Karsten Fogh

Eicosanoids in skin of patients with atopic dermatitis: Prostaglandin E2 and leukotriene B4 are present in biologically active concentrations

The biochemical events leading to atopic dermatitis (AD) are unknown. Certain eicosanoids derived from arachidonic acid are potent mediators of skin inflammation and modulators of certain T-lymphocyte activities. The purpose of the present study was to determine whether eicosanoids are present in biologically active concentrations in the skin of adult patients with AD. The levels of the cyclooxygenase product, prostaglandin E2 (PGE2) and the lipoxygenase products, leukotriene B4 (LTB4), 12- and 15-hydroxyeicosatetraenoic acid were determined in biopsy specimens obtained by keratome from lesional, perilesional, and clinically unaffected skin of patients with AD. Methods for identification of eicosanoids included reversed-phase high-performance liquid chromatography combined with radioimmunoassays. Eicosanoid levels were at the same level in normal skin and in uninvolved skin of AD. Compared with uninvolved skin, both lesional and perilesional skin contained markedly elevated concentrations of PGE2 and LTB4: PGE2, 97.2 +/- 15.6 ng/gm of lesional skin and 128.3 +/- 27.2 ng/gm of perilesional skin; LTB4, 5.2 +/- 1.6 ng/gm of lesional skin and 3.2 +/- 0.6 ng/gm of perilesional skin. Compared with uninvolved skin, the levels of 12- and 15-hydroxyeicosatetraenoic acid were elevated sevenfold and elevenfold, respectively, in lesional skin, but did not reach biologically active concentrations. The results demonstrate that the inflammatory mediators PGE2 and LTB4 are present in lesional skin of atopic subjects in biologically active concentrations. Because these mediators are able to induce cutaneous inflammation and to modulate cellular immunity, they may be involved in the biochemical processes leading to AD.

Improvement of psoriasis vulgaris after intralesional injections of 15-hydroxyeicosatetraenoic acid (15-HETE)

Psoriatic skin lesions are characterized by elevated levels of 5- and 12-lipoxygenase products (leukotrienes B4, C4, and D4, and 12-hydroxyeicosatetraenoic acid [12-HETE]), which can stimulate epidermal proliferation and induce skin inflammation. 15-Hydroxyeicosatetraenoic acid (15-HETE) has the potential to inhibit the activity of 5- and 12-lipoxygenases. The purpose of the present study was to determine the therapeutic effect of intralesional injections of 15-HETE. 15-HETE was formed by oxidation of arachidonic acid by soybean lipoxygenase, purified by reversed-phase high-performance liquid chromatography, and identified by mass spectrometric analysis. Thirteen patients took part in the investigation. Plaques with a diameter of approximately 1 cm were injected with 0.1 ml of 10 mumol/L 15-HETE, 0.1 ml of 1 mumol/L 15-HETE, or 0.1 ml of saline weekly. After 3 weeks the effect was evaluated clinically and histologically by an observer uniformed of the treatment given. We found that plaques injected with 10 mumol/L 15-HETE had cleared completely in four patients and improved considerably in seven. In one patient minimal improvement only was seen and in one patient no change was observed. Injection of 1 mumol/L 15-HETE was without effect in 11 patients and improvement was observed in two patients. Of the plaques injected with saline, minimal improvement was observed in one patient; otherwise the plaques had not changed. Injection of 0.1 ml of 10 mumol/L 15-HEPE (identical to 15-HETE except for five double bonds instead of four) induced only minimal improvement in one of four patients. The results imply that 15-HETE by a dose-dependent and stereospecific mechanism can improve psoriasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin D3 analogues

A vitamin D3 analogue having at least one hydroxyalkyl substituent on the A-ring, preferably the 1-position which is useful in inhibiting cell proliferation.

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophilsin vitro. Evidence of formation of antiinflammatory compounds

Enzymatic transformation of then-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B4 (LTB4), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of othern-6 fatty acids on the formation of LTB4 by human neutrophils and to determine if thesen-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 μM) and different concentrations (0–100 μM) of then-6 fatty acids linoleic acid (LA) and dihomo-gammalinolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB4 was dose dependently inhibited by both LA (IC50=45 μM) and DGLA (IC50=40μM). This inhibition of LTB4 formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB4 formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB4 formation (IC50=7.5 μM and IC50=0.2 μM). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB4 formation (IC50=0.75 μM). The results show that the addition of LA and DGLA to neutrophils results in an inhibition of LTB4 formation and simultaneously to the formation of 13-HODE and 15-HETrE, that also inhibits LTB4 formation. Therefore, dietary supplementation or topical application of LA and DGLA or preferentially their respective 15-LO products, may have a therapeutic effect in inflammatroy diseases in which LTs are suspected to play a pathogenic role.

