copper also stimulated secretion of the lysosomal enzyme, beta-glucuronidase (GUR). Copper nitrate (10(-4) M), but not zinc sulphate, also stimulated eicosanoid formation and lysosomal enzyme release. Our results are consistent with the idea that copper stimulates eicosanoid formation via an effect on PL activity.

Enhanced prostaglandin E2 and thromboxane B2 release from resident peritoneal macrophages isolated from morphine-dependent rats.

Resident peritoneal macrophages from morphine‐addicted rats (4 days) released more prostaglandin (PG) E2 and thromboxane (Tx) B2, but not 6‐keto‐PGF1a, than cells from control animals. This effect, which was due to an enhancement of endogenous AA turnover, was not related to any changes in cAMP synthesis or lysososomal enzyme secretion. [D‐Ala2]‐Met‐enkephalin had no effect on eicosanoid release in vitro. Both morphine and PGE2 have been shown to depress macrophage functions. We suggest that morphine‐stimulated macrophage PGE2 synthesis, and the consequent inhibition of phagocytosis, could contribute to the decreased resistance to infections associated with opiate addiction.

Continuous monitoring of prostacyclin production by the isolated, intact, rat aorta using a bioassay technique

Isolated rat aortae were perfused with Krebs buffer in vitro and the synthesis of prostacyclin-like material (PGI2-L) was continuously monitored by measuring the contraction of a superperfused rat stomach strip exposed to the aortic perfusate. PGI2-L release was high after initiation of the aorta perfusion but then gradually declined and stabilized at a basal rate of production that was maintained for at least 180 min. Levels of 6 keto prostaglandin F1 alpha(6ketoPGF1 alpha), the stable breakdown product of PGI2, in the aortic perfusate reflected the changes in biological activity. The concentration of PGE2 in the aortic perfusate remained constant throughout the experiment while the level of thromboxane (Tx)B2 (the stable product of TxA2) decreased with time to below the level of detection of the radioimmunoassay (RIA) used. All biological activity was abolished by heating the aortic perfusate for 30 min at 37 degrees C, while perfusing with 30 microM indomethacin inhibited aorta PGI-L formation. Analysis (by linear regression) of the relationship between PGI2-L formation and the body weight or age of the animals used revealed that PGI2-L synthesis was better related to body weight (r2 = 0.90) than age (r2 = 0.75).

Differential regulation of the cyclooxygenase pathway in starch elicited rat peritoneal macrophages by prostaglandin E2, U-44069, a stable endoperoxide analogue and dibutyryl-cyclic AMP

The basal and carrageenin-stimulated release of thromboxane (TX) B2, the stable product of TXA2, 6-ketoPGF1 alpha, the stable breakdown product of prostacyclin (PGI2) and prostaglandin (PG) E2 from 24 h starch elicited rat peritoneal macrophages was inhibited by dibutyryl-cyclic AMP (db-cAMP). PGE2 also inhibited the release of TXA2 and 6-keto-PGF1 alpha whereas the stable endoperoxide analogue, U-44069, stimulated PGE2 and 6-keto PGF1 alpha release but inhibited TXB2 release. The effects of all three mediators tested were related to an increase of Mø intracellular cAMP content.

Effects of short- and long-term feeding of L-carnitine and congeners on the production of eicosanoids from rat peritoneal leucocytes

The effect of short- and long-term feeding with L-carnitine, L-acetyl carnitine and L-propionyl carnitine on the production of eicosanoids from in vitro stimulated carrageenan-induced rat peritoneal macrophages was investigated. Both young (4 weeks) and old (18 months) rats were used. A lower number of cells was isolated from the peritonea of treated than control young rats after 4 d feeding, but after 60 d no differences were observed. A similar reduction in cell number was found when old animals were given L-acetyl carnitine or L-propionyl carnitine (acutely) or L-acetyl carnitine or L-carnitine (chronically). Plasma carnitine levels were higher in young rats given carnitine both chronically and acutely. Carnitine derivatives were without effect. In contrast, levels of total carnitine in the plasma of old rats given L-carnitine and L-acetyl carnitine for 4 d and 60 d were higher than in controls. There was no correlation between total plasma carnitine level and effects on prostaglandin, thromboxane and leukotriene B4 (LTB4) production. In young rats the most important changes were observed in relation to the production of prostacyclin (PGI2), measured as 6 keto-prostaglandin F1 alpha. Prostacyclin production was higher in the groups given carnitine or its derivatives. The net result of the changes in PGI2 was that the 6 keto-prostaglandin F1 alpha: thromboxane B2 and the 6-keto-prostaglandin F1 alpha: LTB4 ratios tended to be higher in cells from young animals following short-term feeding with L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)