Anti-inflammatory effect of FK-506 on human skin mast cells.

FK-506 and the structurally related macrolide rapamycin are high-affinity ligands for a specific binding protein (FK-506 binding protein). We examined the effects of FK-506 and rapamycin on the release of pre-formed (histamine) and de novo synthesized inflammatory mediators (prostaglandin D2) from mast cells isolated from human skin tissue. FK-506 (0.1 to 100 nM) concentration-dependently inhibited (5 to 65%) histamine release from skin mast cells activated by anti-IgE. FK-506 was more potent in skin mast cells than in basophils (IC40 = 2.15 +/- 0.78 nM versus 5.12 +/- 1.34 nM; p < 0.001), whereas the maximal inhibitory effect was higher in basophils than in skin mast cells (88.77 +/- 2.44% versus 67.30 +/- 3.98%; p < 0.01). FK-506 had little or no inhibitory effect on histamine release from skin mast cells challenged with compound A23187 and substance P, respectively, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 (0.1 to 100 nM) also inhibited (up to 65%) the de novo synthesis of prostaglandin D2 from skin mast cells challenged with anti-IgE. Despite its structural similarity to FK-506, rapamycin (10 to 300 nM) had little or no effect on the release of histamine from skin mast cells induced by anti-IgE, A23187, and substance P. However, rapamycin competitively antagonized the inhibitory effect of FK-506 on anti-IgE-induced histamine release from skin mast cells with a dissociation constant of about 14 nM. These data indicate that FK-506, but not rapamycin, is a potent anti-inflammatory agent acting on skin mast cells presumably by binding to the FK-506 binding protein. It thus appears that binding to the FK-506 binding protein is necessary, but not sufficient, to deliver an inhibitory signal to skin mast cells.     

Studies on the contact sensitization of man with simple chemicals: V. Clonal priming allows direct in vitro assessment of autologous HLA-associated factors required for immune response to dinitrochlorobenzene.

Human lymphocytes from dinitrochlorobenzene (DNCB)-sensitized human subjects primed in first culture with dinitrophenylated-antigens yield a population of cells which respond in an accelerated manner to the same or similar antigen in second culture. Using this "clonal priming" approach, we have demonstrated that such a primed population showed maximal proliferative response to dinitrophenylated autologous cells. These DNP primed clones also showed responses to some dinitrophenylated allogeneic leukocytes. The magnitude of the accelerated blastogenic response with allogeneic leukocytes varied in most instances in relation to the degree of sharing of HLA-A, B, and DRw antigens with the original autologous stimulator. These findings show that the self-specific factors recognized in conjunction with the dinitrophenyl antigen are closely but not invariably associated with established major histocompatibility (MHC)-associated serologic typing results. While DNP primed clones fail to respond to unmodified autologous leukocytes, they often show significant responses to unmodified allogeneic leukocytes. If such accelerated responses to unmodified allogeneic leukocytes are not the result of nonspecific activation of allogeneic responding lymphocyte clones, these findings further indicate that DNP modified self can resemble some alloantigens.     

Immune response to human keratoacanthoma

Immune reactivity to human keratoacanthoma was investigated by microcytotoxicity tests and immunofluorescence. IgM and complement were consistently present in lesions; IgG and fibrin were infrequent. No evidence of in vivo bound immunoglobulin was found on the surface of keratoacanthoma cells by membrane immunofluorescence. Neither patients' sera nor peripheral blood leukocytes showed significant cytotoxicity against autochthonous tumour cells in microtitre assays. This study fails to support the view that regression of human keratoacanthoma is mediated by immunological mechanisms.     

Intraepidermal accumulation of polymorphonuclear leukocytes in annular pustular psoriasis

A case of annular pustular psoriasis is described. The annular zone of the lesion provides a model for studying the dynamics of intraepidermal accumulation of polymorphonuclear leukocytes in psoriasis. It could be shown that the unstimulated accumulation of polymorphonuclear leukocytes was limited to the clinically involved skin. The appearance of polymorphonuclear leukocytes proved to be associated with acanthosis. The micropustule formation following topical application of leukotriene B4 was markedly decreased in lesional, prelesional and postlesional skin, compared to the response in the clinically uninvolved skin of patients with chronic plaque psoriasis. Although leukotriene B4 might have an initiating role in the intraepidermal accumulation of polymorphonuclear leukocytes other factors are a conditio sine qua non for the peripheral spreading of the lesion in annular pustular psoriasis.     