15-Hydroxyeicosatetraenoic Acid (15-HETE) Specifically Inhibits LTB4-Induced Chemotaxis of Human Neutrophils

15-Hydroxyeicosatetraenoic acid (15-HETE), a 15-lipoxygenase (15-LO) product of arachidonic acid has the potential to inhibit leukotriene formation. In the present study the effect of 15-HETE on leukotriene B4 (LTB4)-induced polymorphonuclear leukocytes (PMN) and monocyte chemotaxis was investigated. LTB4-induced chemotaxis of PMNs was inhibited in a dose-dependent manner by 15-HETE. Maximum inhibition (68%) occurred at a 15-HETE concentration of 10(-4) M. The 15-LO product of eicosapentaenoic acid (15-HEPE) was approximately 10 times less potent in inhibiting LTB4-induced PMN chemotaxis. LTB4-induced chemotaxis of monocytes was unaffected by both 15-HETE and 15-HEPE. Using N-formyl-methionyl-leucyl-phenylalanine (FMLP) and complement split product C5a as chemoattractants, 15-HETE did not decrease PMN chemotaxis. Furthermore, 15-HETE itself did not affect random migration of leukocytes. The present results demonstrate that 15-HETE inhibits LTB4-induced chemotaxis of PMNs in vitro in a specific and selective way. Because 15-HETE not only inhibits formation, but also the effect of LTB4, it may be important in regulating LTB4-induced inflammation.

Effect of dihomogammalinolenic acid and its 15-lipoxygenase metabolite on eicosanoid metabolism by human mononuclear leukocytes in vitro: selective inhibition of the 5-lipoxygenase pathway

SummaryThe purpose of the present study was to determine the effect of the n-6 fatty acid, dihomogammalinolenic acid (DGLA, 20∶3, n-6) on arachidonic acid (AA) (C20∶4) metabolism by human peripheral mononuclear leukocytes (HPML). After incubation of HPML with A23187 (5 ΜM) and DGLA, the cyclooxygenase (CO) and lipoxygenase (LO) products were separated and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with radioimmunoassay. DGLA led to no change in PGE2 formation, but at similar concentrations there was a dose-dependent decrease in LTB4 formation (IC50=45.0 ΜM). The inhibition of LTB4 formation by DGLA was associated with a dosedependent increase in its 15-LO metabolite 15-hydroxyeicosatrienoic acid (15-HETrE) and its CO metabolite prostaglandin E1 (PGE1). Incubation of HPLM with 15-HETrE (0–1.5 ΜM) alone did not result in a change in PGE2 formation, whereas 15-HETrE was a much more potent inhibitor of LTB4 formation (IC50=0.5 ΜM) than DGLA. These results show that the addition of DGLA to HPML results in a selective inhibition of LTB4 formation, presumably via its metabolite (15-HETrE).

Recent Developments in Vitamin D Analogs

Within the past decade it has been shown that psoriasis can be treated topically with analogs of vitamin-D3. Impaired differentiation and increased proliferation of keratinocytes are key features in psoriatic lesions together with a local activation of T lymphocytes. Evidence has accumulated showing that analogs of vitamin D3 increase differentiation and inhibit proliferation of keratinocytes. Therefore, analogs of vitamin D3 have been investigated in a number of trials showing improvement of psoriasis. It has been shown that vitamin D analogs are better than their vehicle and show the same potency as potent topical steroids. However, vitamin D analogs have been proven efficacious and without side effects also when used on long term basis. Vitamin D analogs can be used both as monotherapy and in combination topical steroids, UVB, PUVA, retinoids and cysclosporine. The vitamin D3 analog calcipotriol has been investigated in most detail and is available as an ointment, a creme and as a scalp solutation. From clinical studies involving thousands of patients, it can be concluded that calcipotriol is efficacious, safe, well tolerated and can be used on a long term basis. Other analogs are available, however, these analogs have not been studied in greater details yet.