Epidermal cell-polymorphonuclear leukocyte cooperation in the formation of leukotriene B4 by transcellular biosynthesis.

The cellular origin of Leukotriene B4, a potent pro-inflammatory agent that is present in psoriatic lesions, has not been completely ascertained. The present study was performed in order to assess the possible contribution of epidermal cells to leukotriene B4 synthesis through 5-lipoxygenase or by means of transcellular metabolism of the epoxide intermediate leukotriene A4 from activated polymorphonuclear leukocytes. The metabolism of exogenous arachidonic acid in fresh human epidermal cell, polymorphonuclear leukocyte or mixed suspensions was determined by means of high-performance liquid chromatography. Epidermal cells transformed arachidonic acid mainly into 12-hydroxy-eicosatetraenoic acid and prostaglandin E2. Formation of prostaglandins F2 alpha and D2, 12-hydroxy-eptadecatrienoic acid, and 15- and 11-hydroxy-eicosatetraenoic acids was also detected. We did not detect any eicosanoid derived from 5-lipoxygenase pathway. Mixed suspensions of polymorphonuclear leukocytes and epidermal cells (ratio 1:4) produced 1.72 times more leukotriene B4 than leukocytes alone under the same experimental conditions. Epidermal cells incubated with 5 microM authentic leukotriene A4 for 3 min yielded 2.954 +/- 0.27 pmoles/10(6) cells of leukotriene B4, which was characterized by co-elution with authentic standard and its ultraviolet absorption spectrum. These data demonstrate the existence of a leukotriene A4 epoxide hydrolase activity in human epidermal cells. Our results suggest that epidermal cells could cooperate in leukotriene B4 biosynthesis by transcellular metabolism of leukotriene A4 in lesions of psoriasis, and possibly other inflammatory dermatoses characterized by increased leukotriene B4 levels and prominent polymorphonuclear leukocyte infiltrates.     

Prostaglandins and chemotaxis: enhancement of polymorphonuclear leukocyte chemotaxis by prostaglandin F2alpha.

Prostaglandins E1, E2, F1alpha, and F2alpha did not demonstrate direct in vitro chemotactic properties either to rabbit peritoneal polymorphonuclear leukocytes or to rabbit or human polymorphonuclear leukocytes isolated from blood. Prostaglandin F2alpha, however, enhanced the chemotactic responsiveness of human polymorphonuclear leukocytes to the chemotactic agent casein.     

Increased hydroxyl radical generation by normal polymorphonuclear leukocytes incubated in sera from patients with leukocytoclastic vasculitis

Summary The effect of sera from patients with untreated leukocytoclastic vasculitis was investigated on the generation of oxygen intermediates by normal polymorphonuclear leukocytes. Sera from untreated patients induced increased hydroxyl radical generation, which is one of the most potent oxidants capable of causing tissue damage. It is suggested that vascular injury may be mediated in part by enhanced production of hydroxyl radical by polymorphonuclear leukocytes. Circulating immune complexes in the sera of the patients are considered to be one of the factors responsible for enhanced generation of hydroxyl radical.     

Anti-oxidant effects of gold compounds

The anti-oxidant efficacy, in vitro, of the gold compounds auranofin (AF) and gold sodium thiomalate (GST) was examined by studying their effects on the generation of reactive oxygen species (ROS) using zymosan-stimulated polymorphonuclear leukocytes (PMNs) and a cell-free, xanthine-xanthine oxidase system. The oxygen species investigated were the superoxide radical anion (O2-), hydrogen peroxide (H2O2) and the hydroxyl radical (OH.). AF had an inhibitory effect on ROS production by PMNs. In particular, OH. generation was significantly suppressed in a dose-dependent fashion. AF did not inhibit ROS production in the cell-free system. GST produced only a small degree of inhibition at higher concentrations. These findings suggest that AF may play an important role in the inhibition of respiratory bursts and the generation of inflammatory reaction products. Since the products of the respiratory burst, especially potent oxidants such as OH. and H2O2, are thought to be important inflammatory mediators, it is postulated that the blockade of toxic ROS generation by AF affects rheumatoid as well as dermatological inflammation and tissue damage.     

Production of superoxide free radicals by peripheral blood leukocytes in patients with psoriasis treated by the Re-PUVA-C method

The investigations were carried out in 20 patients with severe forms of psoriasis, and in 18 healthy subjects. The generation of free superoxide radicals by polymorphonuclear leukocytes of peripheral blood was evaluated by Bellavite et al. (1983) method by use superoxide dismutase of Sigma firm. The obtained results may confirm alterations in PMNL functions in patients with psoriasis observed by many authors. It may confirm the probability of damage of membrane structures and alterations in their bactericidal functions.     

Potentiation of the generation of reactive oxidants by human phagocytes during exposure to benoxaprofen and ultraviolet radiation in vitro

The effects of ultraviolet (UV) radiation on the spontaneous membrane-associated oxidative metabolism of human polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (MNL), co-incubated in the presence and absence of the non-steroidal, anti-inflammatory drug (NSAID) benoxaprofen at various concentrations, were investigated in vitro. Assays of superoxide generation and luminol-enhanced chemiluminescence (CL) were used to detect the production of reactive oxidants by PMNL and MNL. Benoxaprofen in the absence of UV radiation caused a dose-related activation of superoxide production and CL by PMNL. Exposure of PMNL to UV radiation in the absence of benoxaprofen caused a slight increase in superoxide generation and an unsustained slight increase in CL within 10 s. Exposure of both MNL and PMNL to UV radiation in the presence of benoxaprofen had a marked synergistic effect on both superoxide generation and CL. These effects were also achieved with UVA irradiation but were not detected when adherent cell depleted MNL or sodium fluoride (NaF) (10-2 M) pulsed PMNL, or PMNL from children with chronic granulomatous disease, were used. The pro-oxidative effects of benoxaprofen and UV radiation alone and in combination are dependent on intact phagocyte membrane-associated oxidative metabolism. It is postulated that the pro-oxidative interactions which occur between human phagocytes, benoxaprofen and ultraviolet radiation cause the dermatological side-effects of benoxaprofen.     

Serum eosinophil chemotactic factor levels in patients with bullous pemphigoid, drug reactions and atopic eczema.

The sera of 4 patients with bullous pemphigoid, of 5 patients with drug reactions and of 13 patients with atopic eczema were examined for the occurrence of low molecular weight eosinophil chemotactic factor (ECF) by fractionation on a Sephadex G 25 column. Almost all patients had a peripheral eosinophilia, and many had raised total serum IgE levels. ECF was demonstrated in the sera of all 4 patients with bullous pemphigoid and in 4 of 5 patients with systemic drug reactions. In contrast, the sera of the 13 patients with atopic eczema did not contain any ECF activity, nor did the 13 control sera. These findings suggest that the ECF from phagocytosing polymorphonuclear leukocytes (PMN) and/or the mast cell derived ECF-A contribute to the elevated serum ECF levels in patients with bullous pemphigoid and drug reactions. A correlation between serum ECF and IgE levels and peripheral eosinophilia could not be established.     

The effect of cyclosporin on human epidermal keratinocytes in vitro

Cyclosporin A has been shown to be effective in the treatment of severe, recalcitrant psoriasis, but it is uncertain whether the mode of action is primarily by immune suppression or by other mechanisms. Cyclosporin-dependent growth-inhibition has recently been demonstrated in vitro using several non-human and transformed epithelial cell lines. In this study the effect of cyclosporin on human epidermal keratinocytes and skin fibroblasts was investigated. Secondary cultures of human epidermal keratinocytes were grown on collagen-coated dishes in the presence of increasing concentrations of cyclosporin. Inhibition of growth was observed at 6-8 microM. An almost identical dose-response curve was obtained for the cytotoxic drug, cis-platin. Short-term exposure (I h) to cyclosporin did not have any effect on epidermal cell growth, suggesting that direct membrane-related effects were not involved. Analysis of cellular proteins by SDS-PAGE indicated no effect of continuous cyclosporin exposure on in vitro differentiation. The observation that human epidermal keratinocyte growth is inhibited by cyclosporin suggests that a topical form of therapy for psoriasis may be an effective alternative to oral treatment.     