New Vitamin D Analogs in Psoriasis

Psoriasis is a common inflammatory and hyperproleferative skin disease characterized by infiltrated plaques of the skin and may involve nails, scalp and intertreginous areas. Recent years of research has shown that psoriasis can be treated topically with analogs of vitamin-D(3). Impaired differentiation and increased proliferation of epidermal keratinocytes are key features in psoriatic lesions together with a local activation of T lymphocytes. Evidence has accumulated that analogs of vitamin D(3) increase differentiation and inhibit proliferation of keratinocytes. Topical treatment with analogs of vitamin D(3) have in a number of trials shown improvement of psoriasis. Vitamin D analogs show the same efficacy as potent topical corticosteroids and do not produce skin atrophy during long-term therapy. Vitamin D analogs can be used both as monotherapy and in combination with topical corticosteroids, UVB, PUVA, acitretin, methotrexate and cyclosporine. The vitamin D(3) analog calcipotriol has been investigated in most detail and is available as an ointment, a cream and as a scalp solution. From clinical studies involving several thousands of patients, it can be concluded that calcipotriol is efficacious, safe and well-tolerated even on a long term basis.

Upregulation of vitamin D receptor levels by 1,25(OH)2 vitamin D3 in cultured human keratinocytes.

Abstract The natural biologically active form of vitamin D 3 , 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), possesses antiproliferative, prodifferentiating and immunomodulatory properties. The actions of 1,25(OH) 2 D 3 are mediated through the intracellular vitamin D receptor (VDR), and the level of VDR is believed to determine the cellular responsiveness to vitamin D 3 . In the present study we examined the effects of 1,25(OH) 2 D 3 on the expression of VDR and its message in cultured human keratinocytes. Western analysis showed the mean VDR content to be higher in undifferentiated cultures (175 pg/μg protein) than in differentiated cultures (90 pg/μg protein). Incubation with 1,25(OH) 2 D 3 induced an increase in the VDR in both undifferentiated and differentiated keratinocytes. The VDR increase was detectable after 2 h and maximal (approximately two-fold stimulation) after 8 h. The 1,25(OH) 2 D 3 -induced stimulation of VDR levels was dose dependent with a maximum at 10 –7 M . The VDR mRNA levels as determined by the ribonuclease protection assay showed a peak (50% stimulation) after approximately 2 h. Although this increase in VDR mRNA was not statistically significant, the results indicate that the ligand-induced upregulation of VDR involves increased transcription. The upregulation of VDR levels may increase the responsiveness to 1,25(OH) 2 D 3 and may, therefore, be an important mechanism for regulating the effects of 1,25(OH) 2 D 3 on keratinocyte proliferation and differentiation.

In vitro inhibition of leukotriene B4 formation by exogeneous 5-lipoxygenase inhibitors is associated with enhanced generation of 15-hydroxy-eicosatetraenoic acid (15-HETE) by human neutrophils

SummaryLeukotrienes, products of the 5-lipoxygenase pathway of arachidonic acid metabolism, have been suggested to play a pathogenic role in psoriasis, because of their ability to induce skin inflammation and to stimulate epidermal proliferation. The 15-lipoxygenase product 15-hydroxy-eicosatetraenoic acid (15-HETE) has no proinflammatory capacity. In contrast, it can inhibit the activity of the 5-lipoxygenase. The purpose of the present study was to study the effect of 5-lipoxygenase inhibitors on the formation of 15-HETE by human neutrophils in vitro. Purified neutrophils were incubated with A 23187 (5 μM) and arachidonic acid (25μM) with and without different inhibitors of 5-lipoxygenase activity (RS 43179, benoxaprofen, NDGA, and CP 66248). Methods for identifying eicosanoids included RP-HPLC and radioimmunoassay. Formation of leukotriene B4 was inhibited in a dose-dependent way, which was strongly correlated with a concomitant increase in the formation of 15-HETE (r=0.97, p<0.01). The cyclooxygenase inhibitor indomethacin did not change 15-HETE formation. The stimulation of 15-HETE formation was not associated with cell damage as assessed by LDH release. Furthermore, identical incubations of T lymphocytes, characterized by a low 5-lipoxygenase activity, did not result in increased 15-HETE formation. These results show that inhibition of 5-lipoxygenase activity can lead to increased formation of 15-HETE. Because 15-HETE inhibits formation of 5-LO products, it may amplify the effect of 5-lipoxygenase inhibitors.