Topical FK506: suppression of allergic and irritant contact dermatitis in the guinea pig

Topical FK506 has recently been shown to have an anti-inflammatory effect in vivo in humans. In this study its effects in contact dermatitis were studied in the guinea pig model. Topical FK506 suppressed both irritant and allergic patch-test reactions. The most prominent suppressive effect was seen when skin sites were pretreated with FK506. Topical FK506 did not impair the induction of contact allergy as assessed by challenges, although it suppressed local lymph node cell accumulation during contact allergy induction. Topical FK506 may hold promise as a treatment for skin disorders that respond to oral FK506 or cyclosporin A.Presented in part orally at the 54th Annual Meeting of the Society for Investigative Dermatology, Washington, D.C., 28 April 1993 (Lauerma et al. (1993) J Invest Dermatol 100: 491A)     

Psoriatic lesions do not respond to oral administration of lobenzarit disodium, an inhibitor of interleukin 1 production

Interleukin 1 (IL-1) has been implicated in the production of characteristic changes in psoriatic lesions such as epidermal hyperproliferation and accumulation of polymorphonuclear leukocytes within the epidermis. Because lobenzarit disodium, an immunomodulator, inhibits the production of IL-1 in peripheral and peritoneal macrophages, we studied the clinical effects of this compound on psoriatic lesions. A 12-week course of oral lobenzarit disodium treatment did not induce any remarkable changes in psoriasis lesions. This does not support the major pathogenic role of IL-1 in the development of psoriasis.     

Clofazimine-mediated enhancement of reactive oxidant production by human phagocytes as a possible therapeutic mechanism

Clofazimine, at concentrations within the therapeutic range (0.01-5 micrograms/ml), stimulated human polymorphonuclear leucocytes (PMNL) to generate increased amounts of reactive oxidants (RO) when activated with the tripeptide leucoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), calcium ionophore, phorbol myristate acetate and opsonised zymosan. Clofazimine per se did not activate the membrane-associated oxidative metabolism of PMNL, but rather primed these cells to hyperreact to the various stimuli. To investigate the therapeutic significance of these observations clofazimine was administered to patients with various chronic inflammatory diseases (lichen planus, discoid lupus erythematosus and rheumatoid arthritis) and generation of RO by FMLP-activated phagocytes was measured before and during clofazimine administration. A statistically significant potentiation of RO generation by FMLP-activated phagocytes was observed during clofazimine administration. Since RO are immunosuppressive and antimicrobial the therapeutic mechanisms of clofazimine may be related to pro-oxidative interactions of this agent with phagocytes.     

Anti-oxidant effects of retinoids on inflammatory skin diseases

Summary It is well known that retinoids are effective in the treatment of various dermatological disorders. It has been reported that retinoids have inhibitory effects on the generation of superoxide (O ₂ ^- ) by stimulated polymorphonuclear leukocytes (PMNs). In the present study, we investigated the effects of retinoids on the generation of O ₂ ^- and other reactive oxygen species (ROS), including OH, H₂O₂ and chemiluminescence, by zymosan-stimulated PMNs and in the xanthinexanthine oxidase system, because these potent ROS may play an important role in PMN-mediated skin inflammation. It was found that some retinoids had anti-oxidant effects in the PMN system; however, apart from the effect of tretinoin and Ro 10-1670 on OH generation, none of the retinoids studied inhibited ROS generation in the xanthine-xanthine oxidase system. On the basis of these results, we discuss a possible mechanism connected with the role of ROS by which retinoids have favourable effects on several inflammatory skin disorders.     

Analysis of the interaction of human C5a and C5a des Arg with human monocytes and neutrophils: flow cytometric and chemotaxis studies

C5a and C5a des Arg are potent complement-derived mediators that bind receptors on peripheral blood leukocytes and tissue-specific cellular elements to elicit and amplify inflammatory and immunomodulatory reactions. To study the interactions of C5a and C5a des Arg with these cells, fluorescein conjugates of these ligands were prepared by a new technique and their binding to monocytes, neutrophils, platelets, and endothelial cells was studied with flow cytometry. Fluoresceinated C5a produced neutrophil myeloperoxidase release and chemotaxis and also bound rabbit anti-C5a antibody much like native anaphylatoxin; likewise, fluoresceinated C5a des Arg demonstrated retention of biologic and antigenic activities. Both fluorescein-conjugated C5a and C5a des Arg bound to monocytes and neutrophils in a concentration-dependent, saturable, and homogeneous manner, but 10- to 15-fold higher concentrations of C5a des Arg were required to attain saturable binding of these leukocytes. Ligand binding was specifically inhibited by native purified human C5a in a concentration-dependent manner, while it was unaffected by C3a or N-formyl-methionyl-leucyl-phenylalanine-lysine. There was no evidence of a C5a receptor-negative subpopulation of monocytes or neutrophils. Moreover, comparative binding experiments with leukocytes from multiple normal volunteers showed that a greater percentage of monocytes than neutrophils bound C5a at less than saturable concentrations of ligand (P less than 0.05, 0.5 to 5.0 nM). A representative half-maximal binding of fluorescein-conjugated C5a (C5a des Arg) binding to monocytes and neutrophils was 1.2 nM (30 nM) and 2.6 nM (68 nM), respectively. In contrast, fluorescein-conjugated C5a did not specifically bind to human platelets or umbilical vein endothelial cells.     

Anti-inflammatory effect of cyclosporin A on human skin mast cells.

We have examined the effects of cyclosporin A (CsA) and cyclosporin H (CsH), which bind with different affinity to cyclophilin, to evaluate the role of this protein in the release of preformed (histamine) and de novo synthesized (prostaglandin D2[PGD2]) mediators of inflammatory reactions from human skin mast cells (HSMC). CsA (2.4-800 nM)-inhibited (5-60%) histamine release from HSMC challenged with anti-IgE. CsA exerted little, if any, inhibitory effect on histamine release from HSMC challenged with compound A23187 and substance P, whereas it completely suppressed A23187-induced histamine release from human basophils. Inhibition of histamine release from HSMC challenged with anti-IgE was extremely rapid and was not abolished by washing (three times) the cells before anti-IgE challenge. CsA (2.4-800 nM) markedly inhibited (25-70%) the de novo synthesis of PGD2 from HSMC challenged with anti-IgE. CsH, which has an extremely low affinity for cyclophilin, had no effect on skin mast-cell mediator release. These data suggest that CsA is a potent anti-inflammatory agent acting on HSMC, presumably by interacting with cyclophilin.     

Effects of FK506 and Cyclosporin a on proliferation,histamine release and phenotype of murine mast cells

Using mouse peritoneal cavity mast cells, we investigated the effects of FK506 and cyclosporin A (CsA) on cell proliferation and histamine release induced by anti-IgE antibody, calcium ionophore (A23 187), or neuropeptide (substance P). Both FK506 and CsA inhibited cytokine-dependent mast cell proliferation in a dose-dependent manner. The inhibitory effects of these compounds on mast cell proliferation was reversible; the removal of the chemicals from the incubation medium resulted in the reinitiation of mast cell proliferation. Flow cytometric analysis suggested that the inhibitory effect of FK506 and CsA was mostly due to G ₁ /S boundary block, although a significant number of G ₂ -arrested cells were also observed following FK506 treatment. Both FK506- and CsA-treated mast cells showed a similar inhibition of histamine release induced by A23187. However, CsA at higher concentrations inhibited the histamine release induced by anti-IgE antibody or substance P more markedly than FK506. Cellular histamine content was decreased by CsA treatment while FK506 had no effect. The staining properties of peritoneal mast cells changed from connective tissue-type mast cell-like to mucosal mast cell-like during CsA treatment but not during FK506 treatment. Thus FK506 and CsA have different effects on mast cell proliferation as well as histamine release, that might be associated with a phenotypic change of the cells during culture. Received: 28 February 1